Li ni u

Sinh hc phn t l khoa hc nghin cu cc hin tng sng mc phn t. Phm vi nghin cu ca mn hc ny c phn trng lp vi mt s mn hc khc trong sinh hc c bit l di truyn hc v ha sinh hc. Sinh hc phn t ch yu tp trung nghin cu mi tng tc gia cc h thng cu trc khc nhau trong t bo, bao gm mi quan h qua li gia qu trnh tng hp ca DNA, RNA v protein v tm hiu cch thc iu ha cc mi tng tc ny. Hin nay, sinh hc phn t v sinh hc t bo c xem l nn tng quan trng ca cng ngh sinh hc. Nh pht trin cc cng c c bn ca sinh hc phn t nh cc enzyme ct hn ch, DNA ligase, cc vector to dng, lai phn t, k thut PCR... sinh hc phn t ngy cng t nhiu thnh tu ng dng quan trng. Gio trnh sinh hc phn t ny cung cp nhng kin thc c bn cho sinh vin vi cc ni dung chnh sau: - Cu trc v chc nng ca gen - Cu trc genome - Cc qu trnh ti bn, phin m v dch m ca nguyn liu di truyn - iu ha biu hin gen - Sa cha v bo v gen - Ti t hp v chuyn gen Do mi c xut bn ln u nn gio trnh ny kh trnh khi thiu st hoc cha p ng c yu cu bn c. V th, chng ti mong nhn c nhiu kin ng gp ln xut bn sau c hon thin hn. Chng ti chn thnh cm n Qu Nng cao cht lng-D n Gio dc i hc h tr chng ti bin son gio trnh ny, PGS. TS. Nng Vn Hi c bn tho v gp nhiu kin qu bu.

Cc tc gi

Chng 1

Cac ai phn t sinh hoc

I. Nucleic acid Nucleic acid, vt cht mang thng tin di truyn cua cac h thng sng, la mt polymer hinh thanh t cac monomer la nucleotide. Mi nucleotide gm ba thanh phn: nhom phosphate, ng pentose (ng 5 carbon) va mt nitrogen base. Cac nitrogen base thuc hai nhom: cac purine gm adenine (A) va guanine (G), cac pyrimidine gm thymine (T), cytosine (C) va uracil (U). Cac nucleotide c ni vi nhau bng lin kt phosphodiester tao thanh chui dai. Nucleic acid gm hai loai phn t co cu tao rt ging nhau la deoxyribonucleic acid (DNA) va ribonucleic acid (RNA).

1. Deoxyribonucleic acid Phn t DNA la mt chui xon kep gm hai si n. Mi si n la mt chui nucleotide (Hnh 1.1). Mi nucleotide gm ba thanh phn: nhom phosphate, ng deoxyribose va mt trong bn base (A, C, G v T) (Hnh 1.2). Hai si n kt hp vi nhau nh cac lin kt hydrogen hinh thanh gia cac base b sung nm trn hai si: A b sung cho T va C b sung cho G. Mi si n co mt trinh t inh hng vi mt u 5phosphate t do, u kia la 3 hydroxyl t do (quy c la 5 3). Hng cua hai si n trong chui xon kep ngc nhau, nn c goi la hai si i song. Nhng phn tich cu truc hin ai a cho thy cu truc cua DNA khng phai lun lun tng ng vi dang c goi la B ma Watson va Crick a a ra. Do s tac ng cua cac hp cht co khi lng nho hoc protein, dang B co th chuyn sang dang A (nen nhiu hn) hoc la dang Z (xon trai). Chung co th t gp lai hoc xon manh, vi du mt si i DNA co dai la 20 cm c nen trong mt chromosome co kich thc la 5 m.

Hnh 1.1. Chui xon kp ca DNA

Phn t DNA trong nhim sc th cua sinh vt eukaryote dang mch thng, con phn ln t bao prokaryote (vi khun) phn t DNA li co dang mch vong. Tuy nhin, du dang nao thi cac phn t DNA ny u tn tai theo kiu cun cht. Trong t bao eukaryote, DNA kt hp cht che vi cc protein la histone. DNA eukaryote co kich thc rt ln (Vi du: DNA ngi co th dai n 1 m) nn vn t ra la phn t nay phai c nen nh th nao trong mt th tich rt han ch cua nhn. Vic nen c thc hin nhiu mc , mc thp nht la nucleosome va mc cao nht la cu truc nhim sc cht. Tht vy, ng kinh cua chui xon DNA chi la 20 , trong khi si nhim sc cht quan sat di kinh hin vi in t co ng kinh 100 , i khi at 300 . iu nay chng to phn t DNA tham gia hinh thanh nhng cu truc phc tap hn (Hinh 1.3). Si nhim sc cht co ng kinh 100 la mt chui cha nhiu nucleosome. o la nhng cu truc hinh thanh t mt si DNA qun quanh mt loi gm 8 phn t histone (mc t chc cao nht cua DNA). Si c ng knh 100 nay c cu truc phc tap hn si co ng kinh 300 . Trong nhn t bo, cc si va k trn kt hp cht ch vi nhiu protein khc nhau v c vi cc RNA to thnh nhim sc cht, mc t chc cao nht ca DNA.

Hnh 1.2. Cu trc cc nucleotide in hnh

Hnh 1.3. Cu trc nucleosome v nhim sc th. Phn t DNA c cun li trn nhim sc th lm cho chiu di ngn li hn 50.000 ln.

Cac DNA eukaryote co c im khac vi DNA prokaryote. Toan b phn t DNA prokaryote u mang thng tin ma ha cho cac protein trong khi o DNA ca eukaryote bao gm nhng trinh t ma ha (cac exon) xen ke vi nhng trinh t khng ma ha (intron). Cac trinh t ma ha eukaryote chim ngp trong mt khi ln DNA ma cho n nay vn cha ro tac dung c gi l DNA rc (junk DNA). Ty theo mc hin din cua chung trong nhn, cac trinh t DNA c chia lam ba loai: - Cac trinh t lp lai nhiu ln. V du: ng vt co vu cac trinh t nay chim 10-15% genome (h gen). o la nhng trinh t DNA ngn (10-200 kb), khng ma ha, thng tp trung nhng vung chuyn bit trn nhim sc th nh vung tm ng (trinh t CEN) hay u cac nhim sc th (trinh t TEL). Chc nng cua cac trinh t nay cha ro, co th chung tham gia vao qua trinh di chuyn DNA trn thoi v sc (trinh t CEN) hoc vao qua trinh sao chep toan b phn DNA nm u mut nhim sc th (trinh t TEL). - Cac trinh t co s ln lp lai trung binh. Vi du: genome ngi cac trinh t nay chim 25-40 %. Chung a dang hn va co kich thc ln hn (100-1.000 kb) cac trinh t lp lai nhiu ln. Cac trinh t nay phn b trn toan b genome. Chung co th la

nhng trinh t khng ma ha ma cung co th la nhng trinh t ma ha cho rRNA, tRNA va 5S RNA. - Cac trinh t duy nht. L cac gen ma ha cho cac protein, co trinh t c trng cho tng gen. Mt c im cua phn t DNA co y nghia rt quan trong v c s dung vao phng phap lai phn t, la kha nng bin tinh va hi tinh. Bin tinh la hin tng hai si n cua phn t DNA tach ri nhau khi cac lin kt hydrogen gia cac base b sung nm trn hai si bi t do cac tac nhn ha hoc (dung dich kim, formamide, urea) hay do tac nhn vt ly (nhit). Sau o, nu iu chinh nhit va nng mui thich hp, cac si n co th bt cp tr lai theo nguyn tc b sung, hinh thanh phn t DNA ban u, o la s hi tinh.

2. Ribonucleic acid Phn t RNA co cu tao tng t DNA ngoi tr ba im khac bit sau: - Phn t RNA la chui n. - ng pentose cua phn t RNA la ribose thay vi deoxyribose. - Thymine (T), mt trong bn loai base hinh thanh nn phn t DNA, c thay th bng uracil (U) trong phn t RNA. Cu truc va chc nng cua RNA co s bin i ro rt. V c ban RNA chi la cht mang thng tin di truyn virus, sau o ngi ta chng minh rng no khng nhng ong vai tro c ban vic chuyn thng tin di truyn ma con co vai tro cu truc khi tao nn phc h RNA-protein. Theo mt ly thuyt tin ha ma ai din la Eigen, RNA la cht mang thng tin di truyn, thanh vin trung gian cua s biu hin gen, thanh phn cu tao va la cht xuc tac. Nhom OH vi tri th hai cua ribose cn thit cho a chc nng lam nhiu loan s tao thanh si i, qua o lam tng khng bn vng cua lin kt phosphodieste. Trong t bao co ba loai RNA chinh, co cac chc nng khac nhau:

2.1. Cac RNA thng tin (mRNA) mRNA la ban sao cua nhng trinh t nht inh trn phn t DNA, co vai tro trung tm la chuyn thng tin ma ha trn phn t DNA n b may giai ma thanh phn t protein tng ng. Cac RNA co cu truc a dang, kich thc nho hn so vi DNA vi chi cha thng tin ma ha cho mt hoc vai protein va chi chim khoang 2-5% tng s RNA trong t bao. Qua trinh chuyn thng tin c th hin nh sau:

 E. coli, kich thc trung binh cua mt phn t mRNA khoang 1,2 kb.

2.2. RNA vn chuyn (tRNA) tRNA lam nhim vu vn chuyn cac amino acid hoat ha n ribosome tng hp protein t cac mRNA tng ng. Co it nht mt loai tRNA cho mt loai amino acid. tRNA vn chuyn cha khoang 75 nucleotide (co khi lng khoang 25 kDa), la phn t RNA nho nht. Cac tRNA co cu truc dang co ba la. Cu truc nay c n inh nh cac lin kt b sung hin din nhiu vung cua phn t tRNA. Hai vi tri khng co lin kt b sung ong vai tro c bit quan trong i vi chc nng cua tRNA: - Trinh t anticodon gm ba nucleotide. - Trinh t CCA, co kha nng lin kt cng ha tri vi mt amino acid c trng.

2.3. RNA ribosome (rRNA) rRNA la thanh phn c ban cua ribosome, ong vai tro xuc tac va cu truc trong tng hp protein. Ty theo h s lng rRNA c chia thanh nhiu loai: eukaryote co 28S; 18S; 5,8S va 5S rRNA; con cac rRNA E. coli co ba loai: 23S, 16S va 5S. rRNA chim nhiu nht trong ba loai RNA (80% tng s RNA t bao), tip n la tRNA khong 16% va mRNA chi khong 2%. Ngoi ra, t bao sinh vt eukaryote con cha nhng phn t RNA kch thc nho ca nhn (small nuclear, snRNA) chim khong <1% tham gia vao ghep ni cac exon. Ribosome l nhng phn t cn thit cho s tng hp protein, ribosome cua moi t bao u gm mt tiu n vi nho va mt tiu n vi ln. Mi tiu n vi co mang nhiu protein va rRNA (trong rRNA l thnh phn ch yu chim khong 65%) co kich thc khac nhau. Ngi ta cng thy ribosome trong ty th, c s tng hp mt s protein ty th.

Bang 1.1. Cac phn t RNA trong E. coli

2.3.1. Ribosome ca prokaryote T bo c nghin cu v ribosome nhiu nht l E. coli. Ribosome (70S) ca E. coli gm hai tiu n v: tiu n v nh (30S) v tiu n v ln (50S). Cn c vo h s lng, ngi ta phn bit ba loi rRNA: 23S rRNA, 16S rRNA v 5S rRNA. - Tiu n v 30S cha: 1 phn t 16S rRNA (c 1540 nu) v 21 ribosomal protein khc nhau. - Tiu n v 50S cha: 1 phn t 5S rRNA (c 120 nu), 1 phn t 23S rRNA (c 2900 nu) v 34 ribosomal protein. Hai tiu n v nh v ln khi kt hp vi nhau s to ra mt rnh ch tip gip ca chng cho mRNA i qua.

2.3.2. Ribosome ca eukaryote Ribosome ca eukaryote (80S) ln hn ribosome ca prokaryote cng bao gm hai tiu n v: tiu n v nh (40S) v tiu n v ln (60S). - Tiu n v 40S cha: 1 phn t 18S rRNA (c 1900 nu) v 33 ribosomal protein. - Tiu n v 60S cha: 3 phn t rRNA (5S; 5,8S v 28S) v 49 ribosomal protein. Tm li, tt ca RNA trong t bao u c tng hp nh enzyme RNA polymerase. Enzyme nay oi hoi nhng thanh phn sau y: - Mt khun mu, thng la DNA si i. - Tin cht hoat hoa: Bn loi ribonucleoside triphosphate: ATP, GTP, UTP va CTP.

Sinh tng hp RNA ging DNA mt s im, th nht hng tng hp la 53, th hai la c ch keo dai ging nhau: nhom 3-OH u cui cua chui tng hp la vi tri gn kt cua nucleoside triphosphate tip theo. Th ba, s tng hp xay ra do thy phn pyrophosphate. Tuy nhin, khac vi DNA la RNA khng oi hoi mi (primer). Ngoai ra, RNA polymerase khng co hoat tinh nuclease sa cha khi cac nucleotide bi gn nhm. Ca ba loai RNA trong t bao c tng hp trong E. coli nh mt loai RNA polymerase. ng vt co vu, cac RNA khac nhau c tng hp bng cac loai RNA polymerase khac nhau.

II. Protein 1. Cu truc cua protein Amino acid la n vi c s (monomer) cu thanh protein. Tt ca 20 amino acid co mt trong protein u c xy dng theo mt kiu mu chung:

Cng thc tng quat cua L--amino acid

Trong o, gc R (mch bn) cung la phn khac duy nht gia 20 loai amino acid, quy inh tinh cht cua tng loai. Nhom amine (NH2) inh nguyn t C2, theo tn cu la nguyn t C. Vi vy, ngi ta goi la nhom -amine. Cac amino acid tn tai chu yu trong t nhin co nhom amine ng bn trai truc, c goi la amino acid dang L. Dang D-amino acid chi tn ti ring bit, vi du trong thanh t bao vi khun. Cac amino acid ring bit co nhng c tinh khac nhau la do gc R ca chng. Nhng amino acid trung tnh c mt nhm amine v mt nhm carboxyl. Nhng protein cha nhiu amino acid trung tnh l nhng protein trung tnh. Khi chiu dai gc R tng s hnh thnh c tinh ky nc. Nhng protein co cha nhiu amino acid nh valine, leucine, isoleucine co tinh cht c trng l ky nc. Nhng amino acid co tinh acid trong phn gc co mt nhom carboxyl. Protein cha nhiu amino acid co tinh acid la nhng protein acid. Tng t nh vy i vi protein chu yu c hnh thnh bi nhng amino acid co tinh kim la nhng protein kim. Phn gc R cua amino acid co y

nghia quyt inh i vi c tinh cua protein ma chung tao nn. iu nay khng nhng co y nghia i vi tinh cht ha hoc ma ca cu truc cua protein. Thuy phn hoan toan protein, thu c chu yu cac L--amino acid. Mc du protein rt a dang nhng hu ht chung u c cu tao t 20 L--amino acid. Da vao c tinh cua gc R, amino acid c chia lam by nhom chinh sau y: - Amino acid trung tinh mach thng. Bao gm glycine, alanine, valine, leucine va isoleucine. - Cac hydroxyl amino acid mach thng. Bao gm serine va threonine. - Amino acid cha lu huynh mach thng. Bao gm cysteine va methionine. Khi oxy ha hai nhom -SH cua hai phn t cysteine tao thanh cystine co cha cu (-S-S-). - Cac amino acid acid va cac amide. Bao gm aspartic acid va glutamic acid. Trong phn t cua chung co mt nhom amine va hai nhom carboxyl. pH sinh ly (6 7), cac amino acid nay tich in m. Amine ha nhom carboxyl mach bn cua aspartate va glutamate tao thanh cac amide tng ng la asparagine va glutamine. - Cac amino acid kim. Bao gm lysine va arginine. - Iminoacid. Proline. - Cac amino acid thm va di vong. Bao gm phenylalanine, tyrosine va tryptophan. Do co cha vong thm nn cac amino acid nay co mt s phan ng c trng. Cac amino acid c ni vi nhau bi cac lin kt peptide, lin kt nay c hinh thanh do s kt hp nhom amine cua mt amino acid vi nhom carboxyl cua amino acid k tip. Phan ng kt hp giai phong mt phn t H2O. Peptide la mt chui ni tip nhiu amino acid (s lng it hn 30). Vi s lng amino acid ln hn chui c goi la polypeptide. Mi polypeptide co hai u tn cung, mt u mang nhom amine t do, u kia mang nhom carboxyl t do. Protein c dung chi n vi chc nng, nghia la mt cu truc phc tap trong khng gian ch khng phai n thun la mt trinh t amino acid. Chui polypeptide co th un thanh cu truc hinh gy nh trong cac protein hinh si hay cu trc khi cu nh trong cac protein dang cu hay mt cu truc gm ca hai dang trn. Mt protein co th c hinh thanh t nhiu chui polypeptide. Ngi ta thng phn bit cu truc cua phn t protein thanh bn bc nh sau (Hnh 1.4): - Cu truc bc 1. La trinh t sp xp cac gc amino acid trong chui polypeptide. Cu truc nay c gi vng nh lin kt peptide (lin kt cng ha tri). Vi mi mt amino acid co gc khac nhau, cac gc nay co nhng c tinh ha hoc khac nhau, nn mt chui polypeptide cac thi im khc nhau co nhng c tinh ha hoc rt khac nhau. Tuy nhin, v tng quat thi tt ca cac chui polypeptide c xy

dng mt cach co h thng t cac nhom nguyn t CO, CH va NH. Vic xy dng co h thng nay la c s tao nn cu truc bc hai.

Hnh 1.4. Cc mc t chc ca phn t protein

- Cu truc bc 2. La tng tac khng gian gia cac gc amino acid gn nhau trong chui polypeptide. Cu truc c bn vng chu yu nh lin kt hydrogen hnh thanh gia cac lin kt peptide k gn nhau, cach nhau nhng khoang xac inh. Cu truc bc 2 cua phn t protein: xon (-helix), la phin va xon collagen. Loai -helix la si dang xon c, cun xung quanh mt truc, mi vong xon co 3,6 gc amino acid. Nhng si collagen chay song song tao nn nhng bo si dai cua gn. Collagen cung co trong xng va trong cac m ni. Elastin la mt protein, gm nhng si protein tng i ngn, gn kt vi nhau nh lin kt cng ha tri. Nhng chui polypeptide quay theo dang xon c, t dui xon khi co ap lc. - Cu truc bc 3. La tng tac khng gian gia cac gc amino acid xa nhau trong chui polypeptide, la dang cun lai trong khng gian cua toan chui polypeptide. Nhiu chui polypeptide trong c th sng tn tai khng phai dang thng ma gp khuc va qua o tao nn cu truc khng gian ba chiu. Tuy nhin, cu truc nay hoan toan xac inh, chu yu la do trinh t cac amino acid va mi trng. Khi mt chui polypeptide tach ra khoi ribosome sau khi tng hp va c a vo trong t bao cht nh la mi trng tao hinh thi no s hinh thanh nn cu truc t nhin rt nhanh, c bit

i vi cu truc hinh cu, em lai cho protein nhng c tinh sinh ly quan trong. Co th do chuyn ng nhit cua cac chui polypeptide ma cac nhom cua cac gc amino acid tip xuc vi nhau, dn n co th kt hp vi nhau. Trong nhiu protein hinh cu co cha cac gc cysteine, s tao thanh cac lin kt disulfite gia cac gc cysteine xa nhau trong chui polypeptide s lam cho chui bi cun lai ang k. Cac lin kt khac, nh lin kt Van der Waals, lin kt tinh in, phn cc, ky nc va hydrogen gia cac mach bn cua cac gc amino acid u tham gia lam bn vng cu truc bc 3. Cu truc hinh cu cua protein c goi la cu truc bc ba, chnh la cu truc cua enzyme. - Cu truc bc 4. L tng tac khng gian gia cac chui ca cc phn t protein gm hai hay nhiu chui polypeptide hnh cu. Mi chui polypeptide nay c goi la mt tiu n vi (subunit). S kt hp gia cac phn t nay long lo va chu yu la do lin kt hydrogen va ky nc. Bng cach nay hai phn t xac inh co th kt hp vi nhau tao thanh mt dimer. Chng hn: hemoglobin c tao nn t hai chui vi mi chui co 141 gc amino acid va hai chui vi mi chui la 146 gc amino acid. Cu truc cua mt hoc nhiu chui polypeptide co y nghia quan trong i vi ha tan va chc nng cua chung. Cu truc protein c hiu la s sp xp cua nhng chui ring l hoc nhiu chui. Chung phu thuc nhiu vao pH cua mi trng. Protein va chui polypeptide ha tan tt khi nhng nhom a nc hng ra phia ngoai, nhom ky nc hng vao bn trong. Khi mt protein thay i cu truc thi nhng nhom ky nc quay ra ngoai, protein mt kha nng ha tan trong nc, vi du trng hp kt tua khng dang tinh th cua protein sa trong mi trng chua. Lactic acid c san sinh do vi khun lam giam pH sa, lam thay i protein sa. Nhiu nhom ky nc c hng ra bn ngoai, protein mt kha nng tan trong nc. Vi vy, vic thng xuyn duy tri gia tri pH trong t bao cht rt quan trong, vi chi co nh vy chc nng hoat ng cua cac enzyme trong t bao cht mi c am bao.

2. Chc nng cua protein Mi mt hoat ng trong t bao phu thuc vao mt hoc nhiu phn t protein c hiu. Mt trong cac cach phn loai protein la da vao chc nng sinh hoc cua chung. Bng 1.2 tom tt s phn loai protein theo chc nng va a ra mt s vi du ai din cho mi loai.

Bng 1.2. Cc chc nng sinh hc ca protein v mt s v d

2.1. Chc nng enzyme Phn ln protein la enzyme. Hin nay, co hn 3.000 loai enzyme a c bit. Enzyme la cht xuc tac sinh hoc co vai tro lam tng tc phan ng. Mi mt bc trong trao i cht u c xuc tac bi enzyme. Enzyme co th lam tng tc phan 16 ng ln 10 ln so vi tc phan ng khng xuc tac. S kt hp gia enzyme va c cht xay ra vi tri hoat ng cua enzyme.

2.2. Protein iu khin Mt s protein khng thc hin bt ky s bin i ha hoc nao, tuy nhin no iu khin cac protein khac thc hin chc nng sinh hoc, chng hn insulin iu khin nng ng glucose trong mau. o la mt protein nho (5,7 kDa), gm hai chui polypeptide ni vi nhau bng cac lin kt disulfite. Khi khng u insulin thi s tip nhn ng trong t bao bi han ch. Vi vy, mc ng trong mau tng va dn n s thai ng manh me qua nc tiu (bnh tiu ng). Mt nhom protein khac tham gia vao s iu khin biu hin gen. Nhng protein nay co c tinh la gn vao nhng trinh t DNA hoc hoat ha hoc c ch s phin ma thng tin di truyn sang mRNA, vi du cht c ch (repressor) inh chi s phin ma.

2.3. Protein vn chuyn Lam nhim vu vn chuyn cht c hiu t vi tri nay sang vi tri khac, vi du vn chuyn O2 t phi n cac m do hemoglobin hoc vn chuyn acid beo t m d tr n cac c quan khac nh protein trong mau la serum albumin. Cac cht c vn chuyn qua mang c thc hin bng cac protein c hiu, chng hn vn chuyn glucose hoc cac amino acid qua mang (Hinh 1.5).

2.4. Protein d tr Cac protein la ngun cung cp cac cht cn thit c goi la protein d tr. Protein la polymer cua cac amino acid va nitrogen thng la yu t han ch cho sinh trng, nn c th phai co protein d tr cung cp y u nitrogen khi cn. Chng hn, ovalbumin la protein d tr trong long trng trng cung cp u nitrogen cho phi phat trin. Casein la protein sa cung cp nitrogen cho ng vt co vu con non. Hat

thc vt bc cao cung cha mt lng protein d tr ln (khoang 60%), cung cp u nitrogen cho qua trinh nay mm ca ht.

Hnh 1.5. Hai kiu vn chuyn c bn. (a): vn chuyn bn trong hoc gia cc t bo hoc m. (b): vn chuyn vo hoc ra khi t bo.

Protein cung co th d tr cac cht khac ngoai thanh phn amino acid (N, C, H, O va S), vi du ferritin la protein tim thy trong m ng vt kt hp vi Fe. Mt phn t ferritin (460 kDa) gn vi 4.500 nguyn t Fe (chim 35% khi lng). Protein co vai

tro gi lai kim loai Fe cn thit cho s tng hp nhng protein c cha Fe quan trong nh hemoglobin.

2.5. Protein vn ng va co rut Mt s protein mang lai cho t bao kha nng vn ng, t bao phn chia va co c. Cac protein nay co c im: chung dang si hoc dang polymer ha tao si, chng hn actin v myosin. Tubulin la thanh phn c ban cua thoi v sc (si xut hin khi phn chia cac nhim sc th v cac cc).

2.6. Protein cu truc C chc nng tao chc va bao v t bao v m. Chng hn: -keratin la protein khng tan, cu tao nn toc, sng va mong. Collagen la protein hinh si co trong xng. ng vt collagen chim 1/3 protein tng s. Fibroin (-keratin) la thanh phn c ban cua ken tm. Mt chc nng ph bin khac cua protein la cu tao nn mang sinh hoc.

2.7. Protein bao v Trong vic giai c cc kim loai nng, phytochelatin co mt y nghia quan trong, y la nhng polypeptide n gian co ngun gc t glutation va co cng thc chung nh sau: ( -glutamyl-cysteinyl)n-glycine Do co nhiu nhom SH nn chung co kha nng kt hp cht vi cac kim loai nng, lam cho nhng kim loai nng nay khng th gy ri loan trao i cht. S tng hp phytochelatin c kich thich bi nhng kim loai nng nh Cd, Cu, Ag, Bi va Au. Protein bao v co vai tro quan trng trong cc phn ng min dich. ng vt co xng sng co mt c ch phc tap v phat trin cao ngn nga nhng tac nhn vi sinh vt gy bnh. Chc nng nay co lin quan n c tinh cua chui polypeptide. Khi mt protein la (c ngun gc virus, vi khun hoc nm) xm nhp vao mau hoc vao m thi phn ng t v ca c th s xut hin rt nhanh. Protein la c goi la khng nguyn (antigen) cha mt vung c trt t xac inh cac nguyn t c th kt hp vi t bao lympho va kich thich t bao nay san sinh khang th. Nhng t bao lympho tn tai 9 trong h thng min dich vi s lng 10 va trn b mt cua no c nhng vung nhn bit ni ma khng nguyn s c kt hp (Hinh 1.6). Nhng vung nhn bit nay rt khac nhau va c hiu cho tng loi khng nguyn. Trong c th lun co sn mt lng ln cac t bao lympho khac nhau v chung co th tng hp rt nhanh cc khang

th c hiu khi khang nguyn xut hin. Mi loai khng th co mt vi tri kt hp duy nht c trng vi khng nguyn. Kha nng bo v ca h min dich a lam cho protein la cua tac nhn gy bnh tr thanh v hai. Nhng khang th nay c goi la globulin min dich. Chung chim khoang 20% protein tng s trong mau. Mt nhom protein bao v khac la protein lam ng mau thrombin va fibrinogen, ngn can s mt mau cua c th khi bi thng. Ca cac vng cc ca Tri t co protein chng ng (antifreeze protein) co tac o dung bao v mau khi nhit xung di 0 C.

2.8. Protein l/ngoi lai Vi du monellin la mt loai protein c tim thy mt loai cy chu Phi, c coi la cht ngot nhn tao cho con ngi. mt s sinh vt bin nh ho Trai tit ra loai protein keo (glue protein), cho phep no gn cht ln b mt.

III. Lipid Mc du khng mang hoat tinh sinh hoc cao nh protein nhng lipid cung ong mt vai tro c bit trong h thng sng. Chung la nhn t chinh tao nn cac mang sinh hoc ma nu thiu thi moi hoat ng cua protein se khng th phi hp nhip nhang. n vi cu truc cua lipid la cac acid beo. Mi acid beo c cu tao t mt mach carbohydrate (gm cac nguyn t C va H) gn vi mt nhom carboxyl co tinh acid. Cac acid beo khac nhau bi dai cua chung, bi s lng va vi tri cac lin kt i. Cac acid beo khng co lin kt i c goi la cc acid bo bao ha, cac acid beo khng bao ha co it nht mt lin kt i.

Hnh 1.6. S biu din ca khng th v khng nguyn. a: khng th gm 4 chui polypeptide. b: khng th kt hp vi khng nguyn. c: kt hp gia khng nguyn v khng th.

Mang sinh hoc co chc nng la gii han nhng vung trao i cht va tham gia vao vic vn chuyn cac cht. Mang sinh hoc cung co kha nng chuyn i nhng tin hiu. Protein mang cung co th la cac enzyme. Chc nng nay c th hin mang trong cua ty th va lap th. Mang sinh hoc bao gm lp kep lipid vi nhng protein phn b trong o (Hinh 1.7) Cac lipid mang c hinh thanh t mt chui dai acid beo ni vi nhng nhom co c tinh a nc cao va c goi la nhng phn t lng cc vi mt u tng tac vi nc, con u kia thi ky nc.

Bang 1.3. Cu truc mt s acid beo tiu biu trong h thng sng

Hnh 1.7. S biu din mt on ct ca mng sinh hc

IV. Polysaccharide Cac polysaccharide co nhiu chc nng quan trong trong t bao, chung tham gia vao cu tao t bao va la ngun d tr nng lng chu yu. Cac polysaccharide c hinh thanh t nhiu monomer, la cac ng n gian (monosaccharide) ni vi nhau bng lin kt glycoside. Lin kt nay c hinh thanh t s kt hp gia C 1 cua mt phn t ng vi nhom hydroxyl cua phn t k tip. Ngun d tr tinh bt cac t bao ng vt la glycogen, trong khi o thc vt la tinh bt. Mt polymer khac cua glucose la cellulose thi tao nn thnh t bao thc vt va la hp cht hu c hin din nhiu nht trong sinh quyn. Chung ta va im qua ring re tng thanh phn cu tao t bao chinh. Trong thc t, hoat ng cua chung phi hp mt thit vi nhau. Cac nucleic acid trong t bao thng kt hp cht che vi cac protein tao thanh nucleoprotein. DNA cua t bao

eukaryote thi c boc bi nhng protein c hiu la cac histone. Mang t bao cung khng phai chi co phospholipid, chinh cac protein gn trong mang a tao ra nhng c trng ring cua mang sinh hoc. Mt im cn lu y la nu nh cu truc va cac tinh cht ha ly cua nucleic acid, lipid va polysaccharide tng i ng nht thi cac protein lai ht sc a dang ca v cu truc va chc nng. Mt phn t protein thng bao gm nhiu vung mang nhng c tinh khac nhau: vung a nc hay ky nc, vung gn mt ng, vung co hoat tinh xuc tac, vung lin kt vi nucleic acid hay vi mt protein khac. T mi chc nng cua t bao, t s hinh thanh vt cht mang thng tin di truyn, truyn at thng tin di truyn, s chuyn ha nng lng, s lin lac gia cac t bao... u co s tham gia cua cac protein. iu ky diu cua s sng la toan b cac hoat ng v cung a dang y c thc hin bi mt phn t duy nht.

Ti liu tham kho/c thm

1. H Hunh Thy Dng. 1998. Sinh hc phn t. NXB Gio dc, H Ni. 2. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 3. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 4. Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J. 2004. Molecular Cell Biology. 5th ed. WH Freeman and Company, New York, USA. 5. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Loscik R. 2004. Molecular Biology of the Gene. The Benjamin Cummings/Cold Spring Habor Laboratory Press, San Francisco, CA, USA. 6. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company Inc. New York, USA.
1

S (Svedberg): n v o vn tc lng. H s lng ca mt tiu n v ph thuc khng nhng vo khi lng ca tiu n v m cn ph thuc vo hnh dng v rn ca n, iu ny gii thch ti sao s kt hp ca hai tiu n v 50S v 30S li to ra mt ribosome 70S.

Chng 2

Cu trc genome
Genome (h gen, b gen) l thut ng c dng vi cc ngha khc nhau nh sau:

- Nguyn liu di truyn ca mt c th: 1) nhim sc th trong t bo vi khun (hoc mt trong mi loi nhim sc th nu hn mt loi c mt, v d: cc nhim sc th ln hoc b ca Vibrio cholerae), 2) DNA hoc RNA trong mt virion, 3) nhim sc th cng vi mi plasmid c kt hp (v d: nhim sc th v hai plasmid nh trong vi khun Buchnera). - Tt c cc gen (khc nhau) trong t bo hoc virion. - B nhim sc th n bi hoc genome n bi trong t bo. Chui genome hon chnh (ngha l trnh t hon chnh ca cc nucleotide trong genome) c cng b cho mt s loi vi khun. Cc trnh t khc cng c cng b, v d genome ca cy cc di (Arabidopsis thaliana) v genome ngi. Genome cha ton b thng tin di truyn v cc chng trnh cn thit cho c th hot ng. cc sinh vt nhn tht (eukaryote), 99% genome nm trong nhn t bo v phn cn li nm trong mt s c quan t nh ty th v lp th. a s genome vi khun v phn genome cha trong cc c quan t thng c kch thc nh v dng vng khp kn. Ngc li, phn genome trong nhn thng rt ln v phn b trn cc nhim sc th dng thng. D n genome l d n xc nh cu trc di truyn chnh xc ca mt genome c th sng, ngha l trnh t DNA ca tt c cc gen ca n. D n genome ca mt s sinh vt m hnh (model organisms) c hon thnh nh sau: - Cc genome vi khun. Cc trnh t hon chnh ca genome Escherichia coli c xc nh theo phng thc t hp/tp hp (consortium) ca cc phng th nghim. Nm 1995, hai trnh t genome hon chnh ca vi khun Haemophilus influenzae v Mycoplasma genitalium cng c hon thnh. Loi M. genitalium c mt genome n gin (khong 580.067 base), do n da vo vt ch vn hnh nhiu b my trao i cht ca mnh. Loi H. influenzae l mt vi khun c trng hn, v c genome khong 1.830.121 base vi 1.749 gen. - Chui genome hon chnh ca nm men Saccharomyces cerevisiae c hon chnh trong nm 1996, nh mt consortium ca cc phng th nghim. Genome ca chng di 12.146.000 base. - Cc d n genome ng vt nh: chut, cu, ln, giun trn (Caenorhabditis elegans), rui gim (Drosophila melanogaster), hoc thc vt nh: la nc, la m, ng, to, cc di, m ni bt nht trong s l d n genome ngi cng c thc hin. Ngy 12. 2. 2001 genome ngi c cng b vi khong 30.000 gen, t hn nhiu so vi d kin trc y (hng trm ngn gen), v ch gp hai ln giun trn hoc rui gim. Ngi ta xc nh h gen ngi ging 98% so vi tinh tinh v c n 99% l ging nhau gia cc dn tc, cc c th. Do , vn hnh thnh v pht trin

nhn cch, ch s thng minh... phi ch yu trn c s x hi v s rn luyn ca tng ngi pht trin tim nng sinh hc ca bn thn. Trnh t genome ca nhng sinh vt m hnh rt c ngha trong nhng nghin cu ca mt chuyn ngnh khoa hc mi l genome hc (genomics). Da vo y, cc nh sinh hc phn t c th phn tch cu trc, hot ng v chc nng ca cc gen, lm sng t c vai tr ca DNA lp li, DNA khng cha m di truyn, DNA nm gia cc gen... iu c bit c ngha l khi so snh cc genome vi nhau, c th hiu c hot ng ca genome trong cc c th sng, mi quan h gia chng, s a dng sinh hc v mc tin ha. Kt qu bc u so snh genome gia cc loi sinh vt vi nhau cho thy c ba c im ni bt: 1) cc gen phn b trong genome khng theo qui lut, 2) kch thc ca genome thay i khng t l thun (tng quan) vi tnh phc tp ca loi, 3) s lng nhim sc th cng rt khc nhau ngay gia nhng loi rt gn nhau. I. Thnh phn v c im ca genome Genome cha mi thng tin di truyn c trng cho tng loi, thm ch cho tng c th trong loi. Genome c th bao gm cc phn t DNA hoc RNA. i vi sinh vt bc cao, kch thc genome thay i t 109 bp (ng vt c v) n 1011 bp (thc vt). Khc vi t bo tin nhn (prokaryote), cc gen trong genome ca eukaryote thng tn ti nhiu bn sao v thng b gin on bi cc on m m khng mang thng tin di truyn (cc intron). V vy, mt trong nhng vn ang c quan tm l cn phi bit s lng cc gen khc nhau c mt trong genome cng nh s lng cc gen hot ng trong tng loi m, tng giai on pht trin v t l cc gen so vi kch thc genome... 1. Genome ca c quan t Hu ht genome ca c quan t, nhng khng phi lun lun, c dng phn t DNA mch vng n ca mt chui duy nht. Genome ca c quan t m ha cho mt s, khng phi tt c, cc protein c tm thy trong c quan t. Do c nhiu c quan t trong mt t bo, cho nn c nhiu genome ca c quan t trn mt t bo. Mc d bn thn genome ca c quan t l duy nht. Nhng n cu to gm mt chui lp li1 lin quan vi mi chui khng lp li2 ca nhn. V nguyn tc, cc gen c quan t c phin m v dch m bi cc c quan t. 1.1. Genome ca ty th DNA ty th (mitochondrial DNA-mtDNA) l mt genome c lp, thng l mch vng, c nh v trong ty th.

- DNA ty th ca t bo ng vt m ha c trng cho 13 protein, 2 rRNA v 22 tRNA. - DNA ty th ca nm men S. cerevisiae di hn mtDNA ca t bo ng vt nm ln do s c mt ca cc on intron di. Cc genome ty th c kch thc tng s rt khc nhau, cc t bo ng vt c kch thc genome nh (khong 16,5 kb ng vt c v) (Hnh 2.1). C khong mt vi trm ty th trn mt t bo. Mi ty th c nhiu bn sao DNA. S lng tng s ca DNA ty th so vi DNA nhn l rt nh (<1%). Trong nm men S. cerevisiae, genome ty th c kch thc kh ln (khong 80 kb) v khc nhau ty thuc vo tng chng. C khong 22 ty th trn mt t bo, tng ng khong 4 genome trn mt c quan t. nhng t bo sinh trng, t l mtDNA c th cao hn (khong 18%). Kch thc ca genome ty th cc loi thc vt l rt khc nhau, ti thiu khong 100 kb. Kch thc ln ca genome gy kh khn cho vic phn lp nguyn vn DNA, nhng bn ct hn ch (restriction map) trong mt vi loi thc vt cho thy genome ty th thng l mt chui n, c cu to nh mt mch vng. Trong mch vng ny c nhng chui tng ng ngn v s ti t hp gia chng sinh ra cc phn t tiu genome (subgenome) mch vng nh hn, cng tn ti vi genome ch (master genome) hon chnh, gii thch cho s phc tp ca cc DNA ty th thc vt.

Hnh 2.1. DNA ty th ca ngi. Bao gm 22 gen tRNA, 2 gen rRNA, v 13 vng m ha protein.

Bng 2.1 tm tt s phn cng ca cc gen trong mt s genome ty th. Tng s gen m ha protein l kh t, v khng tng quan vi kch thc ca genome. Ty th ng vt c v s dng cc genome 16 kb ca chng m ha cho 13 protein, trong khi ty th nm men S. cerevisiae dng cc genome t 60-80 kb m ha cho khong 8 protein. Thc vt vi genome ty th ln hn nhiu m ha cho nhiu protein hn. Cc intron c tm thy trong hu ht cc genome ca ty th, nhng li khng c trong cc genome rt nh ca ng vt c v. Hai rRNA chnh lun c m ha bi genome ty th. S lng cc tRNA c m ha bi genome ty th dao ng t khng cho n y (25-26 trong ty th). Nhiu protein ribosome c m ha trong genome ty th ca thc vt v sinh vt nguyn sinh, nhng ch c mt t hoc khng c trong genome ca nm v ng vt.

Bng 2.1. Cc genome ty th c cc gen m ha cho cc protein, rRNA v tRNA

1.2. Genome ca lp th DNA lp th (chloroplast DNA-ctDNA) cng l mt DNA genome c lp, thng l mch vng, c tm thy trong lp th ca thc vt. - Genome ca lp th rt khc nhau v kch thc, nhng ln m ha cho khong 50-100 protein cng nh rRNA v tRNA. - DNA lp th di t 120-190 kb. Cc genome ca lp th c phn tch trnh t cho thy c khong 87-183 gen. Bng 2.2 m t cc chc nng c m ha bi genome lp th cy trng.

Bng 2.2. Genome ca lp th cc cy trng m ha cho 4 rRNA, 30 tRNA v khong 60 protein

Ni chung, cc c im ca genome lp th tng t ty th, ngoi tr lp th mang nhiu gen hn. Genome lp th m ha cho tt c cc loi rRNA v tRNA cn thit trong tng hp protein, v cho khong 50 protein, bao gm c RNA polymerase v cc protein ribosome. Cc intron trong lp th c chia thnh hai nhm: 1) nhng intron trn cc gen tRNA thng (mc d khng chc chn) c nh v trong vng anticodon, ging nh cc intron c tm thy trong cc gen tRNA ca nhn nm men S. cerevisiae; 2) nhng intron trong cc gen m ha protein tng t vi cc intron ca cc gen ty th. Vai tr ca lp th l thc hin qu trnh quang hp. Do , nhiu gen ca n m ha cho cc protein ca cc phc hp nh v trong cc mng thylakoid. Mt vi phc hp protein ca lp th ging cc phc hp protein ca ty th: c mt s tiu n v c m ha bi genome ca c quan t v mt s khc c m ha bi genome ca nhn. Nhng cc phc hp cn li c m ha hon ton bi genome lp th. 2. ng hc ca phn ng lai DNA Bn cht chung ca eukaryotic genome c phn nh qua ng hc ca s ti lin kt cc DNA (DNA reassociation kinetics) b bin tnh. S ti lin kt gia cc chui DNA b sung xy ra nh bt cp base, ngc li vi qu trnh bin tnh (denaturation) m nh chng c tch ri (Hnh 2.2) thc hin s ti bn hoc

phin m. ng hc ca phn ng ti lin kt phn nh s khc nhau ca cc chui hin din, v th phn ng ny c th c dng nh lng cc gen v cc sn phm RNA ca chng.

Bng 2.3 m t phn ng ti lin kt. S hi tnh ca DNA (renaturation) ph thuc vo s va chm ngu nhin ca cc chui b sung. Phn ng ca cc DNA ring bit c th c m t bng cc iu kin cn thit cho s hon thnh mt na (halfcompletion). y l tch s ca C0 t1/2 v c gi l C0t1/2. Gi tr ny t l nghch vi hng s tc . Do C0t1/2 l tch s ca nng v thi gian yu cu cho mt na ng, nn mt gi tr C0t1/2 ln hn dn n mt phn ng chm hn.
Bng 2.3. Mt phn ng ti lin kt ca DNA c m t bi C0t1/2

S hi tnh ca DNA thng c dng ng cong C0t, ng cong biu din th phn s ca DNA c ti lin kt (1-C/C0) theo log ca C0t. Hnh 2.3 trnh by ng cong C0t ca mt s genome n gin. Cc ng cong c dng tng t nhau, nhng gi tr C0t1/2 ca mi ng l khc nhau. Cc genome trong hnh 2.3 i din cho cc ngun DNA khc nhau (PolyU:PolyA, thc khun th MS2, thc khun th T4 v vi khun E. coli). C0t1/2 lin quan trc tip vi lng DNA trong genome. iu ny phn nh tnh trng khi genome tr nn phc tp hn, th s c thm mt s bn sao ca mt vi chui c bit trong mt lng DNA c trc. V d: nu C0 ca DNA l 12 pg, th n s cha khong 3.000 bn sao ca mi trnh t trong genome vi khun.

Hnh 2.3. C0t1/2 ph thuc vo phc tp ca genome. PolyU:PolyA, thc khun th MS2, thc khun th T4 v vi khun E. coli.

3. Kch thc ca genome Khng phi tt c cc on DNA trong genome u tng ng vi cc gen (m ha cho protein hoc mt sn phm cn thit cho hot ng sng ca t bo). T nhng nm 1970, bng cc th nghim gy bo ha t bin ngi ta c th xc nh c s gen nm trn mt on nhim sc th. Ngy nay, nh cc k thut phn tch DNA v RNA hin i (Southern blot, Northern blot, microarray...) cc nh khoa hc c th xc nh s gen hot ng trong mt t bo. V d: t bo nm men S. cerevisiae (sinh vt eukaryote bc thp) c khong 4.000 gen hot ng, cn t bo ng vt c v khong 10.000-15.000 gen. Nh vy, nu di trung bnh ca mt gen khong 10 kb th tng s chiu di cc gen hot ng trong mt t bo cng ch chim 1-2% genome. Hay ni cch khc, ch mt phn rt nh genome mang thng tin di truyn cn thit cho hot ng sng ca t bo. Vy phn genome cn li c vai tr g, v tnh phc tp ca loi c lin quan g vi kch thc genome hay khng? lm sng t vn trn, chng ta cn xem xt kch thc genome ca mt s loi gn nhau trong bc thang tin ha (c phc tp loi tng t nhau) cng nh genome ca nhng loi xa nhau (c tnh phc tp khc nhau). Chng hn: - Genome ca ngi c kch thc khong 3,3 109 bp, trong khi genome ca nhng loi lng c di khong 3,1 109 bp hoc thc vt c th ln n 1011 bp. Nh vy, c phi l cc loi lng c c tnh phc tp tng t c th chng ta? - Hay l ngay trong cng mt loi, chng ta cng nhn thy c s mu thun v kch thc genome? V d: rui nh (Musca domestica) c genome khong 8,6 108 bp, ln gp su ln kch thc genome ca rui gim khong 1,4 108bp. Ngoi ra, trong cc loi lng c kch thc genome ca chng cng thay i kh ln t 109-1011 bp. V sao ngay trong cng mt loi m kch thc genome li bin thin nhiu nh vy, c phi rui nh c cu to phc tp hn nhiu so vi rui gim?

T nhng d liu trn, chng ta c th nhn nh rng tnh phc tp ca loi khng lin quan n kch thc ca genome. Tuy nhin, vai tr ca phn genome cn li (phn khng m ha) n nay vn cha c bit nhiu. 4. Tng s gen c bit mt s loi eukaryote C 6.000 gen nm men S. cerevisiae, 18.500 gen giun trn, 13.600 gen rui gim, 25.000 gen Arabidopsis, v c kh nng 30.000 gen chut v < 30.000 gen ngi. Nh chng ta bit, mi quan h gia kch thc genome v s lng gen khng cn na. Genome ca cc sinh vt eukaryote n bo c cng phm vi kch thc vi genome ca vi khun ln nht. Cc eukaryote bc cao c nhiu gen hn, nhng s lng khng tng quan vi kch thc genome. Hnh 2.4 cho thy genome ca loi nm men S. cerevisiae di 13.500 kb v loi nm men S. pombe l 12.500 kb, c khong 6.000 gen v 5.000 gen tng ng. Khung c m trung bnh khong 1,4 kb, v th khong 70% genome c chim gi bi cc vng m ha. S khc nhau ch yu gia chng l ch 5% gen ca S. cerevisiae c intron, so vi 43% ca S. pombe. Genome ca giun trn c khong 18.500 gen. Mc d genome ca rui gim ln hn genome ca giun trn, nhng chng li c s gen t hn. n nay, chng ta cha hiu ti sao rui gim-mt c th phc tp hn nhiu-ch c 70% s gen so vi giun trn. iu ny cho thy khng c mt mi quan h chnh xc gia s gen v tnh phc tp ca c quan.

Hnh 2.4. S lng gen ca sinh vt eukaryote rt khc nhau. Thay i t 6.000-40.000 nhng khng tng quan vi kch thc genome hoc phc tp ca c th.

Cy Arabidopsis c kch thc genome trung gian gia giun trn v rui gim, nhng li c s gen ln hn c hai (25.000). iu ny mt ln na cho thy khng c mt quan h r rng, v cng nhn mnh nt c bit ca thc vt, l c th c nhiu gen hn (do s nhn i ca ng b t tin truyn li) cc t bo ng vt. a s genome Arabidopsis c tm thy trong cc on c nhn i, gi rng c mt s nhn i xa xa trong genome (to ra mt dng t bi). Ch 35% cc gen ca Arabidopsis hin din nh cc bn sao n. Genome ca la ln hn Arabidopsis khong 4 ln, nhng s gen ch ln hn khong 50%, c kh nng khong 40.000 gen. DNA lp li chim khong 42-45% genome. Hn 80% gen tm thy trong Arabidopsis c hin din trong la. Trong s nhng gen chung ny, khong 8.000 c trong Arabidopsis v la nhng khng thy bt k genome ng vt hoc vi khun no (nhng gen c phn tch trnh t). C kh nng y l tp hp cc gen m ha cho cc chc nng c trng ca thc vt, chng hn nh quang hp. II. Tnh phc tp ca genome Kt qu nghin cu ng hc ca cc phn ng lai c tin hnh gia genomic DNA vi cDNA (complementary DNA-DNA b sung), gia DNA vi mRNA cho thy hu ht cc gen hot ng u nm trong thnh phn DNA khng lp li. Nh vy, thnh phn ny c ngha rt quan trng trong vic nh gi tnh phc tp ca genome. Hay ni cch khc, da vo thnh phn DNA khng lp li c th bit c kch thc genome cng nh mc tin ha ca loi. Nu nh kch thc genome ( trng thi n bi) c coi l mt thng s ng hc (k hiu C), th gi tr ny c trng cho tng loi v khng phi lun lun t l thun vi tnh phc tp ca loi. Ngc li, gi tr C phn nh cc c im sau: - S lng DNA m ha cho cc sn phm cn thit i vi hot ng sng ca c th rt nh so vi s lng DNA c trong genome. - C s bin i rt ln ca gi tr C gia mt s loi m tnh phc tp ca chng khng khc nhau nhiu. Genome vi khun c xem l ch cha cc on DNA khng lp li v cc gen thng tn ti bn sao n. Ngc li, genome ca eukaryote thng cha cc gen c hai hoc nhiu bn sao. Hn na, trnh t nucleotide ca cc bn sao ny c th khng ging nhau hon ton mc d sn phm protein m chng m ha c cng mt chc nng. Cc bn sao tng ng ca mt gen c xp chung vo mt nhm gi l mt h gen (gene family). Nh vy, ngoi cc gen c mt bn sao ging nh vi khun, genome ca eukaryote cn cha cc h gen. Hu ht cc gen m ha cho protein c phn lp u nm trong cc h gen khc nhau. Cc gen trong mt h thng hot ng theo thi gian v khng gian. iu c ngha, mi thnh vin trong h thng hot ng mt thi im nht nh trong qu trnh hnh thnh v pht trin

c th hoc hot ng trong cc m chuyn bit. Khi mt thnh vin trong h b t bin (bt hot) th thnh vin khc c th hot ng thay th. Khi nim v gen c hnh thnh khi cc nh di truyn hc c in nghin cu biu hin cc tnh trng mi do gy t bin DNA ca genome vi khun hay thc khun th (bacteriophage). Mt gen c xem l mt on DNA m bt c t bin no xy ra trn u dn n xut hin tnh trng mi. iu ny d hiu i vi nhng genome ch cha cc gen c mt bn sao (nh genome ca prokaryote). Tuy nhin, genome ca eukaryote, khi c nhiu gen cng qui nh mt tnh trng hoc cc gen tng ng trong mt h c th hot ng h tr thay th nhau th t bin trn mt gen khng phi lc no cng quan st c mc phenotype. Mt khc, cc on DNA tng ng vi trnh t m ha (coding sequence, exon) cho mt protein thng b ngt qung bi cc on DNA khng cha thng tin di truyn (non-coding sequence, intervening sequence, intron). Cc intron c phin m cng vi cc exon sang phn t RNA gi l phn t tin thn mRNA (pre-mature mRNA hay pre-mRNA) nhng sau chng b loi b v cc exon c ni li vi nhau to thnh phn t mRNA hon chnh (mature mRNA hay mRNA) c dng cho qu trnh sinh tng hp protein. Qu trnh ct cc intron, ni exon khng tun theo mt trt t bt buc m bin ha a dng to ra cc phn t mRNA khc nhau t mt phn t pre-mRNA (Hnh 2.5). Bn cnh mRNA, cc phn t rRNA v tRNA cng c hnh thnh t cc phn t tin thn cha intron. Ngoi ra, cn c hin tng m di truyn ca gen ny nm xen k vi cc m di truyn ca gen khc (cc gen nm gi ln nhau-overlapping) hoc trng hp dch chuyn khung c ngay trn mt on DNA. Chng ta bit n chc nng ca cc phn t RNA trong hot ng sng ca t bo. Bn cnh ba loi RNA c nghin cu kh k (mRNA, rRNA v tRNA), vai tr ca mt s loi RNA khc mi c pht hin vo nhng nm cui th k 20. Chng kim sot s hot ng ca gen (hin tng bt hot gen-gene silencing), tham gia phn ng c sa thng tin di truyn trn phn t mRNA (hin tng RNA editing) hay quyt nh tnh bn vng ca mRNA (cc ribonuclease) Thm ch c nhng phn t pre-mRNA c tng hp khng phi m ha cho protein m vi mc ch phn ct to ra nhng phn t RNA c kch thc nh hn tham gia qu trnh kim sot hot ng ca cc gen khc. t bin nhng on DNA m ha cho tt c cc loi RNA ny thng gn lin vi vic xut hin tnh trng mi. Do , cn xem xt cc on nucleotide nh cc gen mc d chng khng m ha cho protein.

Hnh 2.5. Cc gen b gin on c biu hin thng qua RNA tin thn. Cc intron c loi b, trong khi cc exon c ni li vi nhau. mRNA ch c cc trnh t ca exon c dch m thnh chui polypeptide.

Ngy nay, theo quan im ca sinh hc phn t, mt gen c xem l mt on DNA m ha cho mt sn phm cn thit i vi hot ng sng ca t bo. R rng rng khng phi ch c DNA m ha cho protein m c cc DNA m ha cho rRNA, tRNA v cc loi RNA khc tham gia vo nhng phng thc kim sot hot ng ca genome cng c xc nh l gen. III. Thay i trt t ca cc on DNA trong genome-Transposon 1. S thay i trt t ca cc on DNA trong genome Nh bit kch thc v cu trc genome ca cc loi rt khc nhau. Nguyn nhn ca s a dng ny l do: 1) trao i cho gia cc cp nhim sc th tng ng xy ra trong phn bo gim nhim dn n s a dng trong loi; 2) trong genome trt t cc on DNA cng nh cu trc cc gen c sp xp li, c trng cho tng c th. Chnh cc yu t di truyn c kh nng di chuyn gia cc v tr trong mt genome hoc gia cc genome khc nhau gp phn lm a dng di truyn gia cc c th trong loi.

Cc yu t di truyn c kh nng di chuyn c xp vo ba nhm chnh ty thuc vo tnh c lp ca chng: - Nhm th nht gm cc yu t c kh nng di chuyn gia cc v tr khc nhau trong genome. - Nhm th hai gm cc yu t c kh nng ghp vo v tch ra khi genome tn ti c lp trong t bo (cc episome nh plasmid F, bacteriophage). - Nhm th ba ch di chuyn di s kim sot ca t bo nhng giai on sinh trng pht trin nht nh sp xp khi ng mt s gen c bit (hnh thnh cassette hot ng nm men S. cerevisiae, k sinh trng n bo Trypanosome). S di chuyn ca cc yu t nhm th hai v th ba lin quan ti ti t hp tng ng hoc ti t hp cc v tr c hiu. Mc d khng c kh nng tn ti c lp bn ngoi genome, nhm th nht t kim sot s di chuyn ca chng trong genome v khng i hi s tng ng gia chng vi v tr ghp vo. Do , c th ni chng di chuyn mt cch t do trong genome v c gi tn chung l cc yu t di chuyn (transposable elements, transposons). Trong khi thc khun th c xem l episome, th thc khun th Mu v mt s retrovirus eukaryote c xem l transposable elements.

2. Cc transposon Cc yu t di chuyn c th c chia lm hai loi da vo cch thc di chuyn ca chng: - Loi th nht c gi l retroelement, chng phi tri qua hnh thc trung gian RNA trong qu trnh di chuyn (RNA genome c sao chp nh reverse transcriptase to ra cDNA ghp vo v tr mi) (Hnh 2.6). - Loi th hai l cc on DNA hoc b tch ra hoc c ti bn to thm bn sao ghp vo v tr mi. Thng thng, loi th hai ny c gi l transposon.

Hnh 2.6. Retroelement

S tn tai cua cac transposon (cn gi l gen nhy) la net c trng cua t bao thc vt. sinh vt eukaryote, cc transposon cn c gi l yu t kim sot (controlling elements). Transposon co th di chuyn t nhim sc th nay sang nhim sc th khac hoc cac vi tri khac nhau trn cung mt nhim sc th. Chung co th nhay vao gia day ma cua mt gen ang hoat ng lam cho gen nay bt hoat hoc ngc lai. cy ng, ngi ta a phat hin 10 nhom transposon v mt s trong chng a c giai ma. Tt c chung u co kich thc khong 4.200 nucleotide va khac nhau chu yu oan ma tn cung cua gen. Khi di chuyn, cc transposon gy ra vic sp xp v t chc li genome ca tng c th nh to cc on DNA mi v tr chng ghp vo v tch ra. Chng c th di chuyn ti v tr bt k v hon ton khng cn mt mi quan h no gia hai v tr mi v c. Khi tch ra transposon c th mang theo cc on DNA bn cnh, gy ra s mt on ti v tr c. Ngc li, khi ghp vo v tr mi, chng gy ra hin tng thm on hoc chuyn on v tr mi. Do , transposon ging nh cc vector chuyn ch DNA t ni ny sang ni khc trong mt genome hoc t genome ny sang genome khc. Ngoi ra, trao i cho gia cc transposon tng ng hai v tr khc nhau trn mt hoc trn hai nhim sc th cng to ra nhng bin i tng t. Nhng bin i dn n vic sp xp li genome, to ra tnh a dng gia chng v tnh c th ring ca tng c th. c bit, s thay i v tr ca cc transposon cn c th nh hng n hot ng ca cc gen phn b xung quanh ngay khi chng khng lm thay i trt t nucleotide nhng gen ny. Tn s ghp ca cc transposon vo

genome khong 10-5-10-7 sau mi th h. Ngc li, tn s tch ra khong 10-6 n 1010 . Cc transposon c th chia lm hai nhm da vo kh nng di chuyn c lp hay phi ph thuc vo s c mt ca transposon khc: - Nhm th nht gm cc on DNA c kh nng di chuyn c lp. Chng cha gen m ha cho cc protein iu khin qu trnh , v d nh enzyme nhn bit hai u transposon tch chng ra khi v tr c v ghp vo v tr mi. Do , qu trnh ny c thc hin hon ton c lp. Nh kh nng ny, chng to ra cc t bin khng bn vng. - Nhm th hai gm cc transposon khng c kh nng t hot ng, tc l chng khng c kh nng di chuyn do khng mang thng tin di truyn m ha cho cc enzyme cn thit. V vy, cc transposon loi ny to ra nhng t bin gen mt cch t pht nhng l t bin bn vng. Vic di chuyn ca transposon nhm ny ph thuc vo s c mt ca transposon c kh nng hot ng c lp cng nhm. Hai transposon c th xp vo cng nhm khi chng c cu trc tng ng nhau, c bit l cc on oligonucleotide phn b hai u transposon. y l v tr enzyme nhn bit v ct ni transposon v tr i v n. Cc transposon n gin nht vi khun c gi l IS (insertion sequences). Chng c th nm trn chromosome hoc trn cc plasmid. Transposon vi khun khng gi mt chc nng no trong t bo. Trnh t nucleotide mt u IS thng lp li nhng ngc chiu so vi u kia (inverted repeat). V d nh GGTAT-Xn-ATACC. Do , khi si i IS tch thnh hai si n th mi si ny c kh nng hnh thnh lin kt b sung ti hai u ca IS to cu trc thn-quai/cn-thng lng (stem-loop). Transposon thng m ha cho cc enzyme transposase lm nhim v nhn bit chui nucleotide lp li ngc chiu (inverted repeat) ct transposon v di chuyn. Khi mt IS c ghp vo v tr bt k ca genome th mt on ngn DNA ti v tr ny c nhn i, tc l hai u ca IS c chn bi cc nucleotide ging nhau, sp xp theo cng mt chiu. V vy, on ngn DNA ny c gi l lp li xui chiu (direct repeat). Chiu di ca chng thng khong 9 bp (Hnh 2.7). Da vo s c mt ca cc on cng chiu v ngc chiu c th xc nh c v tr transposon ghp vo hoc chuyn i. Ngoi cc IS, vi khun cn c cc on DNA c kh nng di chuyn vi kch thc di hn, gi l Tn. Cc Tn thng phn b trn plasmid (phn t DNA dng vng, kch thc thng khng ln) v c kh nng chn vo bt k v tr no trong genome. Chng thng mang thng tin di truyn m ha cho cc protein khng khng sinh. Gia IS v Tn c mi quan h v trnh t cc nucleotide. Cc Tn thng c gii hn hai u bi mt loi IS no . Qu trnh di chuyn ca mt transposon t v tr c (v tr cho, donor) sang v tr mi (v tr nhn, recipient) xy ra theo hai c ch khc nhau:

- C ch sao y bn chnh (transposon c mt c hai v tr). Theo c ch ny, phin bn sau khi c sao chp t v tr cho s c ghp vo v tr nhn. Nh vy, mi ln di chuyn th s lng bn sao c tng ln. Qu trnh ny lin quan n hai loi enzyme: transposase (tc ng vo hai u bn gc transposon) v resolvase (tc ng ln bn sao). - C ch tch ra khi v tr c di chuyn n v tr mi. Theo c ch ny, mt transposon c th tch ra khi v tr c v ghp vo v tr mi. Nh vy, s lng transposon khng thay i. Kiu di chuyn ny ch cn enzyme transposase. Khi transposon chuyn i, v tr c b gy v n c ni li nh c ch sa cha DNA trong t bo.

Hnh 2.7. Mt transposon c on lp li ngc chiu gm 9 nucleotide (123456789) gn vo v tr c 5 nucleotide (ATGCA). on ngn ATGCA c mt c hai u ca IS v c trnh t nucleotide sp xp theo cng mt chiu. Khi transposon ghp vo v tr mi, mt on nucleotide ngn c sao thnh hai bn, mi bn nm mt u ca transposon (direct repeat). Ti v tr nhn, mi si n DNA b ct lch nhau vi nucleotide. Transposon ni vo cc u ct, to ra hai khong trng (gaps). Chng c sa cha theo nguyn tc to cp b sung. Do , on nucleotide nm gia hai vt ct c sao chp thnh hai bn, mi bn mt

u v trnh t sp xp cc nucleotide ging nhau. V vy, chng c gi l lp li xui chiu.

IV. Tng tc ca T-DNA vi genome thc vt

S di chuyn DNA t genome vi khun sang genome thc vt c nghin cu kh k i vi tng tc gia Argobacterium tumefaciens hoc A. rhizogenes vi hu ht cc cy hai l mm. Hin tng di chuyn DNA ny gy nhng bin i v mt di truyn, biu hin vic xut hin cc khi u trn thn cy (A. tumefaciens) hoc mc rt nhiu r t (A. rhizogenes) ti ni b nhim vi khun. A. tumefaciens va A. rhizogenes la hai loai vi khun gy bnh thc vt. Tuy nhin, sau bnh c duy tr li khng ph thuc s tn ti ca vi khun. l do mt s gen ca vi khun c chuyn vo genome cy ch v hot ng gy bnh. Cc gen vi khun c kh nng di chuyn v hot ng trong t bo thc vt nm trn Ti-plasmid (tumor inducing plasmid) ca A. tumefaciens hoc trn Ri-plasmid (hairy-root inducing plasmid) ca A. rhizogenes. Cng nh cc khi u ng vt, cc t bo thc vt c DNA vi khun ghp vo genome b chuyn sang trng thi mi, s pht trin v bit ha ca chng hon ton khc vi cc t bo bnh thng. l do hot ng ca cc gen vi khun trong genome ca thc vt. Bnh thng, nhng gen ny c mt trong genome vi khun nhng chng ch c hot ng (m) sau khi hp nht vo genome thc vt v chu s kim sot ca t bo cy ch. Qu trnh ny c tnh cht c hiu vt ch, tc l mt loi vi khun ch c kh nng gy khi u trn mt s loi cy ch ny m khng tng tc c vi cc loi cy khc. Vic to khi u hay thc hin qu trnh chuyn gen t vi khun sang genome thc vt dn n bin i trng thi sinh l ca t bo thc vt i hi cc iu kin sau: - Phi c hot ng ca cc gen trn ba v ng chvA, chvB, pscA nm trn nhim sc th ca vi khun khi ng vic bm dnh vi khun vo thn cy. - Ti-plasmid hoc Ri-plasmid phi mang vng vir (nm ngoi vng T-DNA). Vng ny mang cc gen cn thit cho vic tch v vn chuyn T-DNA sang t bo thc vt. - Cc gen trn vng T-DNA ca Ti-plasmid hoc Ri-plasmid c ghp vo genome thc vt gy bin i trng thi cc t bo ny. 1. Ti-plasmid v Ri-plasmid Plasmid la cac vong DNA t sinh san c lp bn ngoi nhn. vi khun v ng-thc vt, plasmid lin quan ti yu t gii tinh cua t bao, n kha nng chng chiu cac loai khang sinh... c im quan trong cua plasmid la chung co th lin kt vao nhim sc th nhng cung co th tn tai bn ngoai nhim sc th mt cach c lp.

Cac plasmid cua Agrobacterium mang cc vng T-DNA, vir (virulence region), gen chuyn ha opine, v vng ori khi u sao chp trong t bo vt ch. S khc nhau gia Ti-plasmid v Ri-plasmid ch, vng T-DNA ca Ti-plasmid cha cc gen tng hp auxin, cytokinin v opine. Trong khi , vng T-DNA ca Riplasmid ch mang gen tng hp auxin v opine. y l im khc nhau gy ra hiu qu tc ng khc nhau ln thc vt. C ch gy bnh cua cac Agrobacterium la sau khi xm nhim vao t bao, chung gn oan T-DNA vao b may di truyn cua t bao thc vt, lm ri loan cac cht sinh trng ni sinh, tao ra khi u (trng hp A. tumefaciens) hoc r t (trng hp A. rhizogenes). Kha nng chuyn cc gen nay a c khai thac chuyn gen ngoai lai vao b may di truyn cua t bao thc vt theo y mun. 2. T-DNA Vng T-DNA c nghin cu rt ky. o la mt oan DNA co kich thc 25 kb trong o cha gen ma hoa cho sinh tng hp auxin, cytokinin v opine (trng hp Ti plasmid) hoc auxin v opine (trng hp Ri-plasmid). Trong plasmid, vi tri cua T-DNA c gii han bng bin phi (right border-RB) v bin tri (left border-LB), mi bin c chiu di 25 bp. 3. Vng vir Trong cac vung DNA cua Ti-plasmid v Ri-plasmid, ngoai T-DNA, c nghin cu nhiu hn ca la vung DNA phu trach kha nng ly nhim con goi la vng vir (virulence). San phm hoat ng cua cac gen nm trong vung vir di tac ng kich thich cua cac hp cht phenol tit ra t vt thng la mt loat cc protein c hiu nh virA, virE2, virB, virD, virD2, virC1... Cac protein nay nhn bit cac vt thng cac cy chu thich hp (hu ht la cy hai la mm), kich thich san sinh ra cac oan T-DNA, bao boc che ch cac oan DNA nay va giup chung tip cn vi genome cua cy chu mt cach an toan. 4. Qu trnh chuyn T-DNA vo t bo thc vt Qu trnh ct on T-DNA ra khi plasmid v vn chuyn n vo t bo thc vt trc ht ph thuc vo sn phm ca cc gen vir. Vi khun xm nhim ti bt k v tr tn thng no trn thn cy. Cy c vt thng do s hng ngu nhin ca mng t bo thc vt hoc do vi khun tit ra hn hp nhng cht c m ha bi cc gen vir. Hot ng ca cc gen ny c hot ha bi hp cht phenolic ca cy (v d nh acetosyringone, catechol, cc dn xut ca chalcone...). Ngoi ra, cc monosaccharide nh glucose, arabinose c mt trong mi trng cng lm tng kh nng gy cm ng nhm gen vir. Protein VirA ng vai tr quan trng trong vic quyt nh loi cy ch b nhim bi Agrobacterium. Trong thc t, Agrobacterium khng c kh nng xm nhp vo cy mt l mm, c th protein VirA khng nhn bit cc tn hiu do cy mt l mm tit ra. Ni chung, Agrobacterium chi gy hai cy hai la mm, vi vy ngi ta cho rng

chung chi co th a T-DNA vao h gen cac cy hai la mm. Tuy nhin, gn y nhiu tac gia a chng minh khi nhim vi khun, cac cy mt la mm cung co th san xut opine va v th co th khai thac kha nng bin nap gen cua Agrobacterium vo cy mt la mm. Hin nay, ngi ta cng thnh cng trong vic chuyn gen vo mt s cy mt l mm nh la, ma... qua trung gian A. tumefaciens. Khi cy nhim A. tumefaciens, do T-DNA hp nht vao trong genome cua cy chu bt u hoat ng va san xut ra auxin, cytokinin va opine, toan b sinh trng cua cy bi ri loan, cac t bao phn chia v t chc va tao ra cac khi u. Opine c vi khun s dung nh mt loai thc n nh gen chuyn hoa opine trn Ti-plasmid. C ch ly nhim cua A. rhizogenes i vi cy hai la mm cung tng t, nhng trong vung T DNA cua A. rhizogenes chi co gen san sinh ra auxin, vi th s thay i hinh thai chinh cua thc vt la chung tao ra rt nhiu r t khi bi nhim bnh. gn T-DNA vo t bo thc vt, u tin vi khun A. tumefaciens phi tip xc vi thnh t bo thc vt b tn thng. Qu trnh ny c thc hin nh cc gen chvA v chvB. Gen chvB m ha mt protein lin quan n hnh thnh -1,2 glucan mch vng, trong khi gen chvA xc nh mt protein vn chuyn, nh v mng trong ca t bo vi khun. Protein vn chuyn gip vn chuyn -1,2 glucan vo khong gia thnh t bo v mng sinh cht. -1,2 glucan gi vai tr quan trng vi khun Agrobacterium tip xc vi thnh t bo thc vt. Nu khng c s tip xc ny, s khng c s dn truyn T-DNA. Cc sn phm protein ca vng vir c tc dng cho vic dn truyn T-DNA t vi khun vo t bo thc vt. Cc loi protein rt cn thit cho qu trnh ct T-DNA khi plasmid, cm ng thay i mng t bo thc vt m chng tip xc, tham gia di chuyn phn T-DNA qua mng vi khun ti t bo cht ca t bo thc vt, vn chuyn ti nhn ri cui cng xm nhp vo genome ca cy ch. Thc cht, ch ring T-DNA ca plasmid c chuyn vo genome t bo thc vt, m khng cn phn no khc. Qu trnh dn truyn ch do sn phm ca cc gen vir v gen chv quyt nh m khng lin quan n cc gen khc trn T-DNA. Tuy nhin, chui DNA 25 bp (RB v LB ca T-DNA) c vai tr l v tr cm ng cho cc sn phm ca t hp cc gen vng vir, c bit l protein t gen virE mang chng dn truyn vo t bo thc vt. Chng hot ng nh cc tn hiu nhn bit v khi ng qu trnh dn truyn. Trc ht gen virA trong t hp gen vng vir c phosphoryl ha nh tc ng ca cc hp cht phenol nh acetosyringone gii phng ra t cc t bo thc vt tn thng. Sn phm ca qu trnh ny li tip tc phosphoryl ha gen virG. Sn phm ca gen virG lin tip lm hot ha ton b cc gen vir cn li, m hai gen cui cng c hot ha l gen virB v virE. Trc , khi gen virD c hot ha, sn phm ca n cm ng nhn bit RB v LB ca T-DNA v lm t phn TDNA ra khi DNA ca plasmid thnh cc si n. ng thi, qu trnh phosphoryl ha ny cng lm thay i thm sut mng t bo thc vt, mng t bo b mm ra v b

thng. Cc si n T-DNA c gn vo protein do gen virE tng hp v dch chuyn v pha mng t bo vi khun. Ngay sau , si T-DNA c trt t vi khun vo t bo thc vt. Cu ni chnh l s tip hp (conjugation) gia hai t bo do cm ng sn phm gen virB m thnh. Khi T-DNA c chuyn giao vo t bo thc vt, chng nhanh chng hp nht vo genome t bo thc vt c n nh v di truyn nh cc gen bnh thng khc.

V. Sp xp v khuch i cc gen trong genome 1. Sp xp li cc gen Ni chung, genome c cu trc v t chc bn vng. DNA ca genome thng khng b bin i bi s pht trin v tnh, nhng thnh thong cc trnh t ca chng cng c th b chuyn ch trong gen, b ci bin, khuch i hoc thm ch bin mt, nh l mt trng hp t nhin. Trao i cho trong phn bo gim nhim l mt trong nhng nguyn nhn gy ra bin i ca genome. Tuy nhin, iu ny xy ra ch yu trong t bo sinh dc m khng c trong cc t bo soma. Vic sp xp li genome s dn n nhng thay i sau: - To ra cc gen mi cn thit cho s biu hin trong cc trng hp c bit. - S ti sp xp c th p ng cho vic m hoc ng gen. y cng chnh l c ch ca s iu ha biu hin gen. Mt v d in hnh l hin tng sp xp li cc gen trong genome ca nm men S. cerevisiae v ca k sinh trng Trypanosome chu Phi khi gy chng ng li b vt ch. 1.1. Chuyn i dng giao phi ca nm men Nm men c th tn ti c hai dng n bi hoc lng bi. Cc dng lng bi l d hp t trng hp locus kiu giao phi, v cc t bo n bi c th hoc l MATa hoc MAT. Vic chuyn i trng thi c thng qua giao phi, kt hp cc bo t n bi thnh lng bi v qua vic ti to giao t mi. Tuy nhin, giao phi ch xy ra gia hai loi t bo n bi a v . Cc t bo n bi cng loi khng th kt hp vi nhau to thnh t bo lng bi. MAT (mating type locus) l v tr hot ng hay cassette hot ng: mt vng c bit ca nhim sc th s 3 cha cc gen qui nh dng giao phi (a v ). Cc gen ny cn tn ti hai v tr khc trong genome. Tuy nhin, chng u b bt hot. Hai v tr ny c gi l hai v tr tnh (hay cassette tnh HML v HMR). Mi v tr tnh ch mang cc gen qui nh cho mt dng giao phi. Khi cc gen c sao chp t mt v tr tnh vo v tr hot ng th mRNA mi c tng hp t cc gen .

Nh vy, qu trnh sao chp quyt nh dng giao phi ca nm. Bn gc lun c bo tn v tr tnh v bn th hai xut hin v tr hot ng. Nu v tr tnh c mang t bin th chng s c sao chp vo v tr hot ng. Tuy nhin, nu xy ra t bin cassette MAT th tnh trng mi xut hin khng bn. Mt khi dng giao phi chuyn i th cc gen c v tr MAT b thay th bi bn sao ca v tr tnh khc. Lc , t bin b loi i v tnh trng mi s bin mt. So snh gia cc dng cng si nm (homothallic) v khc si nm (heterothallic) ngi ta nhn thy dng heterothallic c gen HO hot ng v c th chuyn i t ng gia cc kiu giao phi, v v th mt bo t n c th lm tng qun th t phi, trong khi dng homothallic khng c gen HO v duy tr cng mt kiu giao phi trong sut chu trnh sinh trng n bi. Phn tch di truyn cho thy cc gen sau y cn cho vic chuyn i kiu giao phi: - MAT - HO, m ha cho enzyme endonuclease - HML (cho MATa chuyn thnh MAT) - HMRa (cho MAT chuyn thnh MATa) Trnh t cc nucleotide v tr tnh v v tr hot ng ch khc nhau mt on ngn k hiu l Ya v Y (Hnh 2.8). Enzyme HO-endonuclease nhn bit v tr c hiu ti ranh gii phn cch gia Z v Y v ca cassette hot ng MAT v ct c hai si DNA ti . iu c bit l enzyme ny khng ct DNA khi chng hin din cassette tnh. Sau khi on Y ca vng MAT b phn hy ht, on Y ca mt trong hai cassette tnh c dng lm khun mu sao chp vo v tr b phn hy (Hnh 2.8). Nu t bin xut hin vng MAT (on Y), tnh trng mi ch biu hin tm thi. Mt khi dng giao phi chuyn i, cc gen bnh thng c sao chp vo vng MAT v t bin b loi i. 1.2. Chuyn i gen Trypanosome Trypanosome c kh nng ln trnh c h thng min dch ca vt ch thng bng cch thay i khng nguyn b mt (surface antigen) ca chng. Mi loi khng nguyn c tng hp nh hot ng ca mt gen tng ng ti v tr hot ng. Gen ny c th b thay th bi mt gen m ha cho loi khng nguyn khc nm mt v tr tnh no trong genome. Mi v tr tnh cha mt gen trng thi khng hot ng. Gen ny ch c m khi chuyn n v tr hot ng. Trong genome ca Trypanosome, c rt nhiu v tr tnh nhng ch c mt v tr hot ng.

Hnh 2.8. Qu trnh chuyn i dng giao phi t a sang nh trao i gen gia vng MATa v HML trn nhim sc th s 3

Bnh thng, Trypanosome s tri qua mt s ln bin i hnh thi khi c truyn t rui chu Phi sang vt ch. B mt ca t bo Trypanosome c bao bc mt lp n gm 5106 phn t ca mt loi glycoprotein (VSG-variable surface glycoprotein). y chnh l khng nguyn b mt ca Trypanosome khi chng xm nhp vo vt ch. iu ng ch l chng c kh nng thay i khng nguyn b mt, do trnh c phn ng min dch ca t bo vt ch. Qu trnh thay th khng nguyn b mt ph thuc vo s chuyn i cc gen m ha cho chng xy ra mt v tr c bit trong genome (v tr hot ng). Chuyn i gen m ha khng nguyn b mt nhm mc ch hot ha gen m ha khng nguyn b mt mi thay th cho khng nguyn tn ti trc . Khi mt gen ang hot ng b thay th bi mt gen khc s tng ng vi vic xut hin khng nguyn mi v loi b khng nguyn c. - Cu trc ca mt VSG Trypanosome Cu trc chung ca mt VSG c m t trn hnh 2.9 v 2.10. Mt VSG va c tng hp di khong 500 amino acid gm tn hiu N-terminus, tip theo l on peptide quyt nh tnh khng nguyn; on peptide bo th gia cc VSG v ui k nc. Phn t ny c tng hp di dng protein tin thn (pre-protein). Do ,

chng phi tri qua bin i hai u NH2 v COOH tr thnh dng protein hon chnh (mature form). Dng ny c nh vo mng t bo u COOH. Mt loi Trypanosome c th to ra t nht khong 100 VSG t khi nhim cho n khi gy cht vt ch. S gen m ha cho VSG c th nhiu hn 1.000 gen, tt c cc gen ny u nm trong genome. Tuy nhin, ti mt thi im bt k ch c mt gen hot ng tng hp nn mt loi VSG. Do , s thay i khng nguyn tng ng vi s thay i hot ng ca gen. Khi mt gen mi c m, gen hot ng trc n phi b c ch hon ton. Lc , mt khng nguyn mi s thay th khng nguyn tn ti trc n. Hnh 2.9 cho thy chui polypeptide cha khong 500 amino acid. N-terminus cha mt peptide tn hiu cho s vn chuyn qua ER (li ni sinh cht, endoplasmic reticulum) v ti mng plasma, c tch ra khi protein hon chnh. Vng bin thin l khc nhau mi VSG, iu cho thy cc VSG c t hoc khng c tnh ng nht. Hng ti phn C-terminus, chui polypeptide c bo ton tt hn v phn ny c gi l vng tng ng. ui k nc cha mt tn hiu nhn bit gn vi m neo glycolipid (glycolipid anchor) (Hnh 2.10). Khi m neo c gn th 20 amino acid cui cng s c tch ra. Glycoprotein bin i b mt (VSG) c gn vi mng thng qua mt m neo glycolipid cha ethanolamine, mt cu trc glycan mang mt s gc mannose (mannose moiety), mt glucosamine v mt phosphoinositol lin kt vi 1,2dimyristoyglycerol c gai trong mng plasma. Gc glycolipid l yu t quyt nh phn ng lai (cross-reacting) (CRD) c nhn bit bng cc khng th phn ng vi tt c dng bin i ca VSG, nhng ch khi VSG c phng thch khi mng. Mng lin kt vi VSG khng c nhn bit bi cc khng th anti-CRD. S phng thch VSG c xc tc bi hot tnh ca trypanosome-specific phospholipase C c gi nh l hin din mt bn trong (inner face) ca mng plasma. Ngi ta khng bit rng enzyme c th ct lin kt phosphoester trn mt khc ca mng nh th no.

Hnh 2.9. S minh ha chui protein ca mt VSG c trng

Hnh 2.10. Cu trc ca m neo glycolipid ca VSG

- Hot ng ca gen VSG Gen m ha cho mt VSG c gi l bn gen gc (basic copy gene). Cc gen ny c phn thnh hai nhm ty thuc vo v tr ca chng trn nhim sc th. + Cc gen nm telomere (khong 5-15 kb)>200 gen. + Cc gen nm cch telomere hn 50 kb. Tng t nh nm men, cc gen m ha cho VSG nm ri rc trong genome v trng thi khng hot ng. Mt gen bt k c hot ha ch khi n c sao chp vo v tr hot ng (expression site) trong khi nguyn bn ca n vn c bo tn v tr tnh. Bn sao ca bn gen gc vo v tr hot ng c gi l ELC (bn sao hot ng-expression linked copy). V tr hot ng nm gn telomere. Nh vy, vic chn mt bn gen gc sao chp to bn sao hot ng s ph thuc vo v tr ca bn gen gc telomere hoc nm pha trong telomere. C hai gi thit mt gen tr nn hot ha nh sau: - V tr hot ng khng thay i m ch c cc ELC thay th cho nhau. Bn sao ca mt bn gen gc s thay th cho bn sao ca mt gen khc ti v tr , iu ny xy ra tng t nh cc gen cassette tnh c sao chp vo cassette hot ng trong trng hp vi nm men. - V tr hot ng thay i, khi cn tng hp mt VSG mi, gen v tr hot ng c b ngng v gen mt v tr khc (gn telomere) c khi ng.

Mt s phn t mRNA tng ng vi cc VSG khc nhau c phn lp v c xc nh trnh t nucleotide (thng qua cDNA). iu ngc nhin l phn oligonucleotide u 3 ca mi gen VSG u khc vi phn 3 ca cc mRNA c phin m t cc gen . Mt khc, cc gen ny khng c phn 5 ging nh cc mRNA. Nh vy, cc mRNA khng c tng hp hon ton trn khun mu cc gen ny. Phn 3 ca cc mRNA tng ng vi phn 3 ca v tr hot ng ELC, trong khi phn 5 (gm 35 nucleotides) c tng hp t nhng on DNA khc v c gn vo mRNA (hin tng trans-splicing). 2. Khuch i cc gen S lng bn sao ca mt s gen cng c th c tng ln tm thi trong qu trnh pht trin cc t bo soma. Vic tng s lng ca mt gen c bit no ph thuc vo tng iu kin c th ca t bo v xy ra khng ph bin. Cc bn sao c th nm tp trung thnh mt nhm gm bn sao ny ni tip bn sao khc hoc c th tn ti nh nhng on DNA c kh nng ti bn c lp. Chng hn: - S nhn bn ca gen m ha cho rRNA trng ch. Trng ch c ng knh khong 2-3 mm, d tr rt nhiu rRNA. Chng c phin m t rt nhiu gen rDNA. Cc gen ny c nhn ln (khong 2.000 ln) theo c ch vng trn quay (xem chng 4) trong qu trnh pht trin v tn ti di dng cc vng trn khp kn. - Khi nui cy cc t bo ng vt c v trong mi trng c bit, DNA ti mt s v tr trong genome c nhn ln. V d: nui cy cc t bo ung th trong mi trng cha c t methotrexate. Cht ny c ch hot tnh ca enzyme dihydrofolate reductase (DHFR) gi vai tr trong tng hp cc nucleotide ca DNA. Cc t bo ung th nui cy trong mi trng c cht c ny pht trin thnh cc qun lc t bo khng li c t. Khi nng cht c tng dn, nng DHFR cng tng theo, c th t ti 1.000 ln ln hn mc bnh thng. Nng enzyme tng do s lng cc gen m ha cho chng tng. C ch chnh xc ca hin tng ny cha r rng, nhng c th xy ra theo hai cch: - Trao i cho khng cn bng gia hai nhim sc t (chromatid) ca nhim sc th dn n mt s t bo khng c gen dhfr v mt s khc c hai bn sao ca gen ny. Trong mi trng c c t, trao i cho khng cn bng c lp i lp li v cc t bo cha nhiu gen dhfr vn pht trin tt trong mi trng ny. - Cc on DNA (100-1.000 kb) cha 2-4 gen dhfr (~31 kb/gen) c sao chp t nhim sc th bnh thng to ra cc nhim sc th rt nh, khng c tm ng. Cc nhim sc th nh ny ghp vo cc nhim sc th bnh thng khc. Qu trnh ny lp i lp li v qua mt s ln phn bo nguyn nhim, t bo no mang s lng

ln cc gen dhfr cng c iu kin pht trin thun li trong mi trng c cha c t. 3. Bin np gen Mt phng thc tng kh nng di truyn l ng dng cc tng tc vt ch cng sinh-k sinh, trong DNA l c chuyn vo t bo vt ch t mt vi khun. C ch ny tng t vi s tip hp ca vi khun. S biu hin ca DNA vi khun trong vt ch mi ca n s lm thay i kiu hnh ca t bo. V d in hnh l vi khun A. tumefaciens cm ng to khi u t bo thc vt b chng xm nhim. Khi DNA l c a vo t bo eukaryote, n c th tn ti ngoi nhim sc th hoc c hp nht trong genome. Nu xy ra theo trng hp th hai, genome s mang nhng t bin di truyn v nhiu khi DNA l vn tip tc hot ng lm xut hin cc tnh trng mi. Vic a cc gen l vo t bo soma hoc t bo sinh dc m vn duy tr hot ng ca nhng gen c gi l chuyn nhim (transfection). C th biu hin tnh trng mi nh hot ng ca gen l a vo t bo sinh dc c gi l c th chuyn gen. Qu trnh ny c th lm thay i s n nh ca genome. DNA sau khi c tim vo trong cc t bo trng ng vt c th c hp nht trong genome v truyn cho th h sau nh mt thnh phn di truyn bnh thng. Kh nng a mt gen chc nng c trng c th tr thnh mt k thut y hc s dng cho vic cha bnh di truyn. Ti liu tham kho/c thm
1. V Th Thng Lan. 2002. Sinh hc phn t. NXB i hc Quc gia H Ni, H Ni.

2. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA.
3. Cantor CR and Smith CL. 1999. Genomics. John Wiley & Sons, Inc. New York, USA. 4. Dale JW and Von Schanzt M. 2002. From Gene to Genome. John Wiley & Sons, Ltd. West Sussex, UK. 5. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 6. Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J. 2004. Molecular Cell Biology. 5th ed. WH Freeman and Company, New York, USA. 7. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Loscik R. 2004. Molecular Biology of the Gene. The Benjamin Cummings/Cold Spring Habor Laboratory Press, San Francisco, CA, USA.

8. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company Inc. New York, USA.
1 2

DNA lp li m t cc chui hin din hn mt bn sao trong mi genome. DNA khng lp li cha cc chui duy nht: ch c mt bn sao trong genome n bi.

Chng 3

Cu trc v chc nng ca gen

I. nh ngha gen Chng ta c th im qua nhng mc chnh trong lch s nghin cu v gen nh sau: Mendel (1865) l ngi u tin a ra khi nim nhn t di truyn. Johansen (1909) xut thut ng gen (t genos, ngha l sn sinh, ngun gc) ch nhn t di truyn xc nh mt tnh trng no . Sau , Morgan trong nhng nm 1920 c th ha khi nim v gen, khng nh n nm trn nhim sc th v chim mt locus nht nh, gen l n v chc nng xc nh mt tnh trng. Vo nhng nm 1940, Beadle v Tatum chng minh gen kim tra cc phn ng ha sinh v nu gi thuyt mt gen-mt enzyme. Tuy nhin, trng hp hemoglobin l mt protein nhng li gm hai chui polypeptide do hai gen xc nh, do gi thuyt trn buc phi iu chnh li l mt gen-mt polypeptide. Vo nhng nm 1950, DNA (deoxyribonucleic acid) c chng minh l vt cht di truyn. M hnh cu trc DNA ca Watson v Crick c a ra v l thuyt trung tm (central dogma) ra i. Gen c xem l mt on DNA trn nhim sc th m ha cho mt polypeptide hay RNA. Cui nhng nm 1970, vic pht hin ra gen gin on sinh vt eukaryote cho thy c nhng on DNA khng m ha cho cc amino acid trn phn t protein. V th, khi nim v gen li c chnh l mt ln na: Gen l mt on DNA m bo cho vic to ra mt polypeptide, n bao gm c phn pha trc l vng 5 khng dch m (5 untranslation) hay cn gi l vng ngc hng (upstream) v pha sau l vng 3 khng dch m (3 untranslation) hay cn gi l vng cng hng (downstream) ca vng m ha cho protein, v bao gm c nhng on khng m ha (intron) xen gia cc on m ha (exon). Hin nay, c th nh ngha gen mt cch tng qut nh sau: Gen l n v chc nng c s ca b my di truyn chim mt locus nht nh trn nhim sc th v xc nh mt tnh trng nht nh. Cc gen l nhng on vt cht di truyn m ha cho nhng sn phm ring l nh cc mRNA c s dng trc tip cho tng hp cc

enzyme, cc protein cu trc hay cc chui polypeptide gn li to ra protein c hot tnh sinh hc. Ngoi ra, gen cn m ha cho cc tRNA, rRNA v snRNA...

Bng 3.1. Tm tt lch s nghin cu v di truyn hc

II. L thuyt trung tm 1. S xc nh di truyn cu trc bc mt ca protein Cu trc khng gian ca chui polypeptide c xc nh bi trnh t sp xp ca cc amino acid tc cu trc bc mt. Nh vy, mc d c nhiu mc cu trc khng gian khc nhau, nhng cu trc bc mt tc trnh t sp xp cc amino acid chi

phi ton b cc mc cu trc khc. Vic xc nh di truyn phn t protein trng thi t nhin c y hot tnh sinh hc ch quy t li ch yu xc nh cu trc bc mt l .

2. Cc enzyme mt hot tnh do t bin Nhiu nghin cu cho thy, vic mt hot tnh enzyme nhiu khi khng phi do vng mt ca enzyme, m ch do cc bin i trn phn t (modification). C trng hp t bin dn n nhng thay i tinh vi, enzyme vn c hot tnh nhng s biu hin khc nu thay i iu kin. Chng hn: nm mc Neurospora crassa, enzyme tyrosinase do gen T xc nh, xc tc cho phn ng chuyn ha tyrosine thnh dihydroxyphenylalanine. Alelle T+ ca dng hoang di sn xut tyrosinase c hot tnh nhit bnh thng v c 60oC. Mt t bin TS sn xut tyrosine c hot tnh nhit bnh thng, nhng li mt hot tnh 60oC. Nh vy, trong a s trng hp, t bin ca mt gen khng lm bin mt enzyme m ch bin i cu trc dn n thay i hot tnh. Cc t bin ca cng mt gen c th gy ra nhng bin i khc nhau trn enzyme. Cc hin tng chng t rng cu trc ca enzyme chu s kim sot trc tip ca gen.

3. Bn cht cc bin i di truyn ca protein Bn cht chnh l quan h mt gen-mt polypeptide. Nh nu trn, ngi ta khm ph ngi c nhng gen to ra hemoglobin (Hb) khi bin d s to ra nhng hemoglobin bt thng do sai hng cc chui polypeptide hoc (Bng 3.2 v 3.3) v gy ra cc bnh di truyn.

Bng 3.2. Cc loi hemoglobin chui polypeptide

 trng hp ny, th t cc amino acid c th b sai lch. T nhng d liu trn ta thy cc dng t bin to mt Hb bt thng l thay mt amino acid ny bng mt amino acid khc.

Bng 3.3. Cc loi hemoglobin chui polypeptide

Qua hai chui polypeptide v chng ta thy c mt s dng hemoglobin bt thng ngi. Trn mi chui, ch trnh by nhng amino acid b thay i dng t bin. S th t ch v tr ca amino acid trong chui polypeptide. Mi hemoglobin bt thng c th c t cho mt ch cng tn (nu c) ca a phng c tm thy. t bin c biu hin bi s thay th v tr ca mt amino acid ny bng mt amino acid khc. 4. S tng quan ng tuyn tnh gen-polypeptide

4.1. t bin tryptophan synthetase-s ng tuyn tnh gia gen v chui polypeptide Nghin cu trn enzyme tryptophan synthetase xc tc cho phn ng tng hp tryptophan ca E. coli ngi ta nhn thy c nhiu t bin xy ra trn cng mt gen m ha cho tryptophan synthetase. Thc hin ti t hp trong gen (nguyn tc l gen cc v tr cng xa nhau trn nhim sc th cng d ti t hp), ngi ta nhn c cc dng bin d c tnh cht khc nhau, v tnh c khong cch tng i gia nhng im khc nhau ca t bin c xc nh. V tr bin d trn th nhim sc tng ng vi v tr ca amino acid trn chui polypeptide. Nh vy, c th cho rng c s ng tuyn tnh gia gen v chui polypeptide (Hnh 3.1).

Hnh 3.1. Tng quan ng tuyn tnh gia gen v enzyme tryptophan synthetase ca E. coli thng qua cc v tr t bin v cc gc amino acid b thay i

Nhiu dng t bin ca tryptophan synthethase c to ra. Bng c ch ti t hp, nhng khong cch tng i gia nhng im khc nhau ca t bin c xc nh. Sn phm protein ca mi dng t bin c phn tch, v nhng thay i cc amino acid khc cng c xc nh. Ngi ta tm thy mi tng quan hon ton gia nhng khong cch ca cc t bin c tm thy trn gen vi khong cch ca amino acid b thay i trong phn t protein.

4.2. t bin 4.2.1. Khi nim Mt gen (DNA) c 4 loi base v mt phn t protein c 20 loi amino acid1, nhng gia chng c mi tng quan nh th no. u tin, ngi ta cho rng mt base qui nh mt amino acid, nhng nhng tnh ton cho thy khng hp l. V ch c 4 base trong DNA v 20 amino acid trong protein, cho nn mi codon phi cha t nht 3 base. Hai base cng khng th lm thnh mt codon bi v ch c 4 2 = 16 cp hp l

ca 4 base. Nhng 3 base th c th bi v s c 43 = 64 b ba hp l. V s lng b ba hp l ln hn 20, cho nn s c trng hp mt vi codon ch nh cng mt amino acid. V d: UCU, UCC, UCA, UCG, AGU v AGC u cng m ha cho serine. T , ngi ta a ra khi nim m di truyn (tn hiu di truyn). M di truyn cho php c th t trn DNA bit th t trn chui polypeptide. M di truyn khng m h, c ngha vi mt trnh t chng hn ATA ta bit n ghi m cho mt amino acid g, v cng thy rng c nhiu m di truyn xc nh cho mt amino acid (Bng 3.4).

4.2.2. t bin im L t bin ch tc ng mt v tr, ni r hn l mt base. Khi thay i mt base trn DNA s to ra s thay i mt amino acid (Hnh 3.2). t bin d nhin xy ra trn DNA v c sao li trn mRNA trong phin m, ri trn protein trong dch m.

Bng 3.4. M di truyn chung

Ch thch Nhng n v m (codon) c c theo chiu 53. STOP: codon kt thc (cn gi l v ngha).

t bin im c cc dng sau: - t bin sai ngha. Thay i mt amino acid trong protein, c th dn n mt trong ba kt qu sau: + Khng hu qu no c, v amino acid khng nm trong v tr hot ng hoc khng c vai tr trong cu trc enzyme. + C bin i nh chui polypeptide s to ra tnh mn cm yu vi nhit, lm gim s n nh chui polypeptide. + Mt hn hot tnh enzyme nu ng ngay v tr hot ng ca enzyme .

- t bin v ngha. Thay i mt base. Nu l mt codon v ngha s lm ngng ko di (tng hp) chui polypeptide v tr amino acid ny. Tc l nu codon ny nm u s khng c chui polypeptide hot ng. - t bin acridine hoc t bin dch khung. t bin ny do cht acridine mu da cam to ra (hoc cn gi l t bin dch khung, frameshift, do thm vo hoc bt i mt base) (Hnh 3.2 E v D). Nh vy, mt t bin trn khung c khi thm vo (C) hoc mt i (A) thng s dn n xut hin mt codon stop lm ngng chui polypeptide v enzyme s khng c hot tnh.

4.2.3. t bin km hm n nay, ngi ta nhn thy mi sai lch trong vic tng hp protein nu c u xy ra t DNA, cn qu trnh din ra t RNA n polypeptide lun lun ng. Nghin cu mt vi kiu protein t bin ta thy: - t bin sai ngha. Lm xut hin mt bt thng trong trnh t amino acid. Kt qu protein mt hot tnh. Hot tnh ny c th c phc hi, hoc do mt t bin ngc cho li protein cu trc ban u. - t bin v ngha. Lm mt i mt phn chui polypeptide, phn cn li khng c hot tnh, v hot tnh ny c th c li c nh t bin trong mt codon b t bin. Thng thng, nhng gen km hm t bin v ngha khng nm gn v tr ca t bin y. l nhng gen lm bin i h thng dch m khi tng hp protein.

Hnh 3.2. Cc dng t bin im

A: trnh t cc codon v cc amino acid tng ng dng t nhin. B: thay i mt base (C thnh A) lm thay i mt amino acid (Arg thnh Pro) gy nn t bin sai ngha. C: thay i mt base (C thnh G) sinh ra mt codon v ngha (UGA). D: thm mt base (C) gy nn t bin acridine hoc t bin dch khung, c s di khung c. E: mt mt base (A), gy nn t bin acridine, chm dt c.

5. L thuyt trung tm ca sinh hc phn t Tng hp protein trong t bo c cc c im sau: - Cc phn t thng tin nh nucleic acid v protein c tng hp theo khun. Tng hp theo khun va chnh xc va t tn enzyme. Tuy nhin, cn c vo hng lot tnh cht ha hc cc protein khng th lm khun mu cho s tng hp ca chnh chng. V vy, khun mu tng hp nn protein khng phi l protein.

- Sinh tng hp protein tch ri v khng gian vi ch cha DNA. Nhiu nghin cu cho thy tng hp protein c th xy ra khi khng c mt DNA. S kin ny th hin r rng nht nhng t bo eukaryote. Trong nhng t bo ny, hu nh ton b DNA tp trung nhim sc th trong nhn, cn tng hp protein ch yu din ra t bo cht. To xanh n bo Acetabularia khi b ct mt phn cha nhn vn tng hp c protein v sng vi thng nhng mt kh nng sinh sn. R rng, ni cha DNA mang thng tin di truyn v ch sinh tng hp protein tch ri nhau v khng gian. - DNA khng phi l khun mu trc tip tng hp protein, do phi c cht trung gian chuyn thng tin t DNA ra t bo cht v lm khun tng hp protein. Cht phi c c trong nhn v t bo cht vi s lng ph thuc vo mc tng hp protein. - Cht trung gian c xem chnh l RNA nh cc c im sau: + RNA c tng hp ngay trong nhn c cha DNA, sau n i vo t bo cht cho tng hp protein. + Nhng t bo giu RNA tng hp protein nhiu hn. + V phng din ha hc RNA gn ging DNA: chui polyribo-nucleotide thng cng cha 4 loi ribonucleotide A, G, C v uracil (U). N c th nhn c thng tin t DNA qua bt cp b sung. Ni chung, trong t bo khng th tm thy cht no khc ngoi RNA c th ng vai tr trung gian cho tng hp protein. Mi quan h ny chnh l thng tin di truyn i t DNA qua RNA ri n protein v c biu din hnh 3.3. Mi quan h ny cn c gi l l thuyt trung tm (central dogma), c Crick a ra t 1956 n nay v cn bn vn ng. Vo nhng nm 1970, ngi ta pht hin qu trnh phin m ngc t RNA tng hp nn DNA nh enzyme reverse transcriptase. n nay, vic sao chp (tng hp) RNA trn khun mu RNA cng c chng minh nhiu loi virus. Ngoi ra, thng tin t protein cng c th c truyn sang protein (prion ca bnh b in). Ring dng thng tin t protein ngc v mRNA/DNA th cha c tm thy (Hnh 3.4).

Hnh 3.4. Nhng b sung mi vo l thuyt trung tm ca Crick 6. DNA v m di truyn Chng ta bit c s lin quan ng tuyn tnh gia DNA v phn t protein, t d dng d on rng trnh t c hiu ca cc amino acid trn phn t protein s c m ha bng nhm cc nucleotide trn phn t DNA. C tt c 4 loi base, nu cc base c nhm i tc 2 nucleotide m ha cho mt loi amino acid th tt c ch c 16 t hp, khng cho 20 loi amino acid. Nh vy, n v m ha (codon) phi gm 3 nucleotide (xem phn 4.2). Vn tip theo l xc nh chnh xc cc codon no m ha cho tng amino acid. Nirenberg v Matthaei s dng enzyme tng hp nhn to RNA. Khi dng ch mt loi nucleotide l U s nhn c RNA l poly(U), nu ch dng A s nhn c poly(A). Nm 1961, Nirenberg v Matthaei dng poly(U) thay cho khun mu mRNA tng hp protein trong h thng v bo (c amino acid, enzyme tng hp protein, nhng khng c DNA...), sn phm thu c l chui polypeptide polyphenylalanine ch cha mt loi amino acid l phenylalanine. iu chng t codon UUU m ha cho phenylalanine. y l codon u tin c xc nh. Sau , h cng chng minh c rng AAA m ha cho lysine, GGG cho glycine v CCC cho proline. Nm 1964, Khorana tm ra phng php tng hp mRNA nhn to vi trnh t lp li (nh AAG AAG AAG...) v nh n gii quyt xong cc vn cn cha r rng. Bng m di truyn (Bng 3.4) cho thy trong 64 codon, c 3 codon UAA, UAG, UGA khng m ha cho amino acid c gi l v ngha (non-sense), ng thi l codon kt thc (termination) tc du chm cu, chm dt chui polypeptide.

M di truyn c tnh suy bin (degeneration) tc mt amino acid c nhiu codon m ha, ch tr methionine v tryptophane ch c mt codon (tng ng l ATG v TGG). Cc codon ng ngha tc m ha cho cng mt amino acid thng c hai base u tin ging nhau, nhng khc nhau ci th ba. V d: CCU, CCC, CCA v CCG tt c u m ha cho proline. Trn thc t, U v C lun lun tng ng nhau v tr th ba, cn A v G tng ng nhau trong 14 trn 16 trng hp. Tr mt s ngoi l, m di truyn c tnh ph bin (universal) tc ton b th gii sinh vt c chung b m di truyn.

III. Cu trc v chc nng ca gen Khi nghin cu cc quy lut di truyn Mendel v hc thuyt di truyn nhim sc th, gen c quan nim nh mt im trn nhim sc th, va l n v chc nng xc nh mt tnh trng, va l n v t bin, va l n v ti t hp. Cng vi s pht trin ca di truyn hc, khi nim v gen c c th ha thm, cu trc v chc nng ca gen c hiu chi tit hn.

1. Cu trc gen Mt gen thng c xem gm c hai phn, phn mang m di truyn c phin m sang phn t mRNA v phn DNA lm nhim v iu khin hot ng ca gen (Hnh 3.5). C hai phn u c cu trc c bn kh ging nhau mi sinh vt prokaryote v eukaryote. Tuy nhin, gen eukaryote cn c cu trc c th lin quan n cc c ch kim sot khc nhau i vi hot ng ca gen. Cu trc mt gen in hnh ni chung u c promoter, v tr RNA polymerase hot ng khi u phin m. i khi nm rt xa gen, c th thy cc enhancer (vng tng cng) hoc silencer (vng c ch) c vai tr lin quan n qu trnh phin m. di ca mt gen thay i ty theo s lng v di ca cc intron cha trong n.

Hnh 3.5. Cu trc chung ca mt gen

Vng DNA mang m di truyn s c phin m sang phn t mRNA. Qu trnh ny thc hin theo chiu 53 trn si mRNA ang c tng hp. Khng phi mi phin m di truyn trn phn t mRNA u c dch m sang phn t protein. Hai u 5 v 3 ca phn t mRNA gm mt s nucleotide khng c dch m m li lin quan n tnh bn vng ca phn t mRNA hoc tham gia kim sot qu trnh dch m. Hai on ny gi l vng khng dch m 5 v 3 (untranslated region). Vng khng dch m 5 nm trc im khi u dch m (3 nucleotide AUG m ha cho methionine u tin ca chui polypeptide) v vng khng dch m 3 nm sau im kt thc dch m (stop codon c th l UAA, UGA v UAG). Do t bo prokaryote khng c cu trc nhn nn qu trnh phin m (tng hp mRNA) v dch m (tng hp protein) xy ra ng thi. Cn phn t mRNA ca eukaryote c phin m trong nhn, sau phi ct b intron v gn cc exon li, chu bin i ti cc u 5 v 3 trc khi vn chuyn ra ngoi t bo cht dng lm khun mu tng hp protein. Hot ng ca mt gen c nh gi thng qua qu trnh phin m (tng hp mRNA) v qu trnh dch m (tng hp protein). Hot ng ny c kim sot rt cht ch bng cc c ch khc nhau mi giai on, nh bt u v kt thc phin m, qu trnh bin i mRNA, quyt nh tnh bn vng v kim tra li thng tin di truyn trn cc phn t ny... Do cu trc sp xp cc gen prokaryote khc vi gen eukaryote nn s phi hp gia cc c ch iu khin mang tnh cht ring bit cho tng loi genome. Cc gen prokaryote thng sp xp nm gn nhau v chu s iu khin chung ca mt promoter, tc l chng c phin m sang cng mt phn t mRNA. Cu trc ny c gi l operon. Nh vy, mt operon gm hai hay nhiu gen nm cnh nhau trn mt nhim sc th. Thng thng, l cc gen cng tham gia vo mt con ng chuyn ha, v d nh cc gen m ha cho cc enzyme cn thit cho qu trnh chuyn ha glucose. Do c chung promoter iu khin cho mi gen nm trong mt operon cho nn ch c mt loi phn t mRNA c tng hp t mt operon (mang thng tin di truyn ca tt c cc gen nm trong ). Ni cch khc, qu trnh phin m ca cc gen trong mt operon xy ra ng thi v phn t mRNA c trng cho operon c gi l mRNA-polycistron. Tuy nhin, iu cn ghi nh l qu trnh dch m trn cc phn t mRNApolycistron xy ra hon ton c lp vi nhau. Mi on tng ng vi mt gen trn phn t ny u c v tr bm ca ribosome, c m bt u v kt thc tng hp chui polypeptide ring bit. Do , tc tng hp cc protein trn cc phn t mRNApolycistron hon ton khc nhau (Hnh 3.6).

Hnh 3.6. Cu trc operon trong genome vi khun. Mt operon l mt n v phin m n bao gm mt chui cc gen cu trc (structural genes), mt promoter v mt operator.

2. S phn chia nh ca gen Khi nim locus c a ra ch v tr ca gen trn nhim sc th, l v tr ca tt c cc allele ca dy a allele. Bn thn hin tng a allele cho thy gen c cu to phc tp, s bin i ca gen c th dn n nhiu trng th i allele khc nhau.

2.1. Hin tng allele gi Theo quan nim c in gen l n v ti t hp. Nu c th mang hai allele ln a1/a2 ca mt dy a allele s to thnh hai loi giao t l a1 v a2, lai phn tch vi b m ng hp t ln s ch cho kiu hnh t bin a1 v a2 m khng c dng ti t hp hoang di. V d:

Tuy nhin, nhiu th nghim cho thy nu tng s c th th nghim ln 10.000 hoc 100.000, th c th pht hin c dng kiu hnh hoang di do ti t hp. V d: Trng hp locus mt qu trm rui gim, c 18 allele. Khi tng s c th nghin cu ln nhiu ln, ngi ta pht hin cc allele xp thnh 3 nhm A, B v C. Cc allele ca cng mt nhm, khi lai ln nhau, khng cho kiu hnh ti t hp hoang di mt bnh thng, m ch c kiu hnh mt qu trm. Nhng lai allele ca nhm ny vi allele ca nhm khc s c xut hin kiu hnh hoang di do ti t hp. Hin tng ny c gi l allele gi. Hin tng allele gi cho thy gen phn chia nh v mt ti t hp, c th xy ra ti t hp gia cc phn trong gen. Lc u, hin tng allele gi c coi l

trng hp ngoi l, nhng khi tng s c th nghin cu ln nhiu ln th r rng l hin tng ph bin. N c tm thy nhiu i tng khc nhau nh nm men S. cerevisiae, ng, b cu, chut, bacteriophage...

2.2. Locus rII ca bacteriophage T4 Nghin cu chi tit v cc t bin rII ca bacteriophage T4 lm sng t hn v cu trc gen. Bacteriophage T4 dng hoang di r+ c kh nng xm nhim ng thi hai chng E. coli B v K, trong khi cc t bin rII ch xm nhim chng B m khng xm nhim chng K (Hnh 3.7).

Hnh 3.7. Kiu hnh ca cc t bin rII ca phage T4

Benzer (1955) thu c vi nghn t bin rII c ngun gc c lp vi nhau. ng cho lai cc t bin ny vi nhau v cn c vo s xut hin cc dng ti t hp hoang di r+ m lp bn cc im t bin (mutation sites). Trc th nghim ca ng, rII c coi l mt locus. Tuy nhin, th nghim ca ng cho thy cc t bin xp thnh hai nhm rIIA v rIIB. Lai cc t bin rIIA rIIB s c r+, nhng lai rIIA rIIA v rIIB rIIB th kiu hnh t bin l r. Cho n nay, chng ta nh ngha mt gen l nh da trn kiu hnh t bin v v tr trn bn ca n. Bacteriophage l mt m hnh di truyn n gin (genome ca E. coli di khong 4.600.000 bp, trong khi bacteriophage T4 l 165.000 bp v bacteriophage khong 46.500 bp), chng c th sinh sn mt s lng ln rt nhanh (1010 hoc hn th) v d dng phn tch. Cc th nghim thc hin vi t bin rII ca T4 c thit lp da trn c s sau: - Cc gen c mt phm vi v ranh gii hn ch.

- Cc gen c th chia c, c th c s ti t hp gia hai allele trong mt gen n. - Hot ng ca gen c th c phn tch bi s phn tch b sung. Kt qu th nghim cho thy, gen c th phn chia nh v mt t bin. Cc on rIIA v rIIB c gi l cistron, n v chc nng nh nht m bo kh nng xm nhim chng K. Thut ng cistron thc cht l gen, ngy nay n ch c tnh cht lch s, t c s dng. Theo quan nim hin nay, rIIA v rIIB l hai locus. Hai khi nim mi c a ra l muton-n v t bin v recon-n v ti t hp. Benzer tm thy 2.000 im t bin trn on gen c nghin cu, chng phn b khng u nhau, c nhng im tp trung nhiu t bin hn. Chiu di gen khong 900 nucleotide. n v t bin muton y tng ng vi 900/2.000. S t bin ghi nhn c th thp hn so vi thc t nn muton c th tng ng vi mt cp nucleotide. Ging nh vy recon c th tng ng vi mt cp nucleotide. Tm li, gen l n v chc nng, c th chia nh bi cc n v t bin ti t hp.

3. Th nghim chc nng allele Mun nghin cu cu trc bn trong mt gen, phi tm hiu nhiu allele ca gen . Nhiu t bin c kiu hnh ging nhau nhng khng allele vi nhau. Th nghim chc nng allele c s dng xc nh xem hai t bin c allele vi nhau khng. y chnh l th nghim m Benzer dng lp bn locus rII. Th nghim ny cn c gi l th nghim b sung (complementary test) v n cho bit sai hng chc nng hai t bin c b sung tc b tr cho nhau c khng. Phng php th ny cng c gi l th nghim u-lch (cis-trans test). S d nh vy l v php th nghim ny so snh hiu qu kiu hnh ca cc gen t bin hai v tr khc nhau trn nhim sc th tng ng. v tr lch ( trans) cc t bin nm trn hai nhim sc th, cn v tr u (cis) cc t bin nm trn cng mt nhim sc th. Trng hp sai hng hai gen khc nhau nn c th b sung c, cn trng hp sai hng cng mt gen khng b p c s dn n kiu hnh t bin.

Hnh 3.8. Th nghim chc nng allele. I: c kiu hnh t bin do sai hng cng mt gen nn khng b p c. II: c kiu hnh hoang di do sai hng khc gen nn b tr c cho nhau.

4. Gen l n v chc nng nh nht Th nghim chc nng allele c th c thc hin d dng trn cc i tng vi sinh vt vi cc t bin ha sinh, thng l cc t bin khuyt dng (auxotroph mutant: mt kh nng tng hp mt cht no ). V d: nm mc Neurospora crassa c nhiu t bin mt kh nng tng hp adenine (Ade-). Cc t bin ny d pht hin v c khun lc mu . C hai dng t bin Adex v Adey, nu d hp t Adex/Adey c kiu hnh t bin tc l cho khun lc mu , th Adex v Adey l hai allele ca mt gen. Th nghim chc nng allele cho thy cc t bin Ade N. crassa to thnh 9 nhm. iu chng t c 9 gen tng hp adenine loi nm ny: ade1, ade2, ade3... trong ade3 c hai locus nm k st nhau l ade3A v ade3B (Hnh 3.9). Qua nghin cu cc gen sn xut adenine ngi ta nhn thy qu trnh tng hp adenine c lin quan n 9 nhm t bin ca gen ny hoc gen kia, v u cng c mt kt qu l mt kh nng tng hp adenine. Nh vy, khi nim gen khng ch kim tra di truyn c chu trnh tng hp adenine, m gen l n v chc nng nh nht, kim tra mt giai on c bn no ca chu trnh. Nu bin i di truyn lm sai hng chc nng s dn n xut hin t bin mt kh nng tng hp adenine.

Hnh 3.9. V tr cc gen ade trn cc nhim sc th ca nm mc Neurospora crassa

Ti liu tham kho/c thm

1. H Hunh Thy Dng. 1998. Sinh hc phn t. NXB Gio dc, H Ni. 2. Phm Thnh H. 2003. Di truyn hc. NXB Gio dc, H Ni. 3. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 4. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 5. Pierce BA. 2003. Genetics: A Conceptual Approach. W.H. Freeman & Co. New York, USA. 6. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California, USA.

Hai mi amino acid c tm thy trong cc phn t protein l: Alanine (Ala), Arginine (Arg), Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamic acid (Glu), Glutamine (Gln), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) v Valine (Val).
1

Chng 4

Ti bn DNA
I. Chng minh ti bn DNA theo c ch bn bo th 1. C ch ti bn bn bo th 1.1. C ch ti bn prokaryote c im c bn ca s ti bn l ti bn theo phng thc bn bo th (semiconservative replication). Ti bn bn bo th ngha l trong hai chui ca tt c cc phn t DNA bao gi cng c:

- Mt chui ca DNA c (t mt trong hai chui ca DNA m). - Mt chui ca DNA mi (mi c tng hp). Mi mt ln ti bn u c s tch ri ca hai chui ca DNA m, ng thi mi chui m tin hnh sao chp cho mt chui con, chui ny sau li kt hp vi chui m. V tr m xon kp v tng hp DNA mi cng mt lc trn DNA gi l chc ba ti bn (replication fork) do cu trc ca vng ti bn c hnh ch Y. S tng hp DNA mi gn lin vi vic m xon DNA c.

1.2. C ch ti bn eukaryote S ti bn t bo eukaryote phc tp hn so vi t bo prokaryote nhng c ch ca s ti bn eukaryote cng tng t nh prokaryote v tin hnh theo cc nguyn tc: - Hai hng. - B sung, i song song, theo chiu 5 3. - Khng lin tc mt trong hai chui. - Cn nhng RNA primer. Tuy nhin, c mt s im khc nh sau: - Trong khi prokaryote ch c mt im khi u, th s ti bn eukaryote bt u cng mt lc nhiu im khi u. iu ny l cn thit do DNA ca eukaryote c chiu di rt ln. - Vn tc pht trin ca chc ba ti bn eukaryote (khong 50 nucleotide/s) ch bng 1/10 so vi E. coli.

2. Th nghim ca Meselson v Stahl Nhng th nghim ca Meselson v Stahl (1957) chng minh l thuyt ti bn DNA theo kiu bn bo th (Hnh 4.1). Cc tc gi trn nui cy E. coli trong nhiu th h trong mt mi trng cha 15NH4Cl lm ngun cung cp nitrogen duy nht. Bng cch ny, DNA c tng hp c 15N (15N l mt cht phng x nng hn cht phng x thng thng 14N). mt thi im nht nh (thi im 0), cc tc gi ny chuyn nui cy vo mt mi trng cha 14NH4Cl. Tip n, sau tng thi gian u n, h phn tch DNA chit xut t vi khun bng phng php ly tm theo gradient CsCl.

Hnh 4.1. Minh ha s ti bn bn bo th. S trnh by s hp thnh cc si i DNA sau 0, 1, v 2 vng sao chp. H: chui nng (15N), L: chui nh (14N).

Kt qu thc nghim cho thy: - thi im 0: ch c mt phn t tng ng vi DNA nng 15N. - Sau mt th h trong mi trng cha 14N: nhng phn t DNA gm mt chui nng 15N (chui m) v mt chui nh 14N (mi c tng hp). - Sau hai th h trong mi trng cha 14N: c hai phn t lai (gm mt chui nng v mt chui nh) v hai phn t u gm nhng chui nh khng c chui nng.

II. M hnh ti bn DNA-chc ba ti bn 1. M hnh ti bn M hnh ti bn c nghin cu trn th nhim sc ca vi khun E. coli, DNA c dng mch vng si i (Hnh 4.2). t ti bn DNA phi tho ra n gin mt v tr nht nh v ni xut hin chc ba ti bn (Hnh 4.3). Th nghim ca Cairns. S dng nucleotide c nh du bng ng v phng x trong mi trng ang phn chia, ta s bit c tin trnh ti bn do ht bc xut hin di knh hin vi in t.

Hnh 4.2. DNA dng mch vng si i. Chiu di thc t 1,6 mm (4,7106 bp).

2. Chc ba ti bn Hnh 4.4 m t cu trc ca chc ba ti bn. Nh vo phng php phng x nh t ghi, ngi ta nhn thy s ti bn thc hin theo hai hng (bidirectional synthesis). ng thi cng chng minh c vi khun E. coli (prokaryote) c duy nht mt im gc ti bn, l im m hai chui xon kp ca DNA m c tch ra, tng ng vi hai chc ba ti bn pht trin ngc chiu nhau. Chui DNA sau khi tch ra c dng lm khun mu cho s tng hp DNA mi (Hnh 4.5).

Hnh 4.3. S ti bn ca phn t DNA si i mch vng ca E. coli. A: s chuyn ng khng cun li ca cc nhnh trong qu o ti bn, khng c cc v tr quay t do, gy ra s cun li qu cht ca phn khng c ti bn. B: c ch si n b t (nick) pha trc ca chc ba ti bn cho php s quay xy ra.

Hnh 4.4. S cu trc ca cc chc ba ti bn. (a): Chc ba n trnh by si ch (leading strand) c tng hp lin tc v si th (lagging strand) c tng hp gin on. (b): Chc ba i, ph bin trong hu ht mi s ti bn DNA ca genome. (c): Cc hng hnh hc ca s ti bn DNA, mi tn ngn ch s chuyn ng dch m ca chc ba, mi tn di v cong ch s quay vng DNA cn thit quanh cc chc ba.

Hnh 4.5. Hnh nh di knh hin vi in t. Phn t DNA mch vng nh ca E. coli c chiu di thc t 0,01 mm (3.000 bp) ti bn bng kiu . Cc on DNA b m v con c trnh by trong hnh v.

Hnh 4.6 so snh s khc nhau gia ti bn DNA khng nh hng v ti bn theo hai hng. Trong ti bn khng nh hng, ch c mt chc ba ti bn. Trong khi ti bn theo hai hng yu cu hai chc ba ti bn. Mi tn cong ch hng chuyn ng ca cc chc ba. Hu ht DNA ti bn theo hai hng.

Hnh 4.6. Ti bn DNA khng nh hng v theo hai hng

Hnh 4.7 v 4.8 m t phng thc hp nht cc vng ti bn DNA rui gim (D. melanogaster). Qu trnh ti bn din ra ng thi trn hng chc ngn v tr khc nhau ca phn t DNA v to thnh cc vng ti bn, cc vng ti bn sau s m rng theo hai hng cui cng hp nht vi nhau to thnh hai phn t DNA.

Hnh 4.7. Hnh nh di knh hin vi in t ca mt on nucleotide rui gim. Phn t DNA si i di 30 kb cho thy c 7 vng ti bn.

Hnh 4.8. Phng thc hp nht cc vng ti bn DNA ca rui gim. Hai gc ti bn c trnh by trn hnh v, cc mi tn nh ch hng chuyn ng ca cc chc ba ti bn.

3. Ti bn DNA theo vng trn quay Trng hp bacteriophage (thc khun th ) c vt cht di truyn l mt phn t DNA mch thng si i, khi ta chuyn chng vo vi khun th cc u dnh kt DNA (cos) ca n gn li theo dng vng trn. S dnh kt li ny l do hot ng ca enzyme DNA ligase gip to li dng xon. Khi , s ti bn DNA tin hnh theo c ch vng trn quay (Hnh 4.9).

Hnh 4.9. Ti bn vng trn quay bacteriophae . DNA c tng hp mi c mu nht. Si thay th c ti bn trong cc on ngn.

III. Bn cht xon ca DNA-Cc giai on ca s ti bn Hnh 4.10 m t ton b qu trnh ti bn DNA. Qu trnh ny tri qua ba giai on chnh sau: 1. M xon Trc tin ta thy qu trnh m xon ca hai si DNA cn thit phi c mt enzyme rt quan trng l helicase (cn gi l enzyme m xon). Chng hn, trn phn t E. coli ch c mt gc ti bn (k hiu l ori C) di 245 bp. C t nht 8 enzyme hoc protein tham gia vo giai on khi u ca s ti bn. Nhng enzyme-protein ny m xon DNA gc ori C v thit lp mt phc hp tin mi (prepriming complex) chun b cho nhng phn ng ca giai on sau. Mt phc hp khong 20 protein Dna A c kt hp vi mt vng ca ori C bt u cho qu trnh m xon v khi u s ti bn. Phn ng ny cn ATP v mt protein ging nh histone ca vi khun (HU). Tip , protein Dna C gip protein Dna B gn vo vng ori C, Dna B chnh l helicase s m xon DNA theo hai hng to

ra hai chc ba ti bn. Cc phn t protein lin kt si n (single stranded binding protein, protein SSB) gn vo chui n DNA n nh chui ny. Enzyme gyrase (DNA topoisomerase II) m xon to siu xon tri ( Enzyme primase (protein ). Dna G) s xc tc tng hp RNA primer gn vo khun mu DNA.

2. Ko di-Tng hp chui Okazaki Giai on ko di bao gm s tng hp cng mt lc hai chui DNA. Mt chui c ti bn cng hng pht trin ca chc ba s lin tc (si ch), cn chui kia s khng lin tc bao gm cc on Okazaki (si th). Cc nucleotide gn vo u 3 t do, v ko di chui ra nh enzyme DNA polymerase III theo chiu 5 3. Trong giai on ny, nhiu enzyme tham gia vo s tng hp hai chui DNA ti chc ti bn. DNA helicase tip tc tch hai chui DNA m, DNA gyrase m xon, protein SSB n nh nhng chui DNA n c tch ra, DNA ligase gn cc on Okazaki trn si th. Sinh tng hp DNA trong t bao cung mt luc din ra trn rt nhiu v tr (20.000 60.000) sut chiu dai khng l cua DNA khun mu. cac vi tri o, enzyme helicase giup DNA chuyn t dang siu xon sang dang dan (hai si DNA tach ra) vi tc 10 m/gi. Vi tc nay, helicase co th chay sut chiu dai chui DNA trong mt t bao cy u nga (DNA dai tng cng khong 9 m) phai mt hn mt th ky. Trong thc t, cng vic nay chi mt khoang 15-40 gi.

Chui DNA xon kep nh vy c tach i cac vi tri se xay ra sinh tng hp, hinh thanh cac chc ba. chc ba, ca hai chui DNA mach n u c s dung lam khun mu cung mt luc. Trn mt chui, sinh tng hp se din ra theo chiu t 5 3 cng chiu vi chiu pht trin ca chc ti bn. Trn si con lai, sinh tng hp vn din ra theo chiu 53 nhng ngc chiu vi chiu pht trin ca chc ti bn, v chi thc hin c tng oan ngn 1.000 nucleotide (oan Okazaki). Trong khi di chuyn tng qung, enzyme primase s tng hp nhng on RNA primer ngn (11 1 nucleotide) t DNA c tip ni nh DNA polymerase III. Khi on Okazaki mi c hon chnh, RNA primer c tch ra nh DNA polymerase I (hot tnh exonulease 53) v c thay th bi DNA cng nh tc dng ca cng enzyme. Cc khe h cn li gia cc on Okazaki c DNA ligase gn va DNA tr lai dang chui xon kep: mt phn t DNA mi c hinh thanh (Hinh 4.11).

3. Kt thc Cui cng, hai chc ba ti bn gp nhau pha i din ca nhim sc th vng ca E. coli. Ngi ta bit rt t v nhng phn ng ca giai on ny. C th hot ng ca mt loi DNA topoisomerase l cn thit tch hai phn t DNA vng c tng hp. Qu trnh phn i hai phn t DNA trong t bo con khi phn chia t bo cng cha c bit r.

IV. Khi nim mi Ti bn DNA ch xy ra khi c s khi ng mi. Thng mi (primer) l on RNA ngn gn trc u DNA. Quan st chc ba ti bn ta thy DNA polymerase gn nhm phosphate vo u 3-OH t do. Tuy nhin, lc u cha c u 3-OH t do, do ngay im gc ti bn enzyme RNA polymerase (enzyme primase) s hot ng to nn mt on mi ngn c u 3-OH t do, nh DNA polymerase mi bt u hot ng ti bn.

Hinh 4.11. Hai si DNA mi c tng hp theo hai kiu khc nhau

Nghin cu ti bn bacteriophage M13 (DNA mt si vng). Gi DNA + (M13) l si (+). T si (+) to ra dng ti bn ( si ( ny s dng lm khun to ), ) ra si (+) khc. DNA polymerase s khng hot ng, nu khng c mi ghp i vo si (+) u tin. Enzyme RNA polymerase s tng hp on mi bng cch b sung mt s nucleotide vo v tr khi u ti bn, to nn u 3-OH t do. RNA polymerase bao gm cc n v 2, , v . Cht khng sinh rifampicin c tc dng c ch n v , khi RNA polymerase s b c ch v M13 khng c hin tng ti bn. Nh vy, chnh RNA polymerase gi vai tr khi ng sau DNA polymerase mi bt u hot ng ti bn c. V vy, u tin phi c mt on mi gn vi DNA bng lin kt cng ha tr, v on mi ny do RNA polymerase tng hp nn. Tip tc, on mi RNA s b loi ra bi DNA polymerase (khc vi DNA polymerase ti bn trn). DNA polymerase ny bt u gn cc nucleotide vo u 3-OH t do, v sau cng enzyme DNA ligase ni cc on ti bn ny li (Hnh 4.12).

Hnh 4.12. Tng hp cc on Okazaki. Qu trnh ny i hi s gn mi, ko di, loi b RNA, lm y cc khong trng v hn cc im t. Primase tng hp on mi RNA, DNA polymerase III ko di on mi RNA thnh on Okazaki si th, DNA polymerase I s dng dch m im t thay th on mi RNA bng DNA, v cui cng DNA ligase hn im t.

Nh vy, vic ti bn DNA cn cc enzyme sau: - Helicase: m xon tch si DNA ang xon. - SSB: gn trn DNA mt si si lun lun tnh trng m. - RNA polymerase (primase): tc ng hnh thnh on mi RNA. - DNA polymerase I: loi b on mi RNA. - DNA polymerase III (khc vi loi trn) tc ng tng hp DNA bng cch ko di on mi RNA.

- DNA ligase: gn cc on Okazaki tng hp vi nhau. T cc nghin cu v ti bn DNA ca bacteriophage M13, ngi ta a ra s gi thuyt v s bin i DNA mt si ca M13 thnh dng sao chp ti bn (RF). V quan st trn chc ba ti bn ang pht trin, ta c th theo di c s ti bn lin tc v khng lin tc ca cc on DNA mi b sung cho si DNA khun mu.

V. Enzyme ti bn 1. DNA polymerase y l enzyme ch yu ca s ti bn, chu trch nhim tng hp hai chui DNA chc ti bn. C ba loi DNA polymerase khc nhau, c k hiu l I, II v II. C ch v vai tr ca cc enzyme DNA polymerase cng rt khc nhau.

1.1. DNA polymerase I Do Kornberg phat hin nm 1955. DNA polymerase I cua E. coli mang chui polypeptide co khi lng phn t la 109.000 Da vi ba hoat tinh ring bit. Nhim vu chnh cua DNA polymerase I la xuc tac cho qua trinh lp rp cac dNTP tng cai mt vao u 3 t do cua oan mi ang gn trn DNA khun mu. Kt qua la si DNA mi se dai dn v phia u 3 (Hinh 4.13).

Hinh 4.13. Cac thnh phn cn thit cho sinh tng hp DNA

DNA polymerase I cua E. coli cn c hot tnh exonuclease (hot tnh ct cc chui nucleotide cc u t do ca DNA) 35 xc tc cho s thoi ha bc thang t u 3 ca c DNA si i v si n khi khng c dNTPs. Trong trng hp c dNTPs, hot tnh exonuclease trn si i s b c ch bi hot tnh polymerase. Trong qu trnh tng hp DNA, hot tnh exonuclease thc hin chc nng c sa bng cch ct b nhng nucleotide lp rp sai. Ngoi ra, enzyme ny cn c hoat tinh exonulease 5 3 xc tc cho s chuyn ch ca cac nucleotide t u 5 cua im t va b sung tc thi cac nucleotide t u 3 tao ra chuyn ng cua im t doc theo DNA.

1.2. DNA polymerase II va DNA polymerase III Khi u, enzyme DNA polymerase I c xem nh gi vai tr ch yu trc tip trong ti bn DNA. Nhng sau , ngi ta quan st thy E. coli t bin khng c DNA polymerase I m vn c s ti bn, v pht hin rng chnh DNA polymerase III gi vai tr ti bn DNA. Nghin cu tnh cht v so snh ba loi enzyme trn ngi ta nhn thy DNA polymerase III mi l enzyme thc s iu khin tng hp DNA. Trong khi DNA polymerase I ch c vai tr loi b RNA mi nh ct u 53 v thay vo ch RNA mi bng DNA khc, cn DNA polymerase II cha r tc dng, c l n tham gia vo qu trnh sa cha (thay mt on DNA hng bng mt on DNA bnh thng) hoc thay th DNA polymerase I khi c kh khn trong tng hp DNA. V tc lam vic, cac DNA polymerase rt khac nhau. Trong mt giy, DNA polymerase I gn c 10 dNTP trong khi DNA polymerase II chi gn c 0,5 dNTP va DNA polymerase III gn ti 150 dNTP.

2. Cc topoisomer v DNA topoisomerase 2.1. Topoisomer Gi s c hai phn t DNA mch vng c cng trnh t nucleotide. Nhng hai phn t ny c th c s vng (linking number-Lk) khc nhau trong phn t. S vng y c nh ngha l s ln ca mt chui DNA qun xung quanh mt chui khc. Nhng trng hp ny c gi l topoisomer. Topoisomer c cc dng sau:

2.1.1. Dng lng lo (relaxed DNA) dng ny sc cng ca xon kp l ti thiu. l dng cu trc n nh nht ca phn t.

2.1.2. Dng siu xon (supercoiled DNA) Trc ca xon kp c th cun xung quanh mnh to thnh mt siu xon. C hai dng siu xon sau: - Siu xon dng (+). S vng tng, xon kp xon cng chiu (xon phi) to thnh dng siu xon dng (positive supercoil). - Siu xon m (). S vng gim, xon kp xon theo chiu ngc li (chiu tri) to thnh dng siu xon m (negative supercoil), Phn ln nhng phn t DNA trong t nhin dng siu xon m.

2.2. DNA topoisomerase Enzyme DNA topoisomerase (l mt enzyme nuclease thun nghch) c tc dng thay i s Lk ca DNA. Cc DNA topoisomerase c tc dng thm vo hoc loi bt nhng siu xon trong phn t DNA xon kp. C hai loi DNA topoisomerase sau:

2.2.1. DNA topoisomerase I DNA topoisomerase I tn ti c prokaryote ln eukaryote v c tc dng sau: - Ct mt chui DNA v tho mt vng xon sau v tr ct. - Ni li chui DNA b ct bi lin kt phosphodiester mi. DNA sau khi c ti to li c cu trc lng lo hn (Hnh 4.14).

Hnh 4.14. C ch hot ng ca topoisomerase I. Enzyme ct mt si n ca DNA si i, chuyn si nguyn vn qua ch t gy, sau hn ch t gy li. Qu trnh ny tng s Lk ln 1.

2.2.2. DNA topoisomerase II prokaryote, DNA topoisomerase II c tn l DNA gyrase. DNA topoisomerase II c tc dng ct tm thi hai chui ca DNA, c tc dng sp xp li siu xon, to ra siu xon tri ( ca chui DNA xon kp (Hnh 4.15). Phn ng ny cn 1 ATP. ) eukaryote, DNA topoisomerase II cng c tm thy nhng t c nghin cu.

3. Helicase v protein SSB 3.1. Helicase Enzyme helicase (cn c tn l enzyme deroulase) co nhim vu giup chui DNA t dang siu xon sang dang dan thnh hai si n bng cch ct cc lin kt hydrogen gia nhng base b sung. Dang cu truc nay cn thit cho cac oan trn chui DNA ma o co nhu cu sinh tng hp. Phn ng ny cn s c mt ca ATP (Hnh 4.16).

Hnh 4.15. Hot ng ca topoisomerase II chc ba sao chp

3.2. Protein SSB Cac protein SSB lin kt phi hp vi DNA si n nhng khng lin kt vi DNA si i. Chung c xem nh la cac cht phan ng trong tao dong phn t xut phat t ch chung co kha nng pha v s n inh cua cac cu truc th cp bn trong si nucleotide; tng nhanh s tai u cua cac polynucleotide b tr va tng hoat tinh cua cac DNA polymerase bng cach loai bo cac cu truc th cp bn trong si la nhng yu t ngn can s tin trin cua cac enzyme nay (Hnh 4.17). Nh tinh cht nay, cac protein SSB tr thanh cac cht phan ng hu ich trong xac inh trinh t DNA.

Hnh 4.16. Hot ng ca enzyme helicase. DNA helicase tch ri hai si ca chui xon kp. Khi ATP c b sung vo DNA helicase c lin kt vi si n, th helicase di chuyn vi mt phn cc xc nh trn DNA si n. phn cc ngha l DNA helicase c lin kt vi khun mu si th trn chc ba ti bn.

4. DNA ligase Co nhim vu ni hai u 3-OH va 5-PO4 cua hai oan DNA ri thanh mt oan lin tuc. Mt mi han nh vy cn mt nng lng la 2ATP (i vi eukaryote) hoc NAD+ (i vi vi khun).

Hnh 4.17. Lin kt ca protein SSB ln DNA si n. Mt lng gii hn ca SSB lin kt vi 4 trong 9 phn t DNA si n. Khi b sung thm protein SSB, n s lin kt vi cc protein SSB lin kt t trc. Ch sau khi protein SSB bao bc hon ton cc phn t DNA si n ban u, th n mi tip tc lin kt vi cc phn t DNA si n khc.

Hnh 4.18. DNA ligase hn cc im t gia cc nucleotide gn nhau

c im cua DNA ligase la khng lam vic vi cac si DNA n (single strand) ma chi han ni cac oan DNA dang chui xon kep. Khi ni DNA, co th gp 2 trng hp: u so le-u dinh (protruding ends-cohesive ends) hoc u bng (blunt ends). Trng hp u so le, cac nucleotide nm trn phn so le cua hai oan DNA phai tng ng thi DNA polymerase mi hoat ng c.

Ti liu tham kho/c thm

1. Phm Thnh H. 2003. Di truyn hc. NXB Gio dc, H Ni.

2. L c Trnh. 2001. Sinh hc phn t ca t bo. NXB Khoa hc v K thut, H Ni. 3. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 4. Karp G. 2002. Cell and Molecular Biology: Concepts and Experiments. 3rd ed. John Wiley & Sons, Inc. New York, USA. 5. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK.

6. Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J. 2004. Molecular Cell Biology. 5th ed. Freeman and Company, New York, USA. 7. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California, USA. 8. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company, New York, USA.

Chng 5

Phin m
Phin m l qu trnh tng hp RNA t khun mu DNA. Qu trnh ny v phng din ha hc v enzyme rt ging vi qu trnh ti bn DNA. C hai u lin quan n cc enzyme tng hp mt chui nucleic acid mi b sung vi khun mu DNA. Tt nhin, hai qu trnh ny c nhng khc bit quan trng, m ng ch nht l chui mi trong qu trnh phin m c to thnh t cc ribonucleotide thay v cc deoxyribonucleotide. Cc nguyn tc c bn ca qu trnh phin m c thit lp da vo nghin cu trn prokaryote (E. coli) nhng dng nh cc nguyn tc ny cng c tnh ph bin cho c eukaryote. Tuy nhin, do s khc bit v cu trc genome v h thng enzyme nn s phin m prokaryote v eukaryote cng c nhng c trng nht nh. I. Cc c im c bn ca qu trnh phin m 1. S phin m to ra RNA b sung vi mt si DNA Cc ribonucleotide ni tip ca RNA c xc nh da trn nguyn tc b sung vi cc nucleotide trn DNA khun mu. Khi s bt cp chnh xc xy ra, ribonucleotide tip theo c lin kt ng ha tr vi chui RNA ang hnh thnh nh phn ng c enzyme xc tc. Nh vy, sn phm phin m c ko di tng ribonucleotide mt v b sung chnh xc vi si DNA c dng lm khun mu.

Qu trnh phin m d c tnh chnh xc cao nhng vn km hn nhiu so vi qu trnh ti bn DNA (t l mc li l 1/10.000 nucleotide so vi 1/10.000.000 trong ti bn). l do s thiu mt c ch sa sai hu hiu, mc d trong qu trnh tng hp RNA cng c hai dng sa sai tn ti. Tuy nhin, v cc RNA c phin m khng bao gi c sao chp li nn cc sai st xy ra khng nh hng n vic truyn t thng tin cho th h sau. 2. S phin m l mt phn ng enzyme Nhng enzyme chu trch nhim cho qu trnh phin m c t bo prokaryote v eukaryote u c gi l RNA polymerase ph thuc DNA (DNA-dependent RNA polymerase), gi tt l RNA polymerase. RNA polymerase xc tc hnh thnh cc cu ni phospho-diester ni cc ribonucleotide thnh mt chui thng. Enzyme dch chuyn tng bc dc theo DNA khun mu v ko di chui RNA theo hng t 53, ngha l cc ribonucleotide c thm vo u 3 ca chui RNA ang hnh thnh. Cc c cht c s dng tng hp RNA l ATP, GTP, CTP v UTP. Cng ging nh trong s ti bn DNA, nng lng cho phn ng c cung cp t s thy phn cc cu ni giu nng lng ca cc c cht ni trn. 3. S phin m ch sao chp chn lc mt s phn ca genome v to ra nhiu bn sao S la chn vng no phin m khng phi xy ra ngu nhin. Mi vng phin m in hnh bao gm mt hoc nhiu gen, c nhng trnh t DNA c hiu hng dn khi u v kt thc phin m. i vi mt vng c chn phin m, c th c mt n hng trm thm ch c nghn bn sao RNA c to ra. S tng hp phn t RNA sau c bt u trc khi phn t RNA trc hon thnh. T mt gen n c, trong vng mt gi c th tng hp c hn mt nghn phn t RNA (i vi eukaryote). S la chn vng no phin m v mc phin m u c iu ha. V vy, trong nhng t bo khc nhau hoc trong cng mt t bo nhng nhng thi im khc nhau s c nhng nhm gen khc nhau c phin m. 4. Ch mt trong hai si n ca phn t DNA c dng lm khun mu Vic gn ca RNA polymerase vo promoter ca gen s quyt nh vic la chn si no trong hai si n ca DNA lm khun mu. Promoter, ngoi vic mang v tr gn RNA polymerase cn cha ng thng tin xc nh si no trong hai si n ca DNA c phin m v xc nh v tr bt u phin m.

5. S phin m c khi pht khng cn mi RNA polymerase c th khi u s tng hp RNA trn khun mu DNA m khng cn mi nh DNA polymerase. iu ny i hi ribonucleotide u tin c mang n v tr bt u phin m phi c gi n nh trn DNA khun mu trong khi ribonucleotide th hai ang c a n xy ra phn ng trng hp. II. Cc giai on ca qu trnh phin m RNA polymerase tin hnh qu trnh phin m mt gen thng qua nhiu bc v c chia thnh ba giai on: khi u, ko di, v kt thc. 1. Giai on khi u u tin, RNA polymerase gn vi promoter ca gen (cng vi cc yu t khi u) to thnh phc hp promoter-polymerase. Mt khi c hnh thnh, phc hp ny s c s thay i cu trc cn thit cho vic tin hnh giai on khi u. Giai on khi u ny bao gm ba bc nh sau: 1.1. Phc hp ng (closed complex) Khi RNA polymerase va mi gn vi promoter th phc hp to thnh trng thi ng. trng thi ny, DNA vn l chui kp v enzyme gn vo mt pha ca vng xon. 1.2. Phc hp m (open complex) Lc ny DNA xung quanh im bt u phin m c m xon v lin kt gia cc cp base b sung b ph v. Hai si DNA vi di khong 14 bp xung quanh v tr bt u phin m tch ri nhau ra v to nn vng phin m. Vic m DNA gii phng si khun mu. Hai nucleotide u tin c mang n v tr bt u c hot ha, chng xp hng trn khun mu v c ni vi nhau. RNA polymerase bt u chuyn dch dc theo si khun mu, m xon pha trc v tr trng hp v ti gn hai si DNA pha sau. Bng cch ny, cc ribonucleotide tip theo c gn vo chui RNA ang pht trin. on RNA ban u vi s lng ribonucleotide t hn 10 s b loi b v enzyme bt u tng hp li. 1.3. Phc hp tam nguyn bn (stable ternary complex) Mt khi enzyme i c khong hn 10 bp, n c xem l thot khi promoter. Lc ny mt phc hp gm ba thnh phn l enzyme, DNA v RNA c hnh thnh v qu trnh phin m chuyn sang giai on ko di.

2. Giai on ko di Mt khi RNA polymerase tng hp c mt on RNA khong 10 base, n chuyn sang giai on ko di. S chuyn giai on ny i hi nhng bin i hn na v cu hnh ca RNA polymerase lm cho n cng gi cht khun mu hn na. Trong qu trnh ko di, enzyme ny thc hin mt lot nhiu nhim v rt quan trng ngoi vic tng hp RNA. N m xon DNA trc mt v ti gn DNA pha sau, n tch dn chui RNA ang hnh thnh ra khi khun mu khi ang di chuyn dc theo gen, v n cn thc hin chc nng c sa sai ( qu trnh ti bn DNA, cc chc nng ny do nhiu enzyme khc nhau thc hin). 3. Giai on kt thc Khi RNA polymerase phin m ht chiu di ca gen (hoc cc gen), n dng li v gii phng sn phm RNA. Trong mt s t bo, c nhng trnh t c hiu thc y giai on kt thc. Trong mt s t bo khc, ngi ta vn cha r yu t no iu khin enzyme ngng phin m v gii phng RNA khi khun mu. III. Phin m prokaryote 1. Enzyme RNA polymerase prokaryote T bo prokaryote ch cha mt loi RNA polymerase. Enzyme ny c cu to bi nm tiu n v, gm hai tiu n v v cc tiu n v , , . Cc tiu n v ny lin kt vi nhau to thnh enzyme li. Ngi ta lm th nghim tinh sch enzyme li t t bo vi khun ri cho vo dung dch cha DNA vi khun v ribonucleotide th enzyme s gn vi DNA v tng hp RNA. Tuy nhin, RNA thu c trong th nghim ny khng ging vi RNA trong t bo v enzyme gn vo cc v tr khng thch hp trn DNA. Nu cho thm mt loi polypeptide tinh sch c gi l yu t trc khi enzyme gn vi DNA th s phin m s bt u ti nhng v tr chnh xc nh trong t bo. Vic gn yu t vo enzyme li lm tng i lc ca enzyme vi v tr khi ng trn DNA, ng thi lm gim i lc i vi cc vng khc. Khi enzyme li c gn thm yu t s tr thnh dng holoenzyme. 2. Promoter ca gen prokaryote Promoter ca gen vi khun nm ngay trc v tr khi u phin m. Bng cch phn tch cc trnh t DNA ny ca mt s lng ln cc gen vi khun ngi ta nhn thy c hai on ngn tng i ging nhau gia gen ny v gen khc, mi on gm su nucleotide. on th nht nm cch v tr khi u khong 35 bp, l s bin i ca trnh t TTGACA. on th hai nm cch v tr khi u khong 10 bp, l s bin

i ca trnh t TATAAT. Chng ln lt c gi l vng v vng (hp 35 10 Pribnow). i vi cc promoter mnh (v d gen m ha rRNA), ngi ta cn tm thy yu t UP lm tng s gn ca RNA polymerase vo DNA. C mt s promoter thiu vng v c thay bng yu t m rng, bao gm vng chun v thm 35 10 10 mt on ngn u 5 ca n (v d gen gal ca E. coli). Yu t nhn dng cc vng v hoc yu t m rng nh cc cu 35 10 10 trc c bit ca chng. Ring yu t UP khng c nhn dng bi m c nhn dng bi mt vng u C tn cng ca tiu n v , gi l CTD (carboxyl terminal domain). 3. Vai tr ca enzyme RNA polymerase v promoter trong qu trnh phin m Khi khi u s phin m, RNA polymerase gn vi DNA vng mt cch 35 lng lo to thnh phc hp ng. Sau , enzyme gn vo promoter cht hn v phc hp chuyn sang trng thi m. Mt vng DNA c m xon nm gia hai v tr 11 v +3, ngha l bao gm c v tr bt u phin m +1. Si n DNA lm khun mu c bc l sinh tng hp RNA. Mt khi RNA polymerase tng hp c 10 nucleotide (sau ln khi u) th yu t ri khi enzyme v c th n gn vo promoter khc khi u mt qu trnh phin m mi. S tch ri yu t cho php hnh thnh mt knh thot m thng qua phn RNA c tng hp chui ra khi enzyme v c ko di (Hnh 5.1).

Hnh 5.1. Cc giai on phin m prokaryote

4. Tn hiu kt thc Tn hiu kt thc l nhng trnh t trn DNA c vai tr thc y s tch RNA polymerase ra khi DNA v gii phng chui RNA mi c tng hp. vi khun, c hai loi tn hiu kt thc l tn hiu kt thc khng ph thuc Rho v tn hiu kt thc ph thuc Rho. 4.1. Tn hiu kt thc khng ph thuc Rho l mt cu trc c bit bao gm hai trnh t i xng b sung nhau giu GC, tip theo sau l mt lot 8 adenine (chng s c phin m thnh 8 uracil). Nhng trnh t ny khng nh hng n RNA polymerase cho n sau khi chng c phin m. Khi cc trnh t i xng b sung trn RNA s bt cp vi nhau v to nn cu trc hnh chic kp tc (Hnh 5.2). Cu trc kp tc ny ph v phc hp ko di hoc bng cch thc y m knh thot cho RNA trn RNA polymerase hoc bng cch ph v mi tng tc RNA-khun mu DNA

Cu trc kp tc ny ch hot ng hiu qu khi n c theo sau bi mt lot U. Ti thi im cu trc kp tc c hnh thnh, chui RNA ang pht trin vn c gi trn khun mu ch bng cc lin kt gia A-U. V lin kt A-U yu hn GC nn chng d dng b ph v bi hiu qu ca cu trc kp tc, v v th RNA d dng c gii phng. 4.2. Tn hiu kt thc ph thuc Rho Tn hiu ny l nhng thnh phn RNA m mun hot ng, i hi phi c yu t Rho. Yu t Rho l mt protein hnh nhn ( E. coli c khi lng phn t khong 50 kDa) gm su tiu n v ging ht nhau. Yu t ny c hot tnh ATPase: khi gn vo sn phm phin m, n s s dng nng lng t s thy phn ATP ko RNA ra khi khun mu v polymerase. Yu t Rho iu khin phn t RNA c bit nh tnh c hiu. Th nht l tnh c hiu ti nhng v tr n gn, l nhng on khong 40 nucleotide m khng gp li thnh cu trc bc hai, chng thng giu C. Tnh c hiu th hai l yu t Rho gim gn vi bt k sn phm phin m no ang c dch m (ngha l nhng sn phm ang gn ribosome). vi khun, phin m v dch m c song hnh cht ch: dch m khi pht ngay trn RNA ang c tng hp k t khi RNA bt u chui ra khi RNA polymerase. V vy Rho ch kt thc nhng sn phm vn ang c phin m qu u tn cng ca gen hoc operon. IV. Qu trnh phin m eukaryote 1. Cu trc gen eukaryote Cu trc mt gen m ha cho protein ca eukaryote bao gm cc vng sau: 1.1. Vng 5 kim sot biu hin gen Vng ny bao gm cc trnh t nucleotide iu ha biu hin gen v hot ha s phin m, bao gm:

- Promoter. L nhng trnh t nh v u 5 khng dch m ca gen c chc nng xc nh v tr bt u phin m, kim sot s lng mRNA v i khi c tnh c hiu m (tissue-specific). Promoter c th di n vi nghn bp. Vng ny thng cha mt trnh t bo th gi l hp TATA nm cch v tr phin m khong 25-30 bp. Hp ny gip xc nh chnh xc v tr bt u phin m. Ngoi ra, cn c hp CCAAT nm cch v tr bt u phin m khong 75-80 bp. Hp ny t ph bin hn hp TATA, n c chc nng tng hiu qu phin m. Mt s gen qun gia m ha cho cc enzyme hin din tt c cc t bo thng thiu c hai hp ny v promoter rt giu GC. Ngoi ra, cn c cc thnh phn c hiu khc. - V tr gn vng c hiu m. L trnh t DNA tng tc vi protein c hiu m ch huy gen cu trc sn xut ra tng protein c hiu ca tng m. - V tr gn vi vng tng cng phin m (enhancer). Cn gi l gen tng cng, l nhng trnh t DNA c gn vi cc tc nhn hot ha kch thch phin m ca cc gen k bn, chng c th tc ng qua mt khong cch xa v c th tc ng theo hai pha (t 5 hoc 3 ti). 1.2. Vng c phin m Bao gm cc exon v intron nm xen k. y l mt c im phn bit vi gen ca prokaryote. Cc exon v intron u c phin m nhng ch c cc exon l c dch m. Cc intron c bt u bng GT v kt thc bng AG. Cc intron chim phn ln trong mi gen v chng s c loi b khi RNA mi c tng hp, cn cc exon c ni vi nhau to nn mRNA hon chnh (mature mRNA). hai u ca vng c phin m (coding region) cn c vng 5 khng dch m (5 untranslation region) v vng 3 khng dch m (3 untranslation region). Vng 5 khng dch m c tnh t v tr bt u phin m cho n codon khi u ATG. Vng 3 khng dch m bt u t codon kt thc n v tr gn ui poly(A). 1.3. Vng 3 khng dch m Chc nng cha r, mt s gen vng ny mang cc trnh t iu ha chuyn bit. 2. Enzyme RNA polymerase ca eukaryote T bo eukaryote c n ba loi RNA polymerase l RNA polymerase I (pol I), RNA polymerase II (pol II), v RNA polymerase III (pol III). Trong , RNA polymerase II m nhn vic phin m cho hu ht cc gen, ch yu l gen m ha cho protein. RNA polymerase I phin m cc gen tin thn ca rRNA ln v RNA polymerase III phin m cc gen tRNA, mt s gen RNA kch thc nh ca nhn (small nuclear, snRNA) v RNA 5S. Cc RNA polymerase ca eukaryote cng bao gm

nhiu tiu n v, trong c nhng tiu n v tng ng vi cc tiu n v trong enzyme ca vi khun.

Bng 5.1. Cc tiu n v ca RNA polymerase

3. Cc yu t gip RNA polymerase khi u phin m Trong khi RNA polymerase ca prokaryote ch cn thm mt yu t khi u l khi ng phin m th RNA polymerase ca eukaryote phi cn nhiu yu t khi u, cc yu t ny c gi l cc yu t phin m tng qut. Mt khc, s khi u phin m eukaryote cn phi i mt vi hin tng cc DNA c ng gi trong nucleosome v cc dng cao hn ca cu trc chromatin. iu ny i hi phi c nhiu yu t khc thm vo gip khi ng qu trnh phin m. 3.1. Vai tr ca cc yu t phin m tng qut Cc yu t phin m tng qut gip RNA polymerase vo ng v tr trn promoter, gip tch hai si n ca DNA ra phin m c bt u, v gii phng RNA polymerase khi promoter mt khi phin m c khi ng xong i vo giai on ko di. Cc yu t ny c gi l tng qut v chng gn trn tt c cc promoter c s dng bi RNA polymerase II. Do , chng c vit tt l TFII (transcription factor for polymerase II), bao gm TFIIA, TFIIB... Nh m t trn, nhiu promoter ca eukaryote cha hp TATA. Hp ny c nhn dng bi mt tiu n v ca TFIID l TBP (TATA binding protein: protein gn TATA). Phc hp TBP-DNA to nn mt ci nn thu ht cc TFII khc v RNA polymerase n promoter. In vitro, cc yu t phin m tng qut khc n gn vo promoter theo th t sau: TFIIA, TFIIB, TFIIF cng RNA polymerase II, TFIIE v TFIIH. Sau , vng promoter c m xon. Khc vi vi khun, s m xon y cn c nng lng cung cp t s thy phn ATP nh TFIIH (yu t ny c hot tnh ging helicase).

Sau cng xy ra hin tng khi u sy ging nh prokaryote cho n khi c s bin i v cu hnh nhm gii phng RNA polymerase II khi promoter v i vo giai on ko di. Tuy nhin, y c mt bc m khng tm thy prokaryote l s gn thm cc gc phosphate vo ui ca RNA polymerase (ui ny cn c gi l CTD: carboxyl terminal domain). S phosphoryl ha ny cng c xc tc bi TFIIH nh hot tnh protein kinase ca n (Hnh 5.3). 3.2. Vai tr ca cc tc nhn hot ha, phc hp trung gian v enzyme bin i chromatin. Ngoi cc yu t phin m tng qut, RNA polymerase II cn cn s h tr ca cc yu t khc (Hnh 5.4). u tin, cn c cc tc nhn hot ha phin m (transcriptional activator) n gn vo cc trnh t c hiu trn DNA (cc enhancer) hp dn RNA polymerase II n v tr bt u phin m. S hp dn ny cn thit RNA polymerase II v cc yu t phin m tng qut vt qua tr ngi khi gn vi DNA c ng gi trong chromatin. Tip n, s khi u phin m in vivo cn cn s hin din ca cc protein to thnh phc hp trung gian (mediator complex). Phc hp ny cho php tc nhn hot ha tc ng tt ln RNA polymerase II v cc yu t phin m tng qut. Cui cng, s phin m cn cn cc enzyme bin i chromatin, bao gm phc hp ti to m hnh chromatin (chromatin remodeling complex) v enzyme histone acetylase. C hai c tc dng gip cho b my khi u phin m c th gn vo DNA trong chromatin mt cch d dng.

Hnh 5.3. Cc yu t phin m tng qut gip khi u phin m Nh vy, c rt nhiu protein gn vo promoter khi ng s phin m eukaryote. Th t gn ca cc protein ny thay i i vi cc gen khc nhau. Thc t, mt s c th gn vi nhau xa DNA ri c mang n DNA di dng phc hp. V d: phc hp trung gian, RNA polymerase II, v mt s yu t phin m tng qut c th gn vi nhau trong nhn tng ri c mang n DNA.

Hnh 5.4. Cc tc nhn hot ha, phc hp trung gian v cc thnh phn bin i nucleosome trong phin m

4. Cc yu t kch thch RNA polymerase II hot ng trong giai on ko di Mt khi RNA polymerase II bt u chuyn sang giai on ko di th cc yu t khi u c loi b nh yu t phin m tng qut v phc hp trung gian. Thay vo , cc yu t kch thch giai on ko di c thu ht n, bao gm TFIIS v hSPT5. Ngoi ra, cn c cc yu t khc c huy ng phc v cho qu trnh bin i RNA mi c tng hp. Giai on ko di trong phin m c song hnh cht ch vi cc bc bin i RNA mi c tng hp. Nh phn tch trn, c mt bc quan trng din ra khi chuyn t giai on khi u sang ko di l s phosphoryl ha ui CTD ca RNA polymerase II. S phosphoryl ha ny khng nhng gip RNA polymerase II thot khi cc protein v tr bt u phin m m cn cho php cc protein khc n kt hp vi ui gip qu trnh ko di phn t RNA v bin i RNA tin thn. 5. Qu trnh bin i cc RNA mi c tng hp eukaryote, s phin m ch l bc u tin trong mt lot cc phn ng bao gm c s bin i hai u ca mRNA tin thn v loi b cc intron to thnh mRNA hon chnh. 5.1. S gn m vo u 5 Ngay khi RNA polymerase II va mi to ra khong 25 ribonucleotide ca RNA, u 5 ca phn t RNA ny c bin i bng cch gn thm mt ci m l guanine c bin i ha hc. Phn ng gn m c thc hin bi ba loi enzyme l: - Phosphatase c tc dng loi mt gc phosphate khi u 5 ca RNA mi sinh. - Guanylyl transferase gn GMP bng lin kt o ngc (5 vi 5 thay v 5 vi 3) vo u 5 ca RNA ang c tng hp.

- Methyl transferase gn nhm methyl vo guanosine (Hnh 5.5). Trong nhn, m gn vi mt phc hp protein c gi l CBC (CAP-binding complex: phc hp gn m) gip RNA c x l tt v c chuyn ra ngoi. M 5 cn c vai tr quan trng trong qu trnh dch m mRNA trong bo tng. 5.2. S gn ui poly(A) vo u 3 v kt thc phin m S gn ui poly(A) vo u 3 c lin kt vi s kt thc phin m. Cc enzyme cn thit cho cc qu trnh ny c tp trung trn ui CTD ca RNA polymerase II, bao gm CPSF (cleavage and polyadenylation specificity factor: yu t c hiu tch RNA v gn ui polyA) v CstF (cleavage stimulation factor: yu t kch thch tch RNA). Khi RNA polymerase di chuyn n cui gen, n gp nhng trnh t c hiu (c gi l tn hiu polyA). Trnh t ny c phin m thnh RNA, thc y chuyn CPSF v CstF n gn vo on RNA ny. Sau cc protein khc s c tp trung n khi u s tch ri RNA v gn ui poly(A). S gn ui c thc hin bi enzyme poly(A) polymerase (PAP). Enzyme ny thm khong t vi chc n 200-250 adenine vo u 3 ca RNA c tch ra. Enzyme PAP s dng ATP lm c cht v hot ng ging RNA polymerase, tuy nhin khng c khun mu. Ngi ta vn cha r yu t no quyt nh chiu di ca ui nhng qu trnh ny c lin quan n cc protein gn c hiu vi ui poly(A). Sau khi RNA c tch ra v gn ui poly(A), RNA polymerase vn cha kt thc phin m ngay. N cn tip tc di chuyn dc theo khun mu v to ra mt phn t RNA th hai c th di hng trm nucleotide trc khi kt thc. Sau , RNA polymerase c tch ra khi khun mu, gii phng RNA mi v RNA ny s b ging ha.

Hnh 5.5. Phn ng gn m vo u 5 ca RNA 5.3. Qu trnh ct ni gen (splicing) y l qu trnh loi b cc intron v ni cc exon li vi nhau. Qu trnh ny c thc hin phn ln bi cc RNA thay v protein. Cc phn t RNA ny tng i ngn (di 200 nucleotide) c gi l snRNA. C nm loi lin quan n dng ct ni chnh l U1, U2, U4, U5 v U6. Mi snRNA c kt hp vi nhiu protein hnh thnh snRNP. C ba trnh t nm trn intron ng vai tr quan trng trong qu trnh ct ni l: v tr ct ni u 5, v tr phn nhnh l mt trnh t giu cc pyrimidine bao quanh mt nucleotide adenine gn u 3, v v tr ct ni u 3. u tin, v tr ct ni u 5 c nhn dng bi U1 snRNP bng s bt cp cc base b sung. V tr phn nhnh cng c nhn dng bi BBP (branch-point binding protein: protein gn v tr phn nhnh) v U2AF. U2 snRNP n thay th BBP

(nh U2AF gip ). S bt cp base gia U2 snRNA v v tr phn nhnh lm tha ra mt gc A khng bt cp v sn sng phn ng vi v tr 5. Sau U4 v U6 snRNP cng U5 snRNP n gn vi phc hp trn. Dng U1 ri khi phc hp v U6 th ch U1 ti v tr ct ni u 5. U4 c gii phng U6 tng tc vi U2. iu ny lm cho v tr ct ni u 5 v v tr phn nhnh c t k nhau v to iu kin cho phn ng ct ni xy ra. Nucleotide adenine c hiu v tr phn nhnh tn cng v ct intron v tr u 5. ng thi c mt lin kt ng ha tr xy ra gia A v tr phn nhnh v u 5 ca intron to nn mt cu trc hnh thng lng. u 3 ca exon trc ni vi u 5 ca exon k tip v thng lng intron c gii phng (Hnh 5.6). V. Phin m ngc Phin m ngc l qu trnh tng hp DNA da trn khun mu RNA. Qu trnh ny c xc tc bi mt loi enzyme c bit gi l enzyme phin m ngc (RT: reverse transcriptase). Enzyme ny c c hot tnh DNA polymerase v hot tnh RNase H. Cng ging nh DNA polymerase trong qu trnh ti bn DNA, enzyme phin m ngc i hi phi c primer (mi) c bit tng hp nn DNA mi.

Hnh 5.6. Qu trnh ct ni gen

Enzyme phin m ngc c pht hin ln u tin retrovirus. Sau khi cc retrovirus i vo t bo vt ch, genome ca RNA ca n s c phin m ngc

thnh DNA si i, ri tch hp vo DNA ca vt ch. Qu trnh phin m ngc ny kh phc tp, ngi ta c th tm tt thnh 10 bc nh sau: - Bc 1: Mt tRNA ca t bo c tnh c hiu vi retrovirus ng vai tr primer khi pht qu trnh phin m ngc. Primer lai vi vng b sung trn genome ca RNA ca retrovirus gi l v tr gn primer (PBS: primer-binding site). - Bc 2: Mt on DNA c ko di t tRNA c trnh t b sung vi trnh t ca genome RNA ca retrovirus. - Bc 3: Trnh t R u 5 v U5 ca virus b loi b bi RNase H. - Bc 4: i khun mu ln th nht (first template exchange) cn c gi l nhy ln mt: DNA n lai vi trnh t R cn li u 3'. - Bc 5: Mt si DNA c ko di t u 3. - Bc 6: Hu ht RNA virus b loi b bi RNase H. - Bc 7: Si DNA th hai c ko di t RNA cn li ca virus. - Bc 8: C tRNA v phn RNA cn li ca virus b loi b bi RNase H. - Bc 9: i khun mu ln th hai (nhy ln hai): vng PBS ca si DNA th hai n lai vi vng PBS ca si th nht. - Bc 10: Ko di c hai si DNA. R l nhng trnh t lp li trn genome ca retrovirus ( u 5 v 3), U5 v U3 l nhng vng m ha cho nhng tn hiu tch hp u 5 v 3 (Hnh 5.7). Ngy nay, ngi ta cng pht hin enzyme phin m ngc cc virus ng vt khc nh hepadnavirus, v cc virus thc vt nh caulimovirus. Tt c chng c gi l retrovirus. Ngoi ra, ngi ta cn pht hin hot tnh enzyme phin m ngc mt s dng ca myxobacteria v E. coli. Enzyme phin m ngc tr thnh mt cng c khng th thiu trong sinh hc phn t. N gip cc nghin cu vin phin m ngc mRNA ca t bo thnh cDNA (complementary DNA), ri sau c th khuch i, to dng v biu hin bng cc phng php c bit. S pht hin enzyme phin m ngc nhiu loi virus v c bit l mt s vi khun cng c ngha quan trng trong vic nghin cu s tin ha ca h thng sinh gii.

Hnh 5.7. Qu trnh phin m ngc ca retrovirus Ti liu tham kho/c thm
1. H Hunh Thy Dng. 1998. Sinh hc phn t. NXB Gio dc, H Ni. 2. Alberts B, Johnson A, Lewis J, Raff M, Roberts K and Walter P. 2002. Molecular Biology of the Cell. 4th ed. Garland Science Publishing, Inc. New York, USA.

3. Flint SJ. 2000. Principles of Virology: Molecular Biology, Pathogenesis, and Control. ASM Press, Washington, USA. 4. Gelehrter TD, Collins FS and Ginsburg D. 1998. Principles of Medical Genetics. 2nd ed. Williams & Wilkins Company, Baltimore, USA. 5. Griffiths AJF, Wessler SR, Lewontin RC, Gelbart WM, Suzuki DT and Miller JH. 2005. An Introduction to Genetic Analysis. WH Freeman & Co. New York, USA. 6. Karp G. 2002. Cell and Molecular Biology. John Wiley & Sons, Inc. New York, USA. 7. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Losick R. 2004. Molecular Biology of the Gene. Pearson Education, Inc./Benjamin Cummings Publishing, San Francisco, USA.

Chng 6

Dch m
Dch m l qu trnh cc thng tin di truyn cha trong cc trnh t nucleotide ca mRNA c s dng to ra cc chui amino acid trong protein. S tng hp mt protein ring l i hi s tham gia ca hn 100 protein v RNA. B my dch m bao gm bn thnh phn quan trng l mRNA, tRNA, aminoacyl tRNA synthetase v ribosome. Cc mRNA l khun mu cho qu trnh dch m. Dch m l mt trong nhng qu trnh c tnh bo th cao v chim nhiu nng lng ca t bo. Tuy nhin, do cu trc khc nhau gia mRNA ca prokaryote v eukaryote nn qu trnh dch m ca chng cng c nhng im khc bit quan trng. I. M di truyn 1. Cc codon Do ch c bn loi nucleotide khc nhau trong mRNA v c n 20 loi amino acid trong protein nn s dch m khng th c thc hin theo kiu tng ng mt nucleotide-mt amino acid c. Chui nucleotide ca mt gen thng qua trung gian mRNA c dch m thnh chui amino acid ca protein theo nhng quy lut c gi l m di truyn. Ngi ta gii m ton b cc amino acid vo nhng nm u ca thp k 1960. Mi amino acid c m ha bi ba nucleotide lin tip trn DNA (hoc RNA tng ng), b ba nucleotide ny c gi l mt codon. Vi 4 loi nucleotide khc nhau s c 43 = 64 codon khc nhau c phn bit bi thnh phn v trt t ca cc nucleotide. Trong s ny c 3 codon kt thc (stop codon) l UAA, UAG v UGA c nhim v bo hiu chm dt vic tng hp chui polypeptide. Trong 61 m cn li c nhiu codon cng m ha cho mt amino acid (Bng 3.4-Chng 3).

M di truyn c tnh ng nht cho ton b sinh gii tr mt s ngoi l i vi cc codon ty th. DNA ca bo quan ny c mt s codon m ha cho cc amino acid khc vi ngha ca cc codon ny trn DNA trong nhn. V d: - UGA m ha cho tryptophan thay v bo hiu chm dt vic tng hp protein. - AGA v AGG khng m ha cho arginine m bo hiu chm dt tng hp protein. - AUA m ha cho methionine thay v m ha cho isoleucine. 2. Cc quy tc chi phi m di truyn C ba quy tc iu khin s sp xp v s dng cc codon trn mRNA l: - Cc codon c c theo hng 5'3'. V vy chui m ha cho dipeptide NH2-Thr-Arg-COOH c vit l 5'-ACGCGA-3'. - Cc codon khng chng ln nhau v vng dch m ca mRNA khng cha cc khong trng. - Thng tin c dch m theo mt khung c (reading frame) c nh. V mt nguyn tc, cng mt trnh t RNA c th c ba khung c khc nhau. Tuy nhin, trn thc t ch c mt trong ba khung c ny cha thng tin thc s, chnh codon khi u xc nh khung c ng cho mi trnh t mRNA. II. Cc ribosome Ribosome l b my i phn t iu khin s tng hp protein. N c cu to bi t nht l 3 phn t RNA v trn 50 protein khc nhau1 vi khi lng phn t l 2,5 MDa (megadalton) i vi ribosome ca prokaryote v 4,2 MDa i vi ribosome ca eukaryote. 1. Thnh phn cu to ca ribosome Mi ribosome bao gm mt tiu n v ln v mt tiu n v nh. Tiu n v ln cha trung tm peptidyl transferase chu trch nhim cho vic hnh thnh cc cu ni peptide. Tiu n v nh cha trung tm gii m, l ni cc tRNA c gn amino acid c v gii m cc codon. Ngoi ra cn c trung tm gn cc yu t tiu n v ln. Theo quy c, cc tiu n v c t tn theo tc lng ca chng di lc ly tm. n v o tc lng l Svedberg (tn ca nh pht minh my siu ly tm) v c vit tt l S. Ribosome ca prokaryote l ribosome 70S, trong tiu n v ln l 50S v tiu n v nh l 30S. Ribosome ca eukaryote l 80S, vi tiu n v ln l 60S v tiu n v nh l 40S.

Mi tiu n v u c cu to bi cc RNA ribosome (rRNA) v cc protein ribosome. n v Svedberg li c s dng phn bit cc rRNA (Bng 6.1). Trong qu trnh dch m, tiu n v ln v tiu n v nh ca mi ribosome lin kt vi nhau v vi mRNA. Sau mi vng tng hp protein, chng li ri nhau ra. Bng 6.1. Cc thnh phn cu to ca ribosome

2. Khi nim polyribosome Mc d mt ribosome ch c th tng hp mt polypeptide ti mt thi im, nhng mi mRNA c th c dch m ng thi bi nhiu ribosome. Mt mRNA mang nhiu ribosome c xem l polyribosome hay polysome. Mi ribosome n c tip xc vi khong 30 nucleotide, nhng do kch thc ln ca ribosome nn mt cho php trn mRNA l 80 nucleotide cho mi ribosome. 3. Cc v tr gn tRNA trn ribosome Trn ribosome cha ba v tr gn tRNA l v tr A, P v E. Trong : - A l v tr gn aminoacyl-tRNA (tRNA c mang amino acid). - P l v tr gn peptidyl-tRNA (tRNA c mang chui polypeptide). - E (exit) l v tr gn tRNA m c phng thch sau khi chui polypeptide c chuyn sang aminoacyl-tRNA. Mi v tr gn tRNA c hnh thnh ti giao din gia tiu n v ln v tiu n v nh. Bng cch ny, cc tRNA c gn vo c th bt ngang qua khong cch gia trung tm peptidyl transferase ca tiu n v ln v trung tm gii m ca tiu n v nh. u 3' ca tRNA c nm gn tiu n v ln v vng i m gn tiu n v nh.

Hnh 6.1. Cc thnh phn chc nng ca ribosome

4. Cc knh ca ribosome l cc knh cho php mRNA i vo v i ra khi ribosome, v knh cho php chui polypeptide mi sinh i ra khi ribosome. mRNA i vo v i ra khi trung tm gii m ca ribosome thng qua hai knh hp ti tiu n v nh. Trong , knh vo c chiu rng ch cho RNA khng bt cp i qua. c im ny m bo cho mRNA c dui thng khi n i vo trung tm gii m, bng cch loi b mi tng tc bt cp base b sung ni phn t. Mt knh xuyn qua tiu n v ln to li thot cho chui polypeptide mi c tng hp. Kch thc ca knh hn ch c s gp ca cc chui polypeptide ang tng hp. V vy, protein ch c th hnh thnh cu trc bc ba sau khi n c gii phng khi ribosome. III. S hnh thnh aminoacyl-tRNA 1. Bn cht ca s gn amino acid vo tRNA Qu trnh gn amino acid vo tRNA l qu trnh hnh thnh mt lin kt acyl gia nhm carboxyl ca amino acid v nhm 2'- hoc 3'-OH ca adenine u 3' ca tRNA. Lin kt ny c xem l mt lin kt giu nng lng. Nng lng gii phng ra khi lin kt b ph v gip hnh thnh cu ni peptide lin kt amino acid vi chui polypeptide ang c tng hp. 2. S nhn din v gn amino acid vo tRNA S nhn din v gn amino acid vo tRNA tng ng c thc hin bi mt enzyme gi l aminoacyl-tRNA synthetase. Qu trnh ny din ra nh sau: u tin, amino acid c adenylyl ha bng cch phn ng vi ATP, kt qu to thnh amino acid c gn adenylic acid qua cu ni ester giu nng lng gia nhm COOH ca amino acid v nhm phosphoryl ca AMP, ng thi gii phng ra pyrophosphate. Sau , amino acid c adenylyl ha ny (vn ang gn vi synthetase) phn ng tip vi tRNA. Phn ng ny chuyn amino acid n u 3' ca tRNA gn vi nhm OH, ng thi gii phng AMP. Phn ng tng hp ca qu trnh ny nh sau:

Amino acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi 3. Tnh c hiu ca aminoacyl-tRNA synthetase Hu ht cc t bo u c mt enzyme synthetase ring bit chu trch nhim cho vic gn mt amino acid vo mt tRNA tng ng (nh vy c tt c 20 synthetase). Tuy nhin, nhiu vi khun c di 20 synthetase. Trong trng hp ny, cng mt synthetase chu trch nhim cho hn mt loi amino acid. S nhn din amino acid chnh xc l da vo kch thc, s tch in v gc R khc nhau ca cc amino acid. S nhn din tRNA da vo cc trnh t nucleotide khc nhau ca tRNA. T l sai st trong qu trnh gn amino acid vi tRNA tng ng l kh thp. 4. Phn loi aminoacyl-tRNA synthetase C hai loi tRNA synthetase. - Loi I bao gm cc synthetase gn cc amino acid nh Glu, Gln, Arg, Cys, Met, Val, Ile, Leu, Tyr, Trp vo nhm 2'-OH. - Loi II gm cc synthetase gn cc amino acid nh Gly, Ala, Pro, Ser, Thr, His, Asp, Asn, Lys, Phe vo nhm 3'-OH. IV. Cc giai on ca qu trnh dch m Qu trnh dch m c bt u bng s gn ca mRNA v mt tRNA khi u vi tiu n v nh t do ca ribosome. Phc hp tiu n v nh-mRNA thu ht tiu n v ln n to nn ribosome nguyn vn vi mRNA c kp gia hai tiu n v. S tng hp protein c bt u ti codon khi u u 5' ca mRNA v tin dn v pha 3'. Khi ribosome dch m t codon ny sang codon khc, mt tRNA gn amino acid k tip c a vo trung tm gii m v trung tm peptidyl transferase ca ribosome. Khi ribosome gp codon kt thc th qu trnh tng hp chui polypeptide kt thc. Chui ny c gii phng, hai tiu n v ca ribosome ri nhau ra v sn sng n gp mRNA mi thc hin mt chu trnh tng hp protein mi. Qu trnh dch m c chia thnh ba giai on l khi u, ko di v kt thc. 1. Giai on khi u 1.1. prokaryote 1.1.1. Cc yu t khi u (IF: initiation factor) C cc yu t khi u xc tc cho tiu n v nh trong vic hnh thnh phc hp khi u. l IF1, IF2, IF3. Mi yu t khi u c tc dng nh sau: - IF1 gip tiu n v nh gn vo mRNA v ngn cn cc tRNA gn vo vng thuc v tr A trn tiu n v nh. - IF2 l mt protein gn v thy phn GTP. IF2 thc y s lin kt gia fMettRNAifMet v tiu n v nh, ngn cn nhng aminoacyl-tRNA khc n gn vo tiu n v nh. - IF3 ngn cn tiu n v nh ti lin kt vi tiu n v ln v gn vi cc tRNA mang amino acid. IF3 gn vo tiu n v nh vo cui vng dch m trc, n gip tch ribosome 70S thnh tiu n v ln v tiu n v nh.

Khi tiu n v nh c gn ba yu t khi u, n s gn tRNA khi u v mRNA. S gn hai RNA ny l hon ton c lp vi nhau. 1.1.2. Bc 1: Tiu n v nh gn vo codon khi u S lin kt gia tiu n v nh vi mRNA c thc hin thng qua s bt cp base b sung gia v tr gn ribosome v rRNA 16S. Cc mRNA ca vi khun c mt trnh t nucleotide c hiu gi l trnh t Shine-Dalgarno (SD) gm 5-10 nucleotide trc codon khi u. Trnh t ny b sung vi mt trnh t nucleotide gn u 3' ca rRNA 16S. Tiu n v nh c t trn mRNA sao cho codon khi u c t ng vo v tr P mt khi tiu n v ln gn vo phc hp. 1.1.3. Bc 2: tRNA u tin c mang methionine bin i n gn trc tip vi tiu n v nh Mt tRNA c bit c gi l tRNA khi u n gn trc tip vi v tr P (khng qua v tr A). tRNA ny c anticodon (b ba i m) c th bt cp vi AUG hoc GUG. Tuy nhin tRNA ny khng mang methionine cng nh valine m mang mt dng bin i ca methionine gi l N-formyl methionine. tRNA khi u ny c gi l fMet-tRNAifMet. Trong hoc sau qu trnh tng hp polypeptide, gc formyl c loi b bi enzyme deformylase. Ngoi ra, aminopeptidase s loi b methionine cng nh mt hoc hai amino acid k tip u chui polypeptide.

Hnh 6.2. Khi u dch m prokaryote

1.1.4. Bc 3: Hnh thnh phc hp khi u 70S Bc gn thm tiu n v ln to thnh phc hp khi u 70S din ra nh sau: khi codon khi u v fMet-tRNAifMet bt cp vi nhau, tiu n v nh thay i hnh dng lm gii phng IF3. S vng mt IF3 cho php tiu n v ln gn vo tiu n v nh ang mang cc thnh phn trn. Nh c tiu n v ln gn vo, hot tnh GTPase ca IF2-GTP c kch thch thy phn GTP. IF2-GDP to thnh c i lc thp i vi ribosome v tRNA khi u dn n s gii phng IF2-GDP cng nh IF1. Nh vy phc hp khi u cui cng c to thnh bao gm ribosome 70S

c gn ti codon khi u ca mRNA, vi fMet-tRNAifMet ti v tr P, cn v tr A ang trng. Phc hp ny sn sng tip nhn mt tRNA mang amino acid vo v tr A bt u tng hp polypeptide (Hnh 6.2). 1.2. Eukaryote 1.2.1. Bc 1: S hnh thnh phc hp tin khi u 43S Giai on khi u i hi s h tr ca hn 30 protein khc nhau, mc d eukaryote cng c nhng yu t khi u tng ng vi prokaryote. Cc yu t khi u ny c k hiu l eIF. Khi ribosome ca eukaryote hon thnh mt chu trnh dch m, n tch ri ra thnh tiu n v ln v tiu n v nh t do thng qua tc ng ca cc yu t eIF3 v eIF1A (tng t vi IF3 prokaryote). Hai protein gn GTP l eIF2 v eIF5B lm trung gian thu ht tRNA khi u gn methionine (ch khng phi N-formyl methionine nh prokaryote) n tiu n v nh. Chnh yu t eIF5B-GTP l tng ng vi IF2-GTP ca prokaryote. Yu t ny lin kt vi tiu n v nh theo phng thc ph thuc eIF1A. Ri eIF5B-GTP gip thu ht phc hp eIF2-GTP v Met-tRNAiMet n tiu n v nh. Hai protein gn GTP ny cng nhau a MettRNAiMet vo vng thuc v tr P ca tiu n v nh. Kt qu, hnh thnh phc hp tin khi u 43S. 1.2.2. Bc 2: S nhn dng m 5 ca mRNA Qu trnh ny c thc hin thng qua eIF4F. Yu t ny c ba tiu n v, mt tiu n v gn vo m 5', hai tiu n v khc gn vi RNA. Phc hp ny li c gn vi eIF4B lm hot ha mt enzyme RNA helicase ca mt trong nhng tiu n v ca eIF4F. Helicase ny tho xon tt c cc cu trc bc hai c hnh thnh u tn cng ca mRNA. Phc hp eIF4F/B v mRNA li thu ht phc hp tin khi u 43S n thng qua tng tc gia eIF4F v eIF3.

Hnh 6.3. Khi u dch m eukaryote

1.2.3. Bc 3: Tiu n v nh tm thy codon khi u bng cch qut xui dng t u 5' ca mRNA v s hnh thnh phc hp khi u 80S Mt khi c gn vo u 5' ca mRNA, tiu n v nh v cc yu t lin kt vi n di chuyn dc theo mRNA theo hng 5' 3' cho n khi gp trnh t 5'-AUG3' u tin m n nhn dng l codon khi u. Codon c nhn dng bng s bt cp base b sung gia anticodon (b ba i m) ca tRNA khi u v codon khi u. S bt cp ny thc y phng thch eIF2 v eIF3 cho php tiu n v ln gn c vo tiu n v nh. S gn ny dn n phng thch cc yu t khi u cn li thng qua s thy phn GTP di tc dng ca eIF5B. Cui cng, Met-tRNAiMet c a vo v tr P ca phc hp khi u 80S. Lc ny, ribosome trong t th sn sng tip nhn aminoacyl-tRNA vo v tr A (Hnh 6.3).

1.2.4. Nhng yu t khi u dch m gi mRNA eukaryote dng vng Ngoi vic gn vo u 5' ca mRNA, cc yu t khi u cn lin kt cht ch vi u 3' thng qua ui poly(A). iu ny c thc hin bi s tng tc gia eIF4F v protein gn poly(A) bc bn ngoi ui poly(A). Vic tm thy cc yu t khi u dch m c vai tr "vng ha" mRNA theo phng thc ph thuc ui poly(A) gii thch c quan st trc y l mt khi ribosome kt thc s dch m mt mRNA m c vng ha thng qua ui poly(A) th ribosome mi c phng thch ny l ribosome l tng ti khi u dch m trn cng mRNA. 2. Giai on ko di 2.1. Bc 1: Aminoacyl-tRNA c a n v tr A nh yu t ko di EF-Tu Mt khi tRNA gn amino acid th EF-Tu n gn vo u 3' ca aminoacyltRNA. EF-Tu ch c th gn vi aminoacyl-tRNA khi n lin kt vi GTP. EF-Tu-GTP a aminoacyl-tRNA vo v tr A ca ribosome. Ch phc hp aminoacyl-tRNA-EF-TuGTP no c anticodon b sung vi codon ca mRNA ti v tr A th mi c gi li trn ribosome. Sau , EF-Tu tng tc vi trung tm gn yu t ca ribosome nm trn tiu n v ln v thy phn GTP, ri EF-Tu c phng thch khi tRNA v ribosome, aminoacyl-tRNA nm li ti v tr A. 2.2. Bc 2: Hnh thnh cu ni peptide Aminoacyl-tRNA ti v tr A c quay vo trung tm peptidyl transferase v cu ni peptide c hnh thnh. Phn ng ny c xc tc bi peptidyl transferase, ngy nay n c xc nh l rRNA, c bit l rRNA 23S ca tiu n v ln. V vy, peptidyl transferase cn c gi l ribozyme. Trong qu trnh hnh thnh cu ni peptide, cu ni gia amino acid v tRNA v tr A khng b ph v. u 3' ca c hai tRNA c a n gn nhau v nhm amine ca amino acid v tr A tn cng nhm carboxyl ca amino acid v tr P. Kt qu l tRNA v tr A mang mt dipeptide, trong khi tRNA v tr P b kh acyl. Sau xy ra s chuyn dch (xem bc 3): peptidyl-tRNA (ang mang dipeptide) chuyn sang v tr P, v v tr A sn sng tip nhn mt aminoacyl-tRNA mi. Cu ni peptide tip theo c hnh thnh theo cch ging ht trn, trong nhm amine ca amino acid mi lin kt vi nhm carboxyl u C tn cng ca chui polypeptide ang tng hp. Thc cht, y l qu trnh chuyn chui polypeptide ang tng hp t peptidyl-tRNA v tr P sang aminoacyl-tRNA v tr A. V vy, phn ng to cu ni peptide c gi l phn ng peptidyl transferase. Nh vy, chui polypeptide c tng hp theo chiu t u N n u C.

Trong qu trnh ny, khng c s thy phn nucleoside triphosphate. Nng lng c cung cp t s ph v cu ni acyl giu nng lng gia chui polypeptide ang tng hp v tRNA. 2.3. Bc 3: S chuyn dch (translocation) Mt khi phn ng peptidyl transferase xy ra th tRNA trong v tr P khng gn vi amino acid na, v chui polypeptide ang hnh thnh c lin kt vi tRNA trong v tr A. mt vng ko di polypeptide mi xy ra, tRNA v tr P phi chuyn n v tr E v tRNA v tr A chuyn n v tr P. ng thi, mRNA phi di chuyn qua 3 nucleotide ribosome tip xc vi codon tip theo. Nhng s di chuyn ny c gi l s chuyn dch. Bc u tin trong chuyn dch c song hnh vi phn ng peptidyl transferase. Khi chui peptide c chuyn sang tRNA v tr A, u 3' ca tRNA ny hng n vng v tr P ca tiu n v ln, trong khi u anticodon vn cn nm v tr A. Tng t, tRNA v tr P (m khng cn gn chui polypeptide na) nm v tr E ca tiu n v ln v v tr P ca tiu n v nh. hon thnh s chuyn dch phi c s tc ng ca mt yu t ko di gi l EF-G. EF-G ch gn vo ribosome khi c lin kt vi GTP. Sau khi phn ng peptidyl transferase xy ra, s thay i v tr ca tRNA v tr A l v tr gn cho EF-G. Khi EF-G-GTP gn vo v tr ny, n tip xc vi trung tm gn yu t v kch thch thy phn GTP. S thy phn ny lm thay i hnh dng ca EF-G-GDP v cho php n vi ti tiu n v nh thc y s chuyn dch ca tRNA v tr A. Khi s chuyn dch c hon thnh, cu trc ca ribosome gim ng k i lc vi EFG-GDP, iu ny cho php yu t ko di c phng thch khi ribosome. Cng vi vic tRNA v tr A chuyn n v tr P, tRNA v tr P chuyn n v tr E v mRNA dch chuyn ba nucleotide. T v tr E, tRNA c phng thch khi ribosome (Hnh 6.4).

Hnh 6.4. Ko di dch m

2.4. Cc yu t ko di c gn GDP (EF-Tu-GDP v EF-G-GDP) c i GDP thnh GTP trc khi tham gia vo vng ko di mi EF-Tu v EF-G l nhng protein xc tc m ch c s dng mt ln i vi mt vng ko di bao gm a tRNA vo ribosome, hnh thnh cu ni peptide, v chuyn dch. Sau khi GTP c thy phn, hai protein trn phi gii phng GDP v gn vi mt GTP mi. i vi EF-G, do GDP c i lc thp vi EF-G hn GTP nn GDP nhanh chng c gii phng v GTP mi c gn vo. i vi EF-Tu, cn c s tham gia ca yu t hon i GTP gi l EF-Ts. Sau khi EF-Tu-GDP c phng thch khi ribosome, EF-Ts c gn vo EF-Tu v th ch ca GDP. Sau GTP n gn vo phc hp EF-Tu-EF-Ts. Phc hp sau cng c tch thnh EF-Ts t do v EF-Tu-GTP. 3. Giai on kt thc 3.1. Cc yu t gii phng kt thc dch m Chu k gn aminoacyl-tRNA ca ribosome, s hnh thnh cu ni peptide, v s chuyn dch xy ra lin tc cho n khi mt trong ba codon kt thc vo v tr A. Cc codon ny c nhn din bi cc yu t gii phng (RF: release factor) (Hnh 6.5). C hai loi yu t gii phng:

- Cc yu t gii phng loi I nhn din codon kt thc v thc y s thy phn tch chui polypeptide ra khi peptidyl-tRNA ti v tr P. Prokaryote c hai yu t gii phng loi I l RF1 v RF2, trong RF1 nhn din codon kt thc UAG v RF2 nhn din UGA, cn UAA c nhn din bi c RF1 v RF2. Eukaryote ch c mt yu t gii phng gi l eRF1 nhn din c c ba loi codon kt thc. - Cc yu t gii phng loi II kch thch s tch yu t gii phng loi I ra khi ribosome sau khi chui polypeptide c gii phng. Ch c mt yu t gii phng loi II, c gi l RF3 prokaryote v eRF3 eukaryote. Yu t gii phng loi II c iu ha bi GTP.

Hnh 6.5. Kt thc dch m

3.2. S hon i GDP/GTP v thy phn GTP iu khin hot ng ca yu t gii phng loi II Yu t gii phng loi II l mt protein gn GTP nhng c i lc vi GDP cao hn GTP. V vy, phn ln RF3 c gn vi GDP. RF3-GDP gn vi ribosome theo

mt phng thc ph thuc s hin din ca yu t gii phng loi I. Sau khi yu t gii phng loi I kch thch s phng thch chui polypeptide, xy ra mt s thay i hnh dng ribosome, v yu t gii phng loi I kch thch RF3 hon i GDP thnh GTP. S gn GTP vo RF3 dn n s hnh thnh tng tc i lc cao vi ribosome v y yu t gii phng loi I ra khi ribosome. S thay i ny cho php RF3 lin kt vi trung tm gn yu t ca tiu n v ln. S tng tc ny kch thch thy phn GTP. V khng cn yu t loi I na nn RF3-GDP c i lc thp vi ribosome v b phng thch ra ngoi. 3.3. S quay vng ca ribosome Sau khi phng thch chui polypeptide v cc yu t gii phng, ribosome vn cn gn vi mRNA cng vi hai tRNA ti v tr P v v tr E. ribosome tham gia vo qu trnh tng hp polypeptide mi, tRNA v mRNA phi i khi ribosome v hai tiu n v ca ribosome phi ri nhau ra. Tp hp nhng s kin nh vy gi l s quay vng ribosome (ribosome recycling). prokaryote, c mt yu t gi l yu t quay vng ribosome (RRF: ribosome recycling factor). RRF gn vo v tr A, n bt chc tRNA. RRF li ko EF-G n ribosome v EF-G kch thch gii phng nhng tRNA ti v tr P v E. Sau , EF-G v RRF c phng thch khi ribosome cng vi mRNA. IF3 c th tham gia vo s gii phng mRNA ng thi n cng cn cho s tch ri hai tiu n v ca ribosome. Kt qu to ra tiu n v nh gn IF3 v tiu n v ln t do. Ribosome by gi c th tham gia vo vng dch m mi. V. Cc nhn t c ch dch m Qu trnh dch m bao gm rt nhiu bc v tun theo mt quy tc l khng c mt bc no c th xy ra khi bc trc n cha hon thnh. Tuy nhin, iu ny to ra mt im yu trong dch m l nu chng may c mt bc khng hon thnh th ton b qu trnh phi b ngng li. V vy, dch m thng l im ch tc ng ca nhiu loi khng sinh v cc c t (Bng 6.2). Khong 40% cc loi khng sinh l nhng yu t c ch dch m. Cc khng sinh ny tiu dit vi khun gy bnh ngi hoc ng vt do n tc ng lm ngng dch m vi khun nhng khng nh hng n dch m eukaryote, do qu trnh dch m vi khun v eukaryote c nhng im khc nhau c ngha, v d ribosome khc nhau v kch thc v cc thnh phn. Nhiu khng sinh gn chn lc vi ribosome ca vi khun v c ch nhiu bc trong dch m nhng khng nh hng n ribosome ca eukaryote. V d tetracycline gn vo v tr A ca ribosome vi khun v c ch s i vo ca cc aminoacyl-tRNA nhng khng hiu qu trn ribosome ca eukaryote. Nhng khng sinh khc nhau th c ch cc bc khc nhau trong qu trnh dch m nn chng thng c s dng nghin cu s hot ng ca b my dch

m, c bit l puromycin. Do cu trc ba chiu ca puromycin ging vi u 3' ca aminoacyl-tRNA nn n c th gn vo v tr A v c ch s i vo y ca cc aminoacyl-tRNA.

Bng 6.2. S c ch dch m ca mt s khng sinh v c t

Cu ni peptide c th c hnh thnh gia puromycin ti v tr A v chui polypeptide ti v tr P. Tuy nhin, puromycin khng gn vo v tr P nn s chuyn dch khng xy ra v chui polypeptide gn puromycin ri khi ribosome d qu trnh tng hp cha kt thc. Do cu trc ca tRNA l tng t nhau mi sinh vt nn puromycin c ch dch m c vi khun v t bo eukaryote, hu qu l n tiu dit c t bo vt ch cng vi vi khun. Thnh thong, puromycin cng c dng iu tr ung th nhm tiu dit t bo khi u. Ti liu tham kho/c thm
1. Alberts B, Johnson A, Lewis J, Raff M, Roberts K and Walter P. 2002. Molecular Biology of the Cell. 4th ed. Garland Science Publishing, Inc. New York, USA. 2. Griffiths AJF, Wessler SR, Lewontin RC, Gelbart WM, Suzuki DT and Miller JH. 2005. An Introduction to Genetic Analysis. WH Freeman & Co. New York, USA. 3. John LB, Carey JC, Bamshad MJ and White RL. 2003. Medical Genetics. Mosby Publishing, Missouri, USA. 4. Karp G. 2002. Cell and Molecular Biology. John Wiley & Sons, Inc. New York, USA. 5. Pierce BA. 2003. Genetics: A Conceptual Approach. WH Freeman & Co. New York, USA. 6. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Losick R. 2004. Molecular Biology of the Gene. Pearson Education, Inc./Benjamin Cummings Publishing, San Francisco, USA.

Chnh xc l t 3-4 phn t RNA v t 55-82 protein.

Chng 7

Sa cha v bo v DNA
Trn phn t DNA c th xut hin nhiu bin i do sai hng trong qu trnh trao i cht, do cc tc nhn gy t bin vt l v ha hc ca mi trng. Tuy nhin, genome lun c n nh cao nh cc c ch sa cha v bo v DNA. DNA l phn t duy nht, m khi bin i hay b ph hng vn c kh nng c sa cha nh t bo. Cc c ch sa sai rt a dng v c hiu qu cao. Ba qu trnh bao gm sa sai, ti bn v ti t hp DNA lin quan cht ch vi nhau. y cng l mt minh chng v s phi hp cht ch gia nhiu c ch di truyn.

I. Khi qut v cc c ch sa sai Hu ht cc t bin trn phn t DNA thng c khc phc bng hai phng thc chnh: - Sa cha phc hi trc tip (direct reversal repair), hoc: - Ct b sai hng v sa cha li bng cch dng trnh t b sung (damage excision and repair using complementary sequence). Sa cha trc tip thng lin quan n hai loi sai hng trn phn t DNA do tia t ngoi gy ra l: cyclobutane-pyrimidine dimer (CPDs) v pyrimidine (6-4) pyrimidone (6-4 PPs). Hai loi sai hng ny u lm bin dng cu trc xon ca DNA. CPDs v 6-4 PPs c nhn bit v sa cha nh enzyme photolyase. Enzyme ny s dng nng lng nh sng thc hin phn ng lm thay i cc lin kt ha hc nucleotide tr li dng bnh thng. Phn ng sa cha DNA bng photolyase xy ra trong rt nhiu sinh vt prokaryote v eukaryote. Tuy nhin, qu trnh ny khng thy ng vt c v. ngi cng cha pht hin c bt k loi photolyase no. a s sai hng ca DNA c sa cha bng phng thc th hai. C ch ny phi s dng thng tin di truyn cha mt trong hai si n DNA. Khi trnh t nucleotide trn mt si b thay i th si th hai (lin kt b sung vi si th nht) c dng lm khun mu sa cha nhng sai hng . Mt s c ch sa cha nh sau: - H thng sa cha nhn bit cc trnh t DNA khng thch hp vi cc cp base chun v thay th chng.

- H thng sa cha-ct b (excision-repair system) loi i mt on DNA v tr sai hng v sau thay th n. - H thng sa cha-ti t hp (recombinant-repair system) s dng phng thc ti t hp thay th vng si i b sai hng. Cc h thng sa cha cng phc tp nh b my ti bn ca n, iu cho thy tm quan trng ca chng i vi s sng ca t bo. Khi h thng sa cha phc hi mt sai hng ca DNA, th khng c mt hu qu xu no xy ra. Nhng mt t bin c th to ra hu qu xu khi DNA b hng. Hnh 7.1 tm tt mt s c ch sa cha DNA nh sau: - Mt vi enzyme phc hi trc tip cc loi sai hng c bit ca DNA. - Mt s phng thc sa cha bng cch ct b base, sa cha bng cch ct b nucleotide, v sa cha ghp i lch, tt c chc nng ca chng thc hin bng cch loi b v thay th nguyn liu. - Cc h thng chc nng c th ti t hp to ra mt bn sao khng b sai hng thay th cho mt si i b sai hng. - Phng thc ni u khng tng ng ni li cc u si i b hng. - Mt s DNA polymerase khc nhau cn thit trong vic tng hp li cc on DNA thay th.

1. Cc bin i xy ra trn phn t DNA Trn DNA c th xy ra cc bin i ngu nhin nh sau: - Gy hay t mch. Phn t DNA c chiu ngang rt mnh, bn thn n li thng xuyn cun xon v gin xon nn d xy ra t gy mt si. Tuy nhin, kh nng t cng lc hai si him khi gp hn.

Hnh 7.1. Vic sa cha cc gen c th c phn loi theo cc phng thc s dng cc c ch khc nhau phc hi hoc b qua s sai hng ca DNA

- Base b ct mt. Hin tng ny lm base tng ng khng bt cp c. Di tc dng ca nhit c th xy ra qu trnh kh purine (depurination) do thy phn lin kt N-glycosyl. - Gn cc nhm mi vo base bng lin kt cng ha tr lm thay i tnh cht nh trng hp methyl ha (gn nhm CH3 vo mt base). - Bin mt base ny thnh mt base khc lm bt cp sai. V d: qu trnh kh amine (desamination) ca cytosine bin n thnh uracil.

- Cc base c th tn ti hai dng keto v enol nn c th dn n bt cp sai. V d: dng enol ca cytosine c th bt cp vi adenine. - To cc thymine dimer. - Lin kt cho gia cc mch (interstand crosslink). Trn y l cc bin i ngu nhin, nu c tc ng ca cc tc nhn gy t bin cc bin i s xy ra nhiu hn.

2. Khi qut cc c ch sa cha mc phn t S nguyn vn ca phn t DNA mang thng tin di truyn c ngha sng cn i vi t bo, nht l t bo prokaryote. t bo vi khun, c n 50% DNA khng b bin i thm ch sau khi ti bn n 100 triu ln. S n nh cao ca DNA t bo c c nh hng lot cc c ch bo v s nguyn vn v sa cha ngay lp tc bt k sai hng no va xut hin. Cc h thng sa sai rt a dng v c hiu qu rt cao E. coli. C khong 100 locus tham gia trc tip hoc gin tip vo vic bo v DNA v sa sai. Ch vi DNA, t bo phi u t rt ln cho s n nh ca thng tin di truyn. Nu nh s ti bn ch xy ra khi phn bo, th cc c ch sa sai phi hot ng lin tc. Ti bn c thc hin vi c ch v c bn ging nhau cc loi t bo v trong mi ln nhn i DNA. Trong khi , sai hng xy ra rt a dng, nn cc c ch sa sai nhiu hn v vi s lng gen tham gia ln hn. Hn na, cc c ch di truyn c bn nh ti bn, ti t hp DNA u c s tham gia ca cc c ch sa sai v ngc li sa sai cn n ti bn v ti t hp.

3. Bin i lm tng tn s t bin Nh cc c ch sa sai nn bnh thng DNA t bo c n nh rt ln, thng s thay th base c tn s 10-9-10-10. Tuy nhin, ngi ta pht hin c cc dng t bin ngu nhin tng cao hn hn, chng c gi l mutator (nhn t gy t bin). Trong nhiu trng hp, cc kiu hnh mutator lin quan n sai hng trong h thng sa sai. E. coli, cc locus mutator mutH, mutL, mutU v mutS tc ng n cc cu phn ca h thng sa sai do ghp i lch (mismatch-repair) sau ti bn nn lm cho tn s t bin ngu nhin tng cao. Ngoi mutator, s sai hng lm tng tnh nhy cm vi tia t ngoi (ultraviolet) v tng sai hng khi ti t hp.

II. Cc kiu sa cha 1. Quang ti hot ha Sa cha trc tip bng quang ti hot ha (photoreactivation) t khi gp, n bao gm s phc hi hoc loi b n gin cc sai hng v qu trnh ny xy ra ngoi sng. Quang ti hot ha ca cc pyrimidine dimers, trong to c hi cho cc lin kt cng ha tr c phc hi nh enzyme ph thuc nh sng (light-dependent enzyme), l mt v d in hnh (Hnh 7.2). H thng sa cha ny ph bin trong t nhin, v c bit quan trng thc vt. Trong E. coli, n ph thuc vo sn phm ca mt gen n (phr) m ha cho mt enzyme c gi l photolyase. Sau khi x l tia t ngoi gy t bin, nu a ra nh sng th phn ln sai hng c phc hi. Hin tng ny c gi l quang ti hot ha. Nng lng ca nh sng kh kin (t 300 n 600 nm) hot ha photolyase (gen phr E. coli) ct cc vng cyclobutyl pyrimidine dimer (thng l thymine dimer). Enzyme ny hot ng trong cc t bo nhiu loi khc nhau. Vo ban ngy, cc sinh vt thng chu tc ng ca nh sng, nn c ch quang ti hot ha (quang phc hi) c vai tr quan trng trong sa sai DNA. V d: Mycoplasma, sinh vt n gin nht hin nay, ch c vi trm gen nhng mt trong s c dng cho quang ti hot ha. 2. Sa cha ghp i lch Sa cha ghp i lch (mismatch-repair) gia cc si DNA l mt trong nhng mc tiu chnh ca h thng sa sai. Sa cha ghp i lch c tin hnh khi pht hin trn DNA cc base nm cnh nhau m khng bt cp thch hp. Cc ghp i lch tng ln trong sut qu trnh ti bn c sa cha bng cch phn bit gia cc si c v mi v c u tin sa cha trnh t ca cc si c tng hp mi (Hnh 7.3). Ghp i lch c to ra bi cc bin i base, l mt kt qu ca s kh amine (deamination).

Hnh 7.2. Quang ti hot ha

Ghp i lch thng c sa sai bng phng thc sa cha ct b, c khi u bng mt enzyme nhn bit mt base sai hng thc s hoc c s thay i chiu hng khng gian (spatial path) ca DNA.

Hnh 7.3. Sa cha ghp i lch trong ti bn

S nhn bit v sa cha cc sai lch do bt cp base sai trn DNA c pht hin E. coli, nm men v t bo ng vt c v. vi khun E. coli, c ba h thng enzyme khc nhau c s dng sa cha cc sai lch bng cch: - Loi b ch sai trong ti bn (errors in replication). - Loi b ghp i lch bn trong cc on trung gian ca ti t hp (mismatch within recombinant intermediates). - Loi b thymine ca cp GT trong trng hp 5-methylcytosine b mt nhm amine (NH2) thnh thymine.

3. Sa cha ct b C hai loi h thng sa cha ct b, l: - H thng sa cha ct b base (base excision repair) loi b trc tip base sai hng v thay th n trong DNA. V d in hnh l enzyme DNA uracil glycolase, loi cc uracil khng c bt cp (mispaired) vi cc guanine. - H thng sa cha ct b nucleotide (nucleotide excision repair) ct b mt trnh t bao gm cc base sai hng; sau mt on DNA mi c tng hp thay th cc nguyn liu b ct b. Cc h thng ny ph bin hu ht sinh vt.

3.1. Ct b base Vic loi b ch sai c thc hin nh mt hoc vi enzyme N-glycosylase. Nglycosylase nhn bit base bin i hay mt gc amine hoc bin dng cu trc xon do sai lch to ra v thy phn lin kt N-glycosylic gia base vi ng vi pentose. L hng va c to ra do ct b c DNA polymerase lm y li da vo khun mu b sung i din v ligase ni lin chng (Hnh 7.4).

3.2. Ct b nucleotide Vic ct b mt vng c nhiu thymine dimer (do UV) hoc vng lin kt cho gia cc si, c thc hin nh incision nuclease (nuclease to khc trn DNA) nh phc hp Uvr ABC ca E. coli, phc hp ny ct cc on 12-13 nucleotide t mt si DNA. S thy phn c thc hin lin kt phosphodiester th tm u 5 n ch hng v pha 3 l lin kt th t hay nm.

Hnh 7.4. Phng thc ct b base

Cu trc si kp b sung cho nhau ca DNA cho php, nu thng tin b mt do ct b sai hng trn mt si c th chp li c nh da vo si n b sung kia. Tuy nhin, mt s sai hng lin quan cng lc c hai si cng c th c sa cha

nh vo mt on nucleotide tng ng tn ti u trong genome. S mt on hay xen on nucleotide, hay lin kt cho gia cc si DNA c th c phc hi nh s thay th on tng ng bng ti t hp. Thm ch s t i c hai si cng ch, c coi l nng nht trong cc sai hng, c th c gn lin li nh ligase hay ti t hp (Hnh 7.5).

4. c sa i vi cc base bt cp sai C ch c sa i vi cc base bt cp sai (proofreading for base-pair matching) c thc hin trong ti bn DNA. S bt cp cn thn ca DNA polymerase m bo cho s chnh xc ca ti bn. Trc khi thc hin phn ng polymer ha ni vi nucleotide, nucleotide triphosphate mi phi bt cp vi base b sung trn si khun mu. Nu s bt cp sai xy ra, DNA polymerase s loi b nucleotide bt cp sai. Thm ch, trc khi nucleotide mi gn vo, enzyme d li cp base cui nu chng khng bt cp th s polymer ha tip theo b dng. Cp nucleotide u cui 3 bt cp sai s b loi b bi hot tnh exonuclease 35 ca DNA polymerase. Khi s bt cp ca cu trc si kp ng, qu trnh polymer ha mi c tip tc. Hot tnh c sa i vi cc base bt cp sai l c tnh ca nhiu DNA polymerase m bo cho s ko di chnh xc ca si ang c tng hp. Tuy nhin, trong trng hp cn thit, DNA polymerase c th b qua ch sai v chp tip nh c ch ti bn error prone (xem mc 6).

5. Cc h thng sa cha ti t hp Cc v tr c s sai hng, xy ra trong mt phn t con (daughter molecule), s c phc hi nh s ti t hp thu c mt bn sao khc ca trnh t t mt ngun khng b sai hng. Bn sao ny sau c dng sa cha l hng (gap) trn si b hng. Khi DNA polymerase gp thymine dimer hay mt s sai lch khc trn si DNA khun mu, c hai kh nng xy ra: - Polymerase lp dn l hng bng ti bn khng theo khun mu do ti t hp v vt qua ch sai.

Hnh 7.5. Ct b nucleotide. Sa cha v ct b mt on DNA c (cc) base sai hng.

- Polymerase dng li v huy ng khong 1.000 nucleotide pha di (downstream); ch trng gin on c lp y vi si b sung t si i ch em (sister duplex) do ti t hp. Trong c hai trng hp, ch sai lch vn cn v c ct b sau bi sa sai trc tip hay ct b.

Hnh 7.6. Sa sai nh ti t hp v ti bn

6. H thng SOS H thng SOS (cha khong 30 gen khng lin kt v thng b c ch bi protein LexA) s hot ng khi t bo b cc tc nhn gy t bin to nhiu sai hng trn DNA. Trong trng hp DNA b hng ngng ti bn, phn ng SOS s phc hi ti bn bng phng thc ti bn error-prone. vi khun E. coli ngi ta quan st thy s ph hy DNA lm m ra khong 20 gen ca h thng SOS, c kim sot m bi cht c ch LexA (LexA repressor).

Trong trng hp c nhiu sai hng cn cp cu, LexA b kch thch, thay i cu hnh, t ct v mt hot tnh c ch. Lc cc gen ca h thng SOS c m ra. Nu qu trnh sa sai thc hin khng kp th t bo phi chp nhn hoc b t bin hoc cht. Protein RecA khi ng h thng SOS nh sau: - Hp SOS l trnh t DNA (operator) di khong 20 bp c nhn bit bi protein ca cht c ch LexA. - Sai hng ca DNA lm cho RecA khi ng phn ng SOS, cha cc gen m ha cho nhiu enzyme sa cha. - RecA hot ha hot tnh t phn gii t ca LexA. - LexA ngn chn h thng SOS, nhng phn ng t phn gii ca n hot ha cc gen ca h thng ny.

6.1. Sa cha theo c ch error-prone Phn t DNA c th b sai hng nng n khi b chiu tia t ngoi hoc khi chu tc ng ca cc tc nhn gy ung th (carcinogens). Lc , khng phi ch mt si n DNA b hng m c hai si u b t gy v mt i mt s nucleotide. Thng tin di truyn ca on hng b mt hon ton nn khng c si khun mu sa cha theo cc c ch thng thng. Trong trng hp ny, t bo cn li mt gii php duy nht tng kh nng sng st l sa cha DNA mt cch ngu nhin vi t l sai st (t bin) cao nhng d sao vn duy tr c s sng. y chnh l c ch sa cha DNA error-prone (cn c xem nh l s pht sinh t bin SOS). Nh vy, sa cha DNA theo c ch ny cng l mt trong nhng nguyn nhn to nn tnh a dng ca DNA. Khi phn t DNA b t bin, mt s protein c bit s lm nhim v dng qu trnh tng hp DNA sa cha theo cc c ch khc nhau. Bi v nu t bo tip tc phn chia (tc l DNA tip tc c nhn ln) th t l t bin cao s xut hin khin s t bo sng st gim xung. Mt s trng hp sai hng c sa cha bng c ch error-prone: - DNA kt hp vi cc base khng b sung trn si con (bt cp sai). - DNA b sai hng khng c sa cha do DNA polymerase III b gi li trong sut qu trnh ti bn. DNA polymerase V (c m ha bi gen umuD v umuC ca E. coli), hoc DNA polymerase IV (c m ha bi gen dinB ca E. coli) c cm ng trong h thng SOS, tng hp mt on b sung cho si DNA b hng. DNA polymerase V l mt phc hp cha hai protein tiu n v UmuD v mt protein tiu n v UmuC (Trong qu trnh sa cha error-prone, protein RecA, hot ng nh mt co-protease,

gin tip t xc tc ct protein UmuD thnh mt on c hot tnh, UmuD). DNA polymerase IV v V l mt phn ca mt h ln, bao gm cc DNA polymerase l nhng enzyme cn thit trong sa cha DNA b sai hng do chng c hot tnh polymerase. Cc protein tiu n v UmuD (thc t l UmuD) v UmuC c kh nng gn cc nucleotide vo si DNA bt chp khng tn ti si khun mu. Nh vy, chng c th c ch hot tnh t sa exonuclease 35 ca DNA polymerase hoc chnh chng l mt loi DNA polymerase c bit (DNA polymerase V). Khi t bin xy ra trn gen m ha cho UmuD v UmuC, cc t bo vi khun s sng st t hn t bo bnh thng di tc dng ca tia t ngoi. Mt vi plasmid mang gen mucA v mucB, tng ng vi gen umuD v gen umuC, khi c a vo trong vi khun s lm tng tnh khng vi tia t ngoi. Ngoi ra, khi c hai si n DNA u chu t bin nhng nu trong genome cn c t nht mt bn sao ging ht on b hng, lc on b hng c th c phc hi nh c ch trao i cho dng bn sao lm khun mu.

6.2. Protein LexA Hot tnh ca RecA gy ra s phn gii sn phm protein ca gen lexA. LexA l mt protein nh (22 kDa) kh n nh trong cc t bo khng b x l, ni n c chc nng nh mt cht c ch nhiu operon. Phn ng phn gii l khng thng xuyn; LexA c hot tnh protease mun c hot ha bi RecA. Khi RecA c hot ha, n s gy ra phn ng t phn gii ca LexA lm bt hot chc nng c ch ca LexA, v cm ng phi hp tt c operon m n c lin kt. Phng thc ny c minh ha hnh 7.7.

Hnh 7.7. LexA v RecA c quan h i khng thun nghch. Protein LexA c ch nhiu gen c cc chc nng sa cha, recA v lexA. Hot tnh ca RecA dn n phn gii protein LexA v cm ng tt c cc gen ny.

Cc gen ch ca protein c ch LexA c nhiu chc nng sa cha. Mt s gen SOS ny ch hot ng cc t bo b x l. Nhng gen khc hot ng cc t bo khng b x l, nhng mc biu hin c tng ln nh s phn gii ca LexA. Sau khi phn gii LexA, gen c th c biu hin t promoter th hai ging nh promoter u tin. Protein LexA c ch cc gen ch ca n bng cch lin kt vi mt on DNA di 20 bp c gi l hp SOS, bao gm mt trnh t lin ng (consensus sequence: 5CTGTN8ACAG-3) vi 8 v tr bo ton tuyt i (N8). Ging nh cc operator khc, cc hp SOS xp chng ln cc promoter tng ng. locus lexA, i tng ca s c ch t sinh, c hai hp SOS cnh nhau.

6.3. Protein RecA

S cun li trc tip ca protein RecA trong sa cha-ti t hp ch l mt trong nhng hot tnh ca n. N c th c hot ha bi cc x l lm sai hng DNA hoc c ch s ti bn trong E. coli. iu ny gip n khi ng mt chui phc tp nhng thay i kiu hnh c gi l phn ng SOS, yu t cn thit cho s biu hin ca nhiu gen m cc sn phm ca chng c cc chc nng sa cha. Cc hot tnh kp ny ca protein RecA lm cho ngi ta khng bit r s thiu ht trong sa cha cc t bo t bin recA l do mt chc nng trao i si DNA ca RecA hay l do mt vi chc nng khc m s cm ng ca n ph thuc vo hot tnh protease. S sai hng xut hin c th do chiu tia UV (hu ht trong cc trng hp nghin cu) hoc do cc nhn t lin kt cho hoc s alkyl ha (alkylation). S c ch ti bn do mt s phng thc nh thiu ht (deprivation) thymine, b sung thm thuc, hoc cc t bin trong mt s gen dna, c cng hiu qu. u tin l hot ha RecA bng x l sai hng. Chng ta khng bit nhiu v mi quan h gia s sai hng v s thay i t ngt trong hot tnh RecA. Mt bin ng ca cc trng hp sai hng c th cm ng phn ng SOS. Hin nay, ngi ta thin v kin cho rng RecA c hot ha bi mt s cht trung gian trong chuyn ha DNA. Tn hiu cm ng c th cha mt phn t nh c phng thch t DNA; hoc n c th l mt vi cu trc c to thnh trong chnh DNA. iu kin in vitro, hot tnh ca RecA i hi s c mt ca DNA si n v ATP. V th, mt vi tn hiu c th hin din vng si n mt v tr sai hng. S tng tc ca n vi RecA l nhanh: phn ng SOS xy ra trong mt vi pht x l sai hng. Tm li. RecA v LexA l cc mc tiu chung trong hp SOS: RecA khi ng s phn gii ca LexA, nhn t c ch recA v chnh n. V th, phn ng SOS to ra s khuch i ca c hai protein RecA v cht c ch LexA. Kt qu l khng cn mu thun nh lc u na. Vic tng mc biu hin ca protein RecA cn thit cho vai tr trc tip ca n trong cc phng thc sa cha-ti t hp. V s cm ng, nng ca RecA c tng ln 50 ln so vi nng ban u ca n khong 1.200 phn t/t bo. Nng cao cc t bo c cm ng c ngha l c RecA m bo rng tt c protein LexA b phn gii.

III. Bo v DNA: H thng ct hn ch (R)-Bin i (M) Ngoi cc h thng sa sai, t bo cn c h thng bo v DNA. Cc vi khun c h thng min nhim hiu qu DNA l, l: h thng ct hn ch-bin i (restriction-modification system) hin din trong nhiu loi vi khun, trong DNA c bin i bi cc enzyme c hiu.

S bin i xy ra nhm mc ch bo v DNA chng li s phn hy enzyme (ct hn ch) bi cc endonuclease ca chnh chng. S bin i bao gm mt kiu c trng ca phn ng methyl ha cc gc nucleotide trong DNA. Bng cch dng Sadenosylmethionine nh l mt cht cho, cc enzyme methyl ha (methylase) s hot ng trn DNA si i (dsDNA). Cc v tr m c s bin i cng c nhn bit bi cc enzyme hn ch (restriction enzyme, RE) tng ng, tuy nhin cc enzyme hn ch khng th ct DNA trong cc v tr c bin i ny mt hoc c hai si DNA. Cc vi sinh vt prokaryote ln eukaryote u c cc enzyme methyl ha gn nhm CH3 nhng im nht nh trn phn t DNA. Cc enzyme ny c tnh c hiu cao chuyn ha cho tng dng vi khun, v th DNA ca mi dng vi khun ch c methyl ha nhng v tr c bit nht nh. Nh , mi vi khun d dng phn bit DNA ca bn thn chng vi DNA ngoi lai (foreign DNA) (v d: DNA ca bacteriophage) xm nhp vo trong t bo. Cc enzyme ct hn ch l mt loi endonuclease ca mi dng vi khun (xem chng 9), khng ct DNA ca chng v c methyl ha nhng im cn thit m ch ct DNA ngoi lai (ca bacteriophage) do chng khng c methyl ha nhng v tr nht nh. Cc c ch ch yu bin i ca DNA ca n mt cch c hiu v ct hn ch (restriction) cc phn t DNA khng c nh du. H thng R-M ngn chn DNA ngoi lai khng c hot ng chc nng nhng c th thc hin ti t hp. H thng R-M c hai tnh cht cn bn: - Hot tnh restriction (ct hn ch) c hiu chng s xm nhp ca DNA ngoi lai. - Hot tnh bo v DNA ca bn thn n. H thng R-M ln u tin c pht hin khi nghin cu hin tng nhim bacteriophage vo E. coli. S bin i thng l methyl ha nhm 6-NH2 ca adenine nhng trnh t nucleotide c hiu trong h thng kiu II, C5 ca cytosine c methyl ha. Ct hn ch c thc hin do cc h thng endonuclease. Chng nhn bit cc im c hiu t nht mt si DNA khng b bin i. Endonuclease ct t si nhiu im nhn bit v sau cc on ngn c ct bng nucleotide khc. Phn ln cc DNA b bin i c cc c tnh di truyn khng n nh. S methyl ha thng mt qua ti bn trong t bo ch. Cc nghin cu cho thy chng E. coli B c 3 gen lin kt cht vi nhau m ha cho 3 sn phm khuch tn: cc polypeptide phc v cho s ct hn ch, cho s bin i v to s c hiu ca im nhn bit. DNA si kp nhy cm hn vi s bin i v ct hn ch. Cc bacteriophage si n nh M13 v X 174 t b tc ng hn. S bin i ca mt si DNA

min nhim DNA l. Do vy, DNA ti bn bn bo tn c bo v bi methyl ha si khun.

1. Cc methylase ca R-M Methylase l enzyme c hot tnh xc tc gn mt nhm methyl vo mt phn t, v d: DNA methyltransferase c chc nng methyl ha cc gc C trong DNA. Enzyme methylase to ra mt cp adenine methyl ha (hoc cytosine) c v tr i xng cho nhau. Cc methylase c lin quan cht ch vi cc kiu ct hn ch. - Kiu I. Cc RE c th tin hnh c hai s bin i v ct hn ch. Mt RE kiu I bao gm ba tiu n v khc nhau (R, M v S) chu trch nhim tng ng cho vic ct hn ch, bin i v nhn bit trnh t. V d: EcoB v EcoK (t E. coli chng B v K), cc trnh t nhn bit ca hai enzyme ny l TGA(N)8TGCT i vi EcoB v AAC(N)6GTGC i vi EcoK (trong N l mt trong bn loi nucleotide). Nu v tr nhn bit khng c methyl ha trong mt si, th enzyme RE s methyl ha si , nhng nu c hai si u khng c methyl ha th enzyme RE s ct DNA (cn c ATP). Phn ng ct xut hin mt v tr khng c hiu >1000 bp tnh t trnh t nhn bit; r rng l enzyme duy tr lin kt v tr nhn bit ca n, v DNA c di chuyn qua mt v tr th hai trn enzyme hng ti mt v tr ct hn ch. - Kiu II. Cc RE l dng chung nht trong vi khun; chng ch thc hin phn ng ct hn ch-cn s bin i c tin hnh bi mt enzyme ring bit. Trnh t nhn bit cho hu ht cc enzyme ny l cc trnh t palindrome ngn (v d: EcoRI nhn bit GAATTC) v thc hin phn ng ct (khng cn ATP) trong hoc gn k vi v tr nhn bit. Mt vi RE kiu II ct c hai si cng v tr to ra u bng , trong khi cc RE khc li to ra u dnh. Cc RE kiu II l cng c hu ch trong cng ngh DNA ti t hp. - Kiu III. Cc RE ny tng t cc enzyme kiu I. Mi RE kiu III c hai tiu n v; n nhn bit mt trnh t i xng ngn, v to ra mt u so le (cn c ATP) v tr khong 24-26 bp so vi v tr nhn bit. V d enzyme EcoPI, c m ha bi prophage ca bacteriophage P1.

2. Adenine v cytosine methyltransferase E. coli S methyl ha A v C thc hin rng ri tt c cc loi DNA. prokaryote (k cc bacteriophage v plasmid), vai tr th hin r l phn hy DNA ngoi lai. Vai tr ph l phn bit DNA b m vi si con mi c tng hp trong phc hi cc

ch bt cp sai. prokaryote, cc sinh vt khng c h thng ct hn ch, vai tr ch yu ca methyl ha c l s iu ha biu hin ca gen. Bn cnh cc methylase ca h thng R-M, cn c cc methyltransferase khc E. coli. Cc nucleotide c methyl ha khng gn trc tip vo DNA. Cc methylase chuyn nhm methyl t S-adenosine methionine sang adenine v cytosine nhng im c hiu trn DNA.

3. S methyl ha DNA ca eukaryote S methyl DNA ca eukaryote c nhiu im khc vi prokaryote: - Base b bin i ch yu eukaryote l 5-methylcytosine. ng vt c v, ch c 5-methylcytosine l base b bin i, trong 90% l trnh t i xng CpG. - Do cha hiu bit r h thng R-M eukaryote nn ngi ta cho rng c l methyl ha khng gp phn bo v chng s xm nhp ca DNA ngoi lai. S methyl ha ca DNA ng vt c v gp phn vo s iu ha biu hin ca gen v bit ha t bo. Cc nghin cu cho thy c s tng quan thun gia methyl ha thp (hyphomethylation) vi hot tnh ca gen v ngc li gia s methyl ha mnh vi bt hot ca gen. S vng mt ca cc nhm methyl trn DNA ca rui gim chng t rng sinh vt bit ha cao c th hot ng khng cn c ch methyl ha. S methyl ha ng vt c v c th ng vai tr: - Thay i trng thi ca DNA v c ch i lc ca repressor vi operator. - S methyl ha c truyn t theo dng t bo soma. - Methyl ha c th to s khc nhau gia cc m. - 5-azacytidine, cht ng ng vi cytidine, tc ng lm t bo nguyn bo si (fibroblast) bit ha thnh t bo c, c l do c ch c ch methyl ha DNA v nh vy hot ha gen tng ng.

Ti liu tham kho/c thm

1. Phm Thnh H. 2003. Di truyn hc. NXB Gio dc, H Ni. 2. V Th Thng Lan. 2002. Sinh hc phn t. NXB i hc Quc gia H Ni, H Ni. 3. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 4. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK.

5. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California, USA. 6. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company, New York, USA.

Chng 8

iu ha biu hin gen

Nh chng ta bit ba qu trnh thit yu cho s tn ti ca t bo, l: ti bn, phin m v dch m. Tuy nhin, t bo khng th tn ti c lp vi mi trng chung quanh. Nh vy, s ny sinh mt vn quan trng: t bo s iu chnh hot ng ca mnh nh th no cho ph hp vi cc bin i ca mi trng bn ngoi c th tn ti thch ng? Chng ny s cp n cc phng thc iu chnh , tc l cc c ch iu ha s biu hin ca gen cc sinh vt prokaryote v eukaryote. S biu hin ca cc gen chu s kim sot ca cc c ch iu ha. Cc c ch ny gi vai tr rt quan trng cho cc hot ng sng, p li nhng bin i ca mi trng bn trong v bn ngoi c th. Biu hin gen ca cc t bo prokaryote v eukaryote cng c s khc nhau ng k. Vic iu ha c thc hin nhiu mc khc nhau v lin quan n tng giai on pht trin. Theo quan nim v operon, cc gen iu ha (regulatory gene) gi vai tr quan trng trong vic ng v m cc gen cu trc (structural gene) c th biu hin tng hp protein ng lc, ng ni theo nhu cu c th ca t bo. Trong mi t bo, tt c cc gen u khng hot ng ng thi. V d: t bo E. coli c khong 107 phn t protein gm 3.000 loi khc nhau. Nhiu loi protein c n 500.000 phn t, tuy nhin mt s loi khc ch khong 10 phn t. Nh vy, khng phi loi protein no cng c tng hp vi s lng ln nh nhau v t bo phi c nhng c cht tng hp protein mt cch tit kim v hp l nht. Mt s gen hot ng thng xuyn cung cp sn phm lin tc, mt s khc ch biu hin nhng giai on nht nh trong chu trnh sng v c th ch hot ng trong iu kin mi trng khng bnh thng. Mt s protein cn c tng hp vi s lng ln, mt s khc ch cn c mt phn t. Do vy, hot tnh ca gen c iu ha bi nhiu c ch khc nhau c hiu qu tt nht trong vic s dng ngun nng lng ca t bo. I. Cc hin tng iu ha

 duy tr ni cn bng (homeostasis) v s pht trin ca c th, cc sinh vt c cc c ch iu ha khc nhau. Cc kiu iu ha u bt ngun t s biu hin ca cc gen. 1. iu ha thch nghi Mt s amip (ameba) biu hin s thay i hnh thi v sinh l c bit p li cc iu kin mi trng khc nhau. Khi cc amip c cho vo nc, chng chuyn t dng amip sang dng c lng bi. Khi mi trng thiu dinh dng chng c th chuyn thnh cc dng tng t nh biu b. Vi khun trong mi trng dinh dng ti thiu c kh nng tng hp amino acid. Nhng khi b sung amino acid vo mi trng nui, vi khun s ngng tng hp amino acid. Lc ngun amino acid t ngoi b sung vo ht, t bo vi khun li t tng hp li amino acid cho bn thn. Cc bin i nu trn l thun nghch, chng t s thay i chc nng y khng phi do bin d di truyn. Cc hin tng trn cn cho thy vic xut hin hay bin mt cc cu trc mi khng lm nh hng n tim nng di truyn sn c. C th cho rng, c trng hp mt s gen hot ng, nhng cng c trng hp mt s gen ngng biu hin. Cc hin tng c cp trn u do c ch iu ha thch nghi (adaptive regulation) chi phi. 2. Hot ng ni tip ca cc gen Khi bacteriophage xm nhim vi khun, DNA ca n lc u s ti bn, sau cc protein khc nhau mi c tng hp nn to thnh v. Nh vy, c cc gen sm to ra enzyme ti bn DNA v cc gen mun xc nh cc thnh phn v protein. iu chng t c c ch iu ha chc nng ca gen din ra theo mt trnh t nghim ngt. y l kiu iu ha ni tip (sequential regulation). Hot ng ni tip ca cc gen cn th hin r trong qu trnh pht trin c th ca cc sinh vt eukaryote a bo. 3. Bit ha t bo Nhiu sinh vt bc cao nh con ngi cha nhiu t t bo bt ngun t mt hp t do phn chia nguyn nhim. T mt hp t ban u n khi trng thnh, c th ngi c khong 200 loi t bo khc nhau. Mi loi t bo ch biu hin mt phn thng tin ca mnh. Qu trnh chuyn mn ha chc nng ca t bo c gi l s bit ha hay phn ha (differentiation). Tuy c s bit ha, nhng t bo vn gi nguyn vn kh nng di truyn ca mnh. Mt v d rt r l nui cy m t bo thc vt (plant tisue and cell culture): ngi ta c th nui cy mt phn m phn sinh trong mi trng dinh dng tng

hp cho n khi chng pht trin thnh cy in vitro hon chnh (plantlet), cc cy ny sau c a ra trng trong iu kin t nhin v ra hoa kt qu. 4. Khi qut v iu ha prokaryote v eukaryote C s khc nhau ng k gia prokaryote v eukaryote trong iu ha biu hin ca gen. Cc t bo eukaryote c cu to phc tp hn nhiu nn c ch iu ha cng phc tp hn prokaryote. prokaryote, mc ch ca s iu ha biu hin gen l nhm iu chnh h enzyme cho ph hp vi cc tc nhn dinh dng v l ha ca mi trng, m bo c hai yu cu chnh ca t bo l sinh trng v sinh sn. S iu ha y rt linh ng v c tnh thun nghch. eukaryote, do t bo khng tip xc trc tip vi mi trng, nn s iu ha y khng cn nhm mc ch i ph vi cc bin ng ngoi bo. S iu ha eukaryote hng n vic chuyn bit ha tng loi t bo vo tng cu trc v chc nng ring v v th khng mang tnh thun nghch. Ba thnh phn chnh ca s iu ha biu hin gen l: 1) Tn hiu gy ra p ng lm thay i biu hin gen; 2) Giai on c thc hin s iu ha trong qu trnh t ti bn n dch m; v 3) C ch phn t ca s iu ha biu hin gen. 4.1. S biu hin ca gen prokaryote B my di truyn ca sinh vt prokaryote l mt DNA mch vng cha mt s lng gen gii hn c phin m trng thi tip xc trc tip vi t bo cht (Hnh 8.1). Chu trnh t bo ngn v khng c s bit ha t bo. V th, hot ng ca cc gen c iu ha do cc nhu cu ca t bo khi cn thit. Tc ng ca cc nhn t mi trng lm nhng gen tng ng c m phin m, dch m tng hp protein hay c hiu qu ngc lm dng li.

Hnh 8.1. S biu hin gen prokaryote

4.2. S biu hin ca gen eukaryote Khc vi prokaryote, nhim sc th ca eukaryote c cu trc phc tp. Ngay trn cu trc nhim sc th c s tham gia ca cc protein histone c vai tr iu ha biu hin ca gen. S iu ha biu hin gen eukaryote phi qua nhiu mc iu ha phc tp hn so vi prokaryote v qua nhiu giai on nh: nhim sc th tho xon, phin m, bin i hu phin m, mRNA ri nhn ra t bo cht, dch m v bin i hu dch m (Hnh 8.2). Ngoi ra, a s eukaryote c c th a bo v mi t bo c biu hin sng khng phi t do, m chu s bit ha theo cc chc nng chuyn bit trong mi quan h hi ha vi c th. Cc vi khun thng phn ng trc tip vi mi trng v biu hin gen thun nghch, nh c ng lactose th m operon phn hy, khi ht ng th operon ng li. Trong khi , cc t bo eukaryote c nhng con ng bit ha khc nhau v s chuyn ha l n nh thng xuyn trong i sng c th. Ngoi s bit ha t bo, cc c th eukaryote a bo cn tri qua qu trnh pht trin c th vi nhiu giai on phc tp ni tip nhau, trong c nhng gen ch biu hin phi v sau th dng hn. Tt c nhng im nu trn cho thy s iu ha biu hin ca gen eukaryote phc tp hn nhiu, m hin nay li c bit t hn prokaryote.

Hnh 8.2. S biu hin gen eukaryote

II. Cc mc iu ha Cc c ch iu ha s biu hin ca gen c th tc ng mt hay nhiu mc khc nhau. S iu ha c th xy ra mc gen bng s kim sot thi gian v tc phin m. Cc c ch khc c th hot ng lc dch m hoc sau dch m. 1. Mc cht nhim sc Ngay trn cht nhim sc c th thc hin cc kiu sau: - DNase ct mt s vng trn genome lm tho xon cc gen biu hin. Hai vng c lu l cc vng nhy cm (sensible) v siu nhy cm (hypersensible).

- Cc vng nhy cm c lin quan n cc gen c hot tnh cao v nhng gen qua biu hin ri (nh cc gen hot ng phi). Cc vng siu nhy cm lin quan n cc gen c hot tnh rt cao (nh cc gen histone). - DNA Z (DNA tri) l dng cu trc siu xon c th lin quan n ng m gen. - Methyl ha cc base. cc prokaryote s methyl ha c th thc hin i vi A v C, cn eukaryote s methyl ha ch thc hin vi C v tr th 5. Methyl ha lm gen ngng hot ng. V d: nhim sc th X bt hot ngi thuc loi siu methyl ha. Ni chung, s thay i cu hnh (reconfiguration) c th nh hng n s biu hin ca gen. 2. Mc phin m y l s iu ha nh hng trc tip n vic m hoc ng ca gen. Kiu iu ha ny thng gp trong iu ha trao i cht, cng nh cc qu trnh bit ha t bo. - S tc ng ca cc trnh t cis (gn k, lin k) nm trn cng mch DNA nh enhancer (vng tng cng) lm tng s phin m. - iu ha bi cc nhn t trans (cch qung, t xa) do cc nhn t khng nm cng trn mt mch DNA. - Chn la promoter thch hp. - S suy yu/suy thoi. 3. Mc hu phin m S iu ha c th biu hin mc tc ng ln mRNA, chng ta gp trng hp trn khi mRNA b ct b cc intron v gn cc exon li vi nhau to thnh mRNA hon chnh (RNA processing). Nh vy, cc h thng nh hng n s hon chnh ca mRNA c th kim tra gin tip biu hin ca gen tng ng. Cc mRNA ca eukaryote cn c nhng on khng m ha lin quan ti thi gian tn ti v ra khi nhn vo t bo cht. - Splicing khc nhau. - im polyadenine ha khc nhau (polyadenylation). - t bin trn phn t mRNA. - Bn chu k phn hy ca mRNA. - S bo tn cc RNA trong t bo. 4. Mc dch m

S bin i ca cc nhn t khi u IF (inititation factor). L cc protein kt hp vi tiu n v ca ribosome vo giai on khi ng ca qu trnh dch m.

5. Mc hu dch m y c s iu ha hot tnh ca protein. Sau khi mch polypeptide c tng hp, cc protein nhiu khi phi tri qua cc bin i th cp trc khi biu hin hot tnh (chc nng). V d: trypsin l enzyme phn gii protein trong d dy ch c c hot tnh sau khi cht tin thn ca n (pro-enzyme khng c hot tnh) b ct mt mt on polypeptide. Cc protein c th chu nhng bin i lp th nh s kt hp cc enzyme vi mt s sn phm c bit c th lm thay i cu trc khng gian ca chng dn n mt hot tnh. - Cc qu trnh glycosylation, phosphorylation tc l gn thm cc nhm cht nh ng, phosphor protein c hot tnh/chc nng sinh hc. - Peptide tn hiu l on gm khong 20 amino acid nm gn pha u N ca polypeptide, c vai tr gn polypeptide v ribosome ang tng hp mch ny vi mng li ni sinh cht. Trong b my Golgi, polypeptide c phng thch ra ngoi. - S phng thch ra protein c chc nng sinh hc t mt phc hp, nh t proinsulin thnh insulin. III. iu ha biu hin gen prokaryote Cc gen c phin m to RNA, c gi l cc gen cu trc. Cc protein c dch m t mRNA c th l enzyme hoc khng phi enzyme. Trong s cc protein khng phi enzyme c cc protein iu ha (regulatory protein), chng tng tc vi cc trnh t DNA c hiu kim sot hot tnh phin m ca cc gen cu trc. Cc gen tng hp cc protein iu ha c gi l cc gen iu ha (regulatory gen). Pha trc mi gen cu trc (hoc mt nhm gen) c mt trnh t promoter, ni RNA polymerase nhn bit (Hnh 8.3). C ch iu ha prokaryote ch yu c thc hin thng qua operon. y l khi nim ch tn ti prokaryote.

Hnh 8.3. Phng thc chung iu ha biu hin gen prokaryote

1. Cu trc ca promoter Thc cht ca khi s phin m l quan h trc tip gia RNA polymerase v promoter. Khi RNA polymerase gn vo promoter, n s phin m to phn t RNA. Phn ln promoter E. coli v cn bn c cng cu trc: Nu base u tin c phin m thnh mRNA (lun l purine, thng l adenine) c nh s +1, th tt c cc base pha 5 hay pha trc so vi n khng c phin m l s tr ( Ngay pha trc +1 c 6 base thng vi trnh t ). TATAAT xung quanh 10, v trnh t TTGACA (trnh t lin ng-consensus sequence) xung quanh C hai trnh t phi hp nhau cho php RNA polymerase 35. gn vo v khi s dch m, trnh t to iu kin u tin cho vic gn vo. 35

2. Cu trc ca operon Operon l n v phin m gm t nht mt promoter v mRNA bc tip theo m ha cho cc trnh t ca mt hay nhiu chui polypeptide. Tuy nhin, operon c th c mt hay nhiu im iu ha khc vi promoter. Cc gen khng chu s iu ha do tc ng mi trng, to sn phm thng xuyn, c gi l cc gen cu trc. S lng sn phm ca cc gen ny c th dao ng ph thuc vo i lc tng

i ca cc promoter ca chng i vi RNA polymerase. Cc promoter c i lc mnh (strong promoter) to ra nhiu sn phm ca gen hn cc promoter c i lc yu. Cc gen m sn phm protein ca chng c tng hp p li vi cc nhn t mi trng, thng c iu khin bi mt hay nhiu protein iu ha. Trnh t DNA bn trong operon, ni m protein c ch gn vo, c gi l operator (im iu hnh). Vic gn protein c ch ln operator ngn cn s phin m ca tt c cc gen cu trc trn cng mt operon. S kim sot nh vy i vi vi gen gi l kim sot m. Cc operon ca vi khun thng to ra cc mRNA a gen, nhng mRNA ca eukaryote ch mt gen. Cc protein cn thit cho biu hin gen c gi l cht hot ha. Chng c th gn vi cc im khi s nm bn trong ca promoter ca operon hay im tng cng hoc c th gn nhng trnh t xa operon. Vic gn ca protein iu ha vo im khi u (initiator) hay enhancer, kch thch s phin m ca cc gen cu trc, c gi l c ch kim sot dng. S kch thch cc gen iu ha phn ng c th l t cc phn t tng i nh nh ng, amino acid n cc phn t ln hn nh cc phc hp hormone steroid v cc protein th th (receptor). Cht lm cho gen phin m c gi l cht cm ng, c tc ng ngc vi cht km hm. Cc gen cm ng thng tham gia vo cc phn ng thoi dng (catabolic reaction), nh phn hy cc polysaccharide thnh ng n. Cc gen c ch thng tham gia vo cc phn ng bin dng thc hin vic tng hp cc cht nh amino acid t cc tin cht n gin hn. 3. iu ha thoi dng: Kim sot m-cm ng Trong thoi dng, cc cht thc n c phn hy d dng to nng lng hoc cc cht cn thit cho qu trnh tng hp. C ch iu ha y l s c mt ca c cht (v d lactose) dn ti tng hp cc enzyme phn hy. V d in hnh cho trng hp ny l operon lactose ca E. coli. -galactosidase l enzyme c chc nng i. Chc nng u tin ca n l thoi dng lactose thnh glucose v galactose. Chc nng th hai ca n l chuyn lin kt 1-4 ca glucose v galactose thnh lin kt 1-5 ca allolactose. Bnh thng enzyme ny khng hin din nng cao trong t bo, khi vng mt lactose trong mi trng. Ngay sau khi cho lactose vo mi trng nui khi khng c glucose, enzyme ny bt u c to ra. S vn chuyn lactose xuyn qua mng t bo c hiu qu nh protein vn chuyn galactoside permease. Protein cng xut hin vi nng cao khi c lactose trong mi trng. S iu ha ca operon lactose cn ph thuc vo nng glucose trong mi trng. Nng glucose ny li kim sot nng bn trong t bo ca phn t nh cAMP (cyclic adenosine monophosphate), l cht bt ngun t ATP v lm tn hiu bo ng cho t bo. T bo c xu hng s dng glucose hn l lactose lm ngun

carbon v glucose c bin dng trc tip cung cp carbon v to nng lng. Cc enzyme bin dng glucose thuc loi cu trc v t bo tng trng ti a vi ngun glucose. Khi ngun glucose cn, t bo phn ng li bng cch to ra c-AMP. Vic tng nng c-AMP trong t bo gy nn hng lot s kin, trong s hin din ca lactose, dn n s phin m cc gen cu trc ca operon lactose. 3.1. Cu trc ca operon lactose H thng lactose (lactose system) bnh thng (Hnh 8.4) gm c gen iu ha (i hoc R) v operon mang trnh t promoter (P) locus operator (O) v ba gen cu trc cho -galactosidase (Z), permease (Y) v transacetylase (A). Nhiu t bin cc locus ny c pht hin. 3.2. Hot ng ca h thng - iu kin cm ng (c lactose). Lactose c chuyn vo t bo rt yu v ch c vi phn t permease lm vic. Khi vo trong t bo, mt s lactose (lin kt 1,4) c chuyn thnh allolactose (lin kt -1,6) nh -galactosidase. Allolactose l cht cm ng, n gn vo protein km hm v gy bin i cu hnh to phc hp allolactose-repressor. Phc hp ny mt kh nng gn operator. Lc ny operon c m, RNA polymerase bt u phin m cc gen cu trc. Ton b s kin din ra nh trn hnh 8.4b. - iu kin khng cm ng (khng c lactose). Gen iu ha ca operon thng xuyn tng hp protein km hm (repressor protein) mc thp, v n c promoter t hiu qu. S tng hp cc protein ny b tc ng do nng lactose trong t bo. Ngc li, promoter bnh thng ca operon lac gn vi RNA polymerase rt c hiu qu. Khi khng c ng lactose, protein iu ha hot ng (active regulator protein) cn gi l protein km hm gn vo promoter hay c trnh t operator v protein km hm chim on ny. Nh vy, s phin m ca tt c cc gen cu trc ca operon lac b dng (Hnh 8.4a). Do s lng permease tng, nn lactose vo t bo vi s lng ln v c phn hy bi -galactosidase. Khi lactose c s dng ht, cc protein km hm gn tr li vo operator lm operon b ng; s phin m cc gen cu trc b dng. Bn thn gen iu ha lacI ch c mt promoter (Pi) v gen cu trc ca protein km hm. Promoter ny yu, khi cc protein km hm c s lng ln, n b cc protein ny gn vo lm dng phin m.

Hnh 8.4. Operon lactose v hot ng ca n

4. iu ha bin dng: Kim sot m-c ch Bin dng (anabolism) l qu trnh tng hp nn cc cht cn thit cho t bo. V d tng hp cc amino acid. Qu trnh tng hp tryptophan bt u t tin cht tryptophan l chorismic acid, tri qua 5 giai on k tip do enzyme xc tc. H thng tng hp amino acid tryptophan E. coli l v d in hnh v operon b km hm do s kim sot m. 4.1. Cu trc v hot ng H thng tryptophan cng c cu trc tng t h thng lactose gm gen iu ha trpR v operon tryptophan (promoter, operator v 5 gen cu trc). Cc gen cu trc xc nh 5 enzyme c xp theo th t tng ng vi chc nng xc tc theo trnh t cc phn ng ca chui bin dng tryptophan (Hnh 8.5).

Hnh 8.5. Operon tryptophan

S khc nhau cn bn vi h thng lactose l gen iu ha. Gen iu ha ca h thng tryptophan tng hp thng xuyn aporepressor protein, l cht km hm m ring n khng c hot tnh. Khi tryptophan d tha n tr thnh cht corepressor (ng km hm) v kt hp vi aporepressor thnh phc hp km hm (holorepressor) c hot tnh. Phc hp ny gn vo operator ca operon tryptophan (trp) lm dng phin m cc gen cu trc. Khi nng tryptophan thp, n tch khi phc hp km hm v aporepressor mt hot tnh. Lc ny cc operator li c m v RNA polymerase dch m 5 gen cu trc tng hp 5 enzyme to tryptophan (Hnh 8.5). S iu ha kiu ny cn gi l iu ha c ch ngc (retro-inhibition) do sn phm cui cng c mi lin h ngc (feed-back). Nh vy, hot ng ca h thng ny ngc li vi h thng lactose: khi c tryptophan th operon b ng, thiu tryptophan th gen c m. 4.2. S suy yu (attenuation)

Kiu iu ha th hai c pht hin operon tryptophan c gi l s suy yu. u 5 ca mRNA a gen (polycistronic) ca operon ny c 5 enzyme. on ny c gi l trnh t leader (trnh t ch huy). Mt phn ca trnh t ny c phin m to leader peptide gm 14 amino acid, m chc nng n nay cha r. C ch kim tra m s tng hp mRNA c bt u nhng kt thc sm vi chiu di mRNA ngn hn c gi l s suy yu. V d: cc t bo E. coli ang tng trng trong mi trng thiu mt amino acid no , nhng cha cc enzyme cn c cho s tng hp ca tt c 20 amino acid cn thit to ra protein. Khi thm mt amino acid (tryptophan) vo mi trng s lm gim ng k s tng hp ca cc enzyme cn cho s to amino acid . Phn ng ny c tnh thch ng v duy tr cc ngun enzyme khng cn cn na khi sn phm cui cng ca chui sinh tng hp (tryptophan) ang c trong t bo (c ch ngc). Operon tryptophan c phng thc iu ha hot ng gen th hai, hon ton c lp vi h thng repressor-operator (cht km hm on iu hnh). operon tryptophan, s tng hp mRNA bt u t 161 base trc codon khi s ca trpE, enzyme cu trc u tin c phin m. t bin do mt on DNA ngay pha trc trpE, gii phng trpE khi s km hm ca phc hp represser-operator. Cc t bin ny lm tng mc biu hin ca c operon ln gp su ln. Tinh sch cc DNA c phin m in vitro cho thy phn ln mRNA kt thc ngay base 139 v khng bao gi t ti trpE. y, c s km hm phin m ngay trc gen cu trc v khi mt n s biu hin ca gen c hiu qu hn. on di ca mRNA nm trc trpE c gi l leader (trpL) v on km hm phin m c gi l attenuator (trpa, ch khng phi trpA). Cui cng, quan st cho thy s suy yu dao ng theo nng ca tryptophan thp, nhiu phn t mRNA c to ra hn qua attenuator v ton b operon c phin m. S suy yu l mt phng thc khc kim sot s biu hin ca gen.

Hnh 8.6. S suy yu xy ra khi cu trc th cp c bit to thnh trong mRNA. mRNA c trnh by y l t trnh t leader ca operon trp. Nu tryptophan trong t bo phong ph v trnh t leader c phin dch nhanh, th mRNA s to thnh cu trc thn v quai (stem-and-loop) hot ng nh l mt tn hiu kt thc phin m.

C ch suy yu thc hin s kt hp gia phin m v dch m. RNA polymerase bt u phin m cc gen m ha cho cc enzyme sinh tng hp cn thit to ra tryptophan. V d: khi hin din tryptophan n nhanh chng t ti im (trn RNA mi c tng hp) gi l attenuator v dng phin m ti y. Attenuator c chc nng nh im kt thc ch khi tryptophan hin din. S phin m dng khi trnh t leader t attenuator. Nu c trytophan trong t bo, s dch m ca cc codon c thc hin, v c phin m v dch m c thc hin hon chnh cho n lc RNA polymerase t ti im attenuator, ni kt thc s phin m. Khi khng c trytophan, dch m dng v phn leader ca mRNA cun li sao cho attenuator khng thc hin chc nng, trong trng hp ny s tng hp ca mRNA ton vn cho operon tryptophan c to ra. Vi s hin din ca cc enzyme , trytophan c tch ly vi s lng ln trong t bo. Attenuator l dng tinh t ca s c ch, iu ha hot ng gen, to c hiu cho s tng hp amino acid trong t bo. C ch repressor iu ha th h thng tryptophan, trong khi h thng c ch attenuation kim sot nng tryptophan mt cch tinh t. S suy yu ca operon Trp cng nhy cm vi nng ca mt s amino acid nh histidine v leucine. 5. Kim sot dng v cm ng C ch iu ha dng, cm ng c tm thy operon arabinose ca E. coli. Arabinose l cht ng, cn ba enzyme (c m ha bi cc gen araB, araA v araD) cho s bin dng. Hai gen khc nm xa operon gp phn a arabinose vo t bo. Gen iu ha araC nm gn cc gen B, A v D. Sn phm ca gen araC l protein km hm ca operon khi khng c ng arabinose. Tuy nhin, khi c ng arabinose trong t bo, n gn vi repressor (protein araC) hnh thnh nn phc hp activator (hot t) to thun tin cho s phin m ca RNA polymerase. Trn thc t, cc nghin cu cho thy c ch iu ha ny rt phc tp. V d: cAMP v protein hot ha thoi dng (catabolite gene activator protein) cn c gi

l CRP (cyclic AMP receptor protein-protein th nhn cAMP) u tham gia vo s iu ha ca h thng arabinose.

Hnh 8.7. Operon arabinose ca E. coli

IV. iu ha hot tnh ca eukaryote Cc c ch iu ha eukaryote c th xy ra 5-6 mc khc nhau (xem mc II). Trong phn ny ch nhn mnh thm mt s c im ca iu ha hot ng gen eukaryote: - cc operon ca prokaryote, cc gen iu ha v cc promoter thng nm gn nhau, nhng eukaryote cc gen iu ha t khi nm gn cc promoter do chng kim sot. - Cc enhancer l nhng trnh t cng nm trn mt phn t vi cc promoter c th c hng trm cp base pha trc hoc pha sau promoter m chng kch thch. - Trnh t iu ha 5 pha trc c promoter eukaryote thng rt di, c khi hng chc kilobase (kb). - C nhiu kiu iu ha dng cc nhn t c tc ng t xa (trans) l cc protein. S phin m c th c kch thch bi cc tn hiu khc nhau. S iu ha hot ng gen prokaryote phn ln p ng li cc tn hiu bn trong. 1. Cc promoter

Tng t nh vi khun, cc promoter ca eukaryote cng nm pha trc im xut pht ca mRNA v c nhng trnh t c bo tn trong tin ha. Hp TATA, nh hng cho RNA polymerase bt u phin m, nm khong di 30 basepair (bp) ng vt c v, v 60-120 nm men. Hp TATA hot ng c hiu qu cng vi hai trnh t tng ng pha trc khong 40 bp l CCAAT v 110 bp l trnh t giu GC. S thay i hp TATA lm gim tc phin m. Hiu qu ca tc phin m c o bng s thay i ca tng base trong promoter, cc thay i base ngoi hp TATA v cc trnh t pha trc khng gy hiu qu i vi tc phin m, trong khi cc thay i nhng yu t c nu trn lm gim ng k tc phin m. Khc vi promoter ca prokaryote, cc promoter ca eukaryote kh m bo nhn bit cc tn hiu mt cch y RNA polymerase khi s phin m in vitro. Hp TATA v cc trnh t pha trc phi c nhn bit bi cc protein iu ha, v chnh cc protein ny gn vi cc im nht nh trn chng v hot ha s phin m. 2. Cc enhancer Enhancer l cc trnh t c tc ng cis (lin k), chng tng tc phin m ng k t cc promoter nm ngay trn cng mt phn t DNA. Tnh c o ca cc enhancer l ch chng c kh nng thc hin mt cch hiu qu khi mt khong cch rt xa, c khi n vi nghn cp base. Ngoi ra, chng c hot ng chc nng bt k hng no, d pha trc hay pha sau promoter. Xt trn mt gc no , cc enhancer tng t promoter. C th, chng c t chc gm mt dy cc trnh t c tc ng cis nhn bit cc nhn t tc ng t xa (trans) (Hnh 8.8). 3. Cc protein l nhn t c tc ng trans Mt vi protein, nhn bit hp CCAAT, c xc nh t bo ng vt c v. Cc nhn t ny c th c phn bit gia cc n v ca cc phn t CCAAT. Cc hp GC c nhn bit bi cc nhn t ph. Cc v d v cc tc ng trans (trans-acting factor) tm thy nm men v ng vt c v. Cc nhn t trans c c im chung l gm hai vng cu trc v chc nng chnh: - Vng gn nhn t trans vo DNA. - Vng tc ng ln s phin m. ng thi cc nhn t trans c bn kiu tc ng nh: - ngn tay km (zinc-finger) - xon-vng-xon (helix-turn-helix) - xon-nt-xon (helix-loop-helix)

- dy ko leucine (leucine-zipper) (Hnh 8.9).

Hnh 8.8. Enhancer ca DNA eukaryote

c tnh chung ca cc cu trc va k l chng lun lun gn ln cc trnh t cis di dng dimer (gm hai dimer). Trnh t cis v nhn t trans thng c cc tng tc c hiu do nhng lin kt yu hnh thnh gia cc phn t ca nhn t trans vi cc base ca trnh t cis. V d, trng hp tng tc gia cc hormone steroid vi cc enhancer. Nhiu protein c tc ng trans tham gia vo tin trnh pht trin ca c th rui gim, chng c vai tr c bit trong xc nh s sp xp cc on thn ca rui. Nh vy, cc gen eukaryote c hot ha bi hai trnh t DNA c tc ng cis l promoter v enhancer, chng nhn bit c cc nhn t protein c tc ng trans, cc nhn t trans cho php RNA polymerase khi s phin m v t tc phin m ti a.

Hnh 8.9. Cc kiu tc ng ca nhn t trans

4. Hormone V d r nht v cc cht iu ha ni ti ca hot tnh gen l hormone. l nhng cht to ra do mt lot t bo c hiu qu n cc t bo khc. Cc hormone thng c vn chuyn n cc phn ca c th nhng ch tc ng n cc t bo c cc receptor tng ng. S tng tc gia hormone vi receptor gy ra tn hiu tc ng n cc vng c trng ca DNA, gy hot ha gen hoc mt nhm gen tng ng. Cc hormone c th kch thch phin m bi mt trong cc c ch sau: - Hormone c th lm cho DNA tch khi histone v to iu kin cho RNA polymerase bt u phin m. - C th lm cht cm ng (inducer) gy bt hot phn t receptor. - C th gn trc tip vi on DNA c hiu to thun li cho vic gn RNA polymerase hoc protein l nhn t phin m (protein transcription factor).

- C th hot ha effector protein lm thnh phc hp gn DNA v kch thch gn RNA polymerase. - C th gn vi protein to phc hp hot ha kch thch gn RNA polymerase. 5. Kim sot cc cht thng gp trong nhn Mt s nhm phn t hin din rt nhiu trong mi t bo eukaryote, nh cc histone, cc cht ca b my dch m, cc cu phn ca mng Vi chng trnh khc nhau c thc hin duy tr s lng ln ca chng trong t bo. Cc chng trnh gm: - S phin m lin tc v lp li trong t bo. - S lp li cc gen. - S khuch i ngoi nhim sc th cc trnh t gen c hiu.

5.1. S phong ph Cc gen m ha cho rRNA v tRNA c nghin cu k. Phn ng lai n gin DNA-RNA cho thy nhim sc th ca Xenopus c vng t chc hch nhn (nucleolus organizer) cha 450 bn sao ca DNA m ha cho 18S v 28S rRNA. Ngc li, trong mi nhn c 20.000 bn sao ca cc gen m ha cho 5S rRNA v cc gen ny khng nm vng t chc hch nhn. Cc gen m ha cho histone cng c vi nhiu bn sao c lp li hng trm ln. S phong ph ca cc gen nu trn m bo s lng cn thit cho dch m khi phi tng hp hng triu phn t protein vo lc cn thit.

5.2. S khuch i gen Mt v d v s khuch i gen trong nhn l cc ch phnh (puff) ca nhim sc th khng l hay a si (polytene chromosome) tuyn nc bt ca rui gim. cc ch phnh ny, DNA nguyn nhim sc (euchromatic) c khuch i khong vi nghn ln. Trong nhn t bo trng ca Xenopus, c hng trm nhn con ngoi nhim sc th (extrachromosome nucleoli) vi kch thc khc nhau, mi nhn con cha cc vng trn rDNA kch thc khc nhau, m vai tr n nay cha r. Cc DNA vng trn ny sn sinh nhiu rRNA mc hp l.

V. S bit ha t bo 1. Cc t bo bit ha mang thng tin ging nhau cc sinh vt bc cao cng nh ngi, c th trng thnh gm nhiu loi t bo khc nhau. Cc t bo ny u bt ngun t mt hp t ban u, nhng qua qu trnh bit ha tr thnh cc t bo c cc chc nng khc nhau. Tuy nhin, thc nghim xc nh rng s lng nhim sc th, s lng DNA v c t l (A+T)/ (G+C) ca cc t bo thuc cc m khc nhau ca cng mt c th u ging nhau. S dng k thut lai DNA cho thy DNA t nhng t bo ca cc m khc nhau ca cng mt c th khng b bin i trong qu trnh bit ha, chng c th t hi tnh (renaturation) vi nhau. Th nghim ghp nhn t bo rut ch vo t bo trng b hng nhn (do chiu tia t ngoi) cho thy 1% t bo ghp nhn pht trin thnh ch trng thnh. iu ny chng t t bo rut tuy bit ha vn gi nguyn vn thng tin di truyn to ra ch trng thnh. Tm li, s lng DNA ca cc t bo bit ha v cn bn ging vi cc hp t ban u v cha nguyn vn thng tin di truyn pht trin thnh c th nguyn vn.

2. Cc t bo bit ha tng hp cc nhm protein khc nhau hiu c s khc nhau gia cc t bo bit ha, cn xt nhiu vn sau: - Th nht, nhiu qu trnh l chung cho tt c cc t bo nn c nhiu protein ging nhau. Nhng protein ny gm s lng nhiu, d phn tch nh phn ln cc protein cu trc ca thnh t bo v nhim sc th; mt s protein cn bn ca cc bo quan (li ni sinh cht, b my Golgi, cc ribosome). Nhiu protein c s lng khng nhiu nh cc enzyme khc nhau, tham gia vo cc phn ng trung tm ca qu trnh trao i cht, u hin din nh nhau tt c cc kiu t bo. - Th hai, mt s protein c s lng phong ph mt s t bo chuyn ha m vic pht hin chng cn c th nghim ring. V d, hemoglobin ch c th pht hin t bo mu. - Th ba, nu nh hn 2.000 loi protein c s lng di do c so snh gia cc kiu t bo bit ha ca cng mt vi sinh vt vi nhau bng in di hai chiu polyacrylamide, th mt s khc bit ng k s c tm thy. D s so snh gia hai dng t bo nui cy (nh cc dng t bo c v thn kinh) hay gia cc t bo ca cng mt m ca chut (nh gan v phi), i a s cc protein c pht hin u c tng hp c hai loi t bo v vi tc tng hp c th khc nhau n 105; ch c vi trm protein hin din vi s lng khc nhau ng k hai kiu t bo.

Cc nghin cu cho thy cc t bo eukaryote bc cao tng hp t 10.000-20.000 protein khc nhau. Phn ln chng c s lng rt t v kh pht hin. Nu cc protein him (minor protein) ny khc nhau gia cc t bo vi mc tng t cc protein di do, th ch mt s lng nh ca chng cng to nn nhiu khc bit chuyn ha ln v hnh thi v sinh l ca t bo. T bo c th thay i s biu hin gen ca chng p li tn hiu bn ngoi. Phn ln cc t bo chuyn ha ca sinh vt a bo c kh nng thay i phng thc biu hin ca gen p li tc ng bn ngoi. V d: nu t bo b tc ng bi glucocorticoid hormone, th s tng hp mt s protein chuyn bit c tng vt (Hnh 8.10). Glucocorticoid c phng thch ra khi b i bng hay hot ng mnh v bo hiu cho gan tng cng to glucose t amino acid hay cc phn t nh khc nhau. Cc protein, c to ra do cc enzyme cm ng nh tyrosine aminotransferase h tr bin tyrosine thnh glucose. Khi hormone khng cn na, s tng hp cc protein ny gim xung mc bnh thng. Cc loi t bo khc nhau phn ng vi glucocorticoid bng nhiu cch khc nhau. V d t bo m, s tng hp tyrosine transferase gim, trong khi mt s loi t bo khc hon ton khng c phn ng vi glucocorticoid. Cc v d ny minh ha cho tnh cht chung ca bit ha l cc kiu t bo bit ha khc nhau thng phn ng li cng mt tn hiu bn ngoi bng nhiu cch khc nhau. Trn c s , mi kiu t bo bit ha c mt c tnh n nh thng xuyn. Cc tnh cht phn nh s biu hin lu bn ca cc nhm gen khc nhau.

Hnh 8.10. Tc dng ca glucocortcoid lm tng mc biu hin ca gen

Nh vy, cc t bo bit ha ch s dng mt phn thng tin. Nhiu loi t bo chuyn ha tng hp ch yu mt s protein, ngoi cc protein cu trc v protein c bn s dng cho cc qu trnh sinh l bnh thng. V d: t bo c tng hp nhiu myosin, mt protein quan trng trong co c, hay t bo biu b (epithelial) tng hp nhiu keratine. Nh vy, cng nh cha thng tin di truyn ging nhau, nhng mi loi t bo bit ha ch s dng mt phn thng tin: tng hp ch yu mt s loi protein .

3. S iu ha mc phin m l ngun gc cn bn ca cc sai khc gia nhng t bo bit ha Nu nhng s bit ha gia cc kiu t bo khc nhau ph thuc vo cc gen chuyn bit m t bo biu hin, th s kim sot biu hin gen c thc hin mc no? Gi thuyt c chp nhn hin nay l trong cc t bo bit ha mt s gen phin m, cn c cc gen khc th khng. Khng c s kin no mu thun vi gi thuyt ny v n gii thch hp l hn c tnh trng bit ha ca cc t bo. i vi phn ln gen, s kim sot phin m c tm quan trng hng u, v ch n mi m bo cho s tng hp khng d tha cc cht trung gian. Vic pht hin cc gen iu ha v cc gen ng hay m gip hiu c s iu ha qu trnh pht trin c th v bit ha t bo. Genome n bi ca t bo ngi c s lng DNA nhiu hn gp 1.000 ln so vi genome ca vi khun. Tuy nhin, s lng gen cu trc ngi ch ln hn 10 ln s gen cu trc vi khun. iu cho thy nhiu gen ngi tham gia vo cc c ch iu ha. Tm li, genome ca t bo cha cc trnh t nucleotide ca DNA thng tin to ra hng nghn cc protein v phn t RNA khc nhau. T bo ch biu hin in hnh mt nhm gen ca n v cc kiu t bo bit ha khc nhau sinh vt a bo c sn sinh ra do cc nhm gen khc nhau c s biu hin khng ging nhau. Hn th na, cc t bo c th thay i phng thc biu hin cc gen ca chng p li nhng thay i ca mi trng, cc tn hiu t cc t bo khc. Mc d tt c cc bc trong s biu hin ca gen v cn bn u c iu ha, nhng i vi phn ln cc gen vic khi s phin m l im kim sot quan trng nht.

Ti liu tham kho/c thm

1. Phm Thnh H. 2003. Di truyn hc. NXB Gio dc, H Ni.

2. L c Trnh. 2001. Sinh hc phn t ca t bo. NXB Khoa hc v K thut, H Ni. 3. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 4. Karp G. 2002. Cell and Molecular Biology: Concepts and Experiments. 3rd ed. John Wiley & Sons, Inc. New York, USA. 5. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK.

6. Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J. 2004. Molecular Cell Biology. 5th ed. Freeman and Company, New York, USA.
7. Walker JM and Rapley R. 2000. Molecular Biology and Biotechnology. Chapman & Hall Limited, London, UK.

8. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California, USA. 9. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company, New York, USA Chng 9

Cng ngh DNA ti t hp

I. M u Vo nm 1973, mt nhm cc nh khoa hc to ra c th sinh vt u tin vi cc phn t DNA ti t hp. Theo , Cohen (H Stanford, M) v Boyer (H California, M) cng cc cng s a c mt on DNA t mt plasmid ny vo mt plasmid khc, to ra mt plasmid hon ton mi, plasmid ti t hp. Sau , h a plasmid ti t hp vo trong cc t bo E. coli. Trong mt thi gian ngn, cc tc gi ny dng cc phng php ging nhau gn cc gen t hai loi vi khun khc nhau, cng nh chuyn cc gen t ch vo vi khun. Cc th nghim ny nh du mt cuc cch mng v cng quan trng trong lch s nghin cu khoa hc ca nhn loi. Cng ngh DNA ti t hp l mt tp hp cc k thut phn t nh v, phn lp, bin i v nghin cu cc on DNA. Thut ng ti t hp c dng thng xuyn do mc tiu ca n l phi hp DNA t hai ngun xa nhau. V d: cc gen t hai ngun vi khun khc nhau c th c lin kt li, hoc mt gen ngi c th c a vo nhim sc th vi khun. Cng ngh DNA ti t hp (cn gi l cng ngh di truyn, cng ngh gen hay k thut gen) hin nay bao gm mt mng li cc k

thut phn t c dng phn tch, bin i v ti t hp hu nh mi trnh t DNA.

1. Tc ng ca cng ngh DNA ti t hp Cng ngh DNA ti t hp bin i su sc phng thc nghin cu gen. Trc y, thng tin v cu trc v t chc ca gen thu c bng cch kim tra biu hin kiu hnh ca chng, nhng nhng k thut mi to ra kh nng t c cc trnh t nucleotide. Trc y, cc nh di truyn phi ch i s xut hin cc t bin ngu nhin hoc cm ng phn tch hiu qu ca s sai khc di truyn, ngy nay h c th to ra t bin cc im nht nh mt cch chnh xc v xem chng thay i kiu hnh nh th no. Cng ngh DNA ti t hp cung cp cc thng tin mi v cu trc v chc nng ca gen v thay i nhiu khi nim c bn ca di truyn hc. V d: trong khi m di truyn c xem l rt ph bin, th by gi chng ta cn bit rng cc m khng ph bin cng tn ti trong DNA ty th. Trc y, chng ta ngh rng t chc ca cc gen eukaryote ging vi prokaryote, nhng by gi chng ta bit rng nhiu gen eukaryote b gin on bi cc intron. Ngy nay, chng ta bit y hn v cc qu trnh ti bn, phin m, dch m, bin i RNA (RNA processing) v iu ha gen thng qua vic s dng cc k thut ti t hp DNA. Cc k thut ny cng c dng trong nhiu trong nhiu lnh vc khc, bao gm ha sinh hc, vi sinh vt hc, sinh hc pht trin, sinh hc thn kinh, tin ha v sinh thi hc. Cng ngh DNA ti t hp cng c ng dng to ra nhiu sn phm thng mi, chng hn: thuc, hormone, enzyme v cc ging cy trng-vt nui. Mt nn cng nghip hon ton mi, cng nghip cng ngh sinh hc, pht trin chung quanh vic s dng cc k thut ny to ra cc sn phm mi. Trong y hc, cc k thut ti t hp DNA c dng thm d bn cht ca ung th, chn on cc bnh di truyn v nhim trng, sn xut thuc v iu tr cc ri lon di truyn.

2. Lm vic mc phn t K thut gen cho thy mt lot c hi, m ra cc phng thc cn thit (m trc y c th khng c) gn nh l hin nhin. Vn c bn l cc gen c kch thc qu nh v c hng ngn gen trong mi t bo. Thm ch, khi quan st trn knh hin vi mnh nht, th DNA xut hin nh l mt si dy b xu, cc nucleotide ring r khng th thy, v khng c mt du hiu no v cc ng nt vt l ch bt u v kt thc ca mt gen.

 minh ha vn ny, chng ta hy xem xt mt v d c trng v di truyn phn t nh sau: Gi thit rng chng ta mun phn lp mt gen c bit ca ngi v t n vo trong vi khun sn xut mt lng ln cc protein ngi c m ha. Vn u tin l tm c gen mong mun. Genome n bi ca ngi cha khong 3,3 t cp base ca DNA. Gi s gen m chng ta mun phn lp di 3.000 bp. Nh vy, gen ch ca chng ta ch chim mt phn triu ca genome; v th tm kim gen ca chng ta trong mt genome s nh th l kh khn hn rt nhiu so vi vic tm kim mt cy kim trong mt ng c kh. Nhng thm ch, nu chng ta c th nh v gen, th chng ta s tch n ra khi genome nh th no? Khng c forcept nh gp mt mnh DNA n, v cng khng c mt ci ko c hc no nh ct ra khi genome mt on gen ring bit. Nu chng ta thnh cng trong vic nh v v phn lp gen mong mun, th bc tip theo chng ta cn a n vo trong t bo vi khun. Cc on DNA mch thng s b thoi bin nhanh bi vi khun; v th gen phi c chn vo trong mt dng n nh. N cng phi n nh ti bn thnh cng hoc n s khng c phn chia tip khi t bo phn chia. Nu chng ta chuyn gen vo vi khun thnh cng trong mt dng n nh, chng ta vn cn phi m bo rng gen c phin m v dch m. S biu hin ca gen l mt qu trnh phc tp i hi mt s cc trnh t DNA khc nm bn ngoi gen. Tt c nhng trnh t ny phi hin din trong cc hng cc v tr thch hp ca chng sn xut protein. Cui cng, cc phng php c s dng phn lp v chuyn gen c hiu qu v cng thp, trong hng triu t bo c hng ti cho cc phng thc ny, ch c mt t bo c th chn lc thnh cng v biu hin gen ca ngi. V th, chng ta phi tm kim nhiu t bo vi khun pht hin c mt t bo mang DNA ti t hp. Trc y, cc vn ny dng nh l khng vt qua c. Nhng ngy nay, cc k thut phn t c pht trin khc phc chng, v cc gen ngi c chuyn d dng vo cc t bo vi khun v chng s c biu hin tt.

II. Endonuclease hn ch Trong t nhin, cc enzyme endonuclease hn ch (restriction endonuclease, RE), gi tt l enzyme hn ch, hin din trong hu ht cc t bo vi khun ngn cn DNA ngoi lai tip qun b my tng hp protein ca t bo. DNA ca chnh chng s c bo v khi tc dng ca enzyme hn ch nh s c mt ca cc enzyme ni bo c th methyl ha (methylation) cc nucleotide c bit, v th cc nucleotide ny khng c nhn bit bi cc enzyme hn ch.

Vic pht hin ra cc enzyme hn ch ca vi khun ct DNA nhng trnh t c bit, gip cho vic thao tc gen d dng hn, do n c th gim chiu di ca cc phn t DNA thnh mt tp hp bao gm cc on ngn hn. Hin nay, cac enzyme han ch c phn lp t cc sinh vt prokaryote. Mi enzyme hn ch ch nhn bit v ct mt trnh t DNA c bit thng cha bn hoc su cp nucleotide. V d enzyme EcoRI tch chit t E. coli ct trnh t GAATTC, enzyme BalI ca Brevibacterium albidum ct trnh t TGGCCA. C hn 900 enzyme hn ch khc nhau c tinh sch t khong 250 chng vi sinh vt. Cc enzyme hn ch ct cc phn t DNA si i theo hai cch khc nhau (Hnh 9.1):

Hnh 9.1. Hai kiu ct ca enzyme hn ch. (a) to ra u so le, v (b) to ra u bng.

- Ct trn mt ng thng i xng to ra cc phn t u bng (u th). - Ct trn nhng v tr nm i xng quanh mt ng thng i xng to ra nhng phn t u so le (u dnh). V mt enzyme hn ch ch nhn bit mt trnh t duy nht, cho nn s v tr ct trn mt phn t DNA c bit thng l nh. Cc on DNA c ct bi enzyme hn ch c th c phn tch theo kch thc bng in di agarose gel nghin cu. Do s tng t ca t chc phn t trong tt c cc c th, cho nn DNA vi khun, DNA thc vt v DNA ng vt c v tng hp nhau v cu trc. V th, mt on DNA t mt dng sng ny c th d dng c pha trn vi DNA ca

mt dng sng khc. S tng t ny cng ph hp i vi plasmid, nhn t di truyn ngoi nhn c tm thy trong nhiu loi vi khun khc nhau. Chng l nhng phn t DNA mch vng ng si i c dng lm vector mang cc on DNA ngoi lai dng trong k thut ti t hp DNA. Ging nh EcoRI, nhiu enzyme hn ch a tao ra cac oan DNA vi u 5 li (protruding). Mt s enzyme hn ch (v d: PstI) a tao ra cac oan DNA co u 3 li. Mt s enzyme hn ch khac (v du: BalI) ct truc i xng tao ra cac oan DNA mang u bng (blunt) (Bang 9.1).
Bang 9.1. Trinh t nhn bit cua mt s enzyme han ch

1. Gn cac u bi ct bi enzyme hn ch

C hai kiu gn khc nhau: gn u bng v gn u dnh, bng cch dung enzyme DNA ligase cua bacteriophage T4 co th gn cac u bng hoc u dinh li vi nhau, tuy nhin trng hp u bng thng cho hiu qua thp hn u dnh. V d: Hnh 9.2 minh ha vic gn cc u dnh c ct bng enzyme HindIII. 2. Isochizomer Noi chung, cac RE khac nhau nhn bit cac trnh t khac nhau (Bang 9.1), tuy nhin cung co mt s trng hp cho thy co nhng enzyme c phn lp t nhiu ngun khac nhau nhng lai ct trong mt trnh t, cac enzyme o c goi la isochizomer. Hn na, mt vai enzyme nhn bit cac chui tetranucleotide ma trong mt s trng hp cac tetranucleotide nay lai xut hin trong cac chui hexanucleotide cua cac enzyme khac. Vi du: - MboI va Sau3A nhn bit trnh t:

Hnh 9.2. Gn cc u dinh

- Trong khi o BamHI nhn bit trnh t:

Trong mt vai trng hp cac oan c tao ra nh mt enzyme hn ch nay khi gn vi cac oan c tao ra nh mt enzyme hn ch khac a hinh thanh cac th lai ma cac enzyme b me khng th nhn bit c. Vi du: SalI ct trnh t (G TCGAC) va XhoI ct trnh t (CTCGAG), cac trnh t c sinh ra nay a gn vi nhau tao thanh th lai (hybrid) ma cac vi tri ct han ch cua chung khng th nhn bit bi cac enzyme SalI va XhoI:

III. Phng thc to dng Cc phng thc c bn ca k thut DNA ti t hp l: (1) Gn mt on DNA vo mt phn t DNA (nh l vector) c th ti bn, v (2) cung cp mt mi trng cho php sao chp phn t DNA c gn. C ba nhm vector c dng ph bin to dng cc on DNA ngoi lai v ti bn (sao chp) trong E. coli; l plasmid, bacteriophage v cosmid. Tt c nhng vector ny phi c mt s tnh cht cn thit sau: - Chng c kh nng t ti bn trong E. coli. - Mang cc gen ch th chn lc d dng phn bit v tinh sch vector ca th ti t hp vi cc dng khc. - Chng c cc vng DNA khng cn thit cho s sinh sn trong vi khun, v th DNA ngoi lai c th c a vo trong cc vng ny. - Chng c th bin np vo t bo vt ch mt cch d dng.

1. Plasmid vector Plasmid la cac thng tin di truyn ngoai nhn c trong nhiu loai vi khun khac nhau. Chung la nhng phn t DNA mach vong, si i, kich thc t 1- 200 kb, co kha nng t sao chep tn tai c lp trong t bao. Cac plasmid co th mang mt s gen cua vi khun va cac gen nay co th biu hin ra protein.

DNA ca plasmid c th c phn lp t nui cy vi khun cha plasmid bng cch b sung cht ty (nh l sodium dodecyl sulfate-SDS) v ly tm s sinh tan (lysate)1. Phc hp nhim sc th vi khun, ln hn plasmid nhiu, s lng xung y ca tube ly tm, plasmid siu xon v cc on nhim sc th mch thng gi li trong th ni. Plasmid siu xon mt ln na c phn tch bng ly tm sau khi x l vi CsCl v EtBr. Plasmid mang cc gen m ha cho cc enzyme thng c li cho vi khun vt ch. Cc plasmid c th mang cc kiu hnh khc nhau nh: khng khng sinh, sn xut khng sinh, phn hy cc hp cht hu c phc tp, sn xut cc enzyme hn ch v enzyme bin i (modification enzymes). Cc plasmid c th c chuyn vo trong vi khun sau khi vi khun c x l t bo c th cho thm qua nht thi i vi cc phn t DNA nh. Qu trnh ny c bit nh l s bin np (transformation). Vi khun c bin np thnh cng c th c chn lc da trn kiu hnh mi m chng nhn c t plasmid, chng hn kh nng khng cc khng sinh. Mt s plasmid hin din trong t bo c s bn sao thp, mt hoc mt vi bn sao trn t bo, do DNA ca plasmid ch sao chp mt hoc hai ln trc khi t bo phn chia. Tuy nhin, cc plasmid khc tn ti mt s bn sao ln hn (10 ti 100 bn sao trn mt t bo) do DNA ti bn lp li cho n khi t c s bn sao thch hp. Cc plasmid c s bn sao ln c gi l plasmid dng xon lng lo (relaxed plasmid), v y l mt trong nhng tnh cht hu ch ca vector to dng. Hnh 9.3 trnh by mt trong cc plasmid vector th h th hai dng xon lng lo, pBR322, di 4.363 bp, vector ny cha hai gen khng khng sinh l ampicillin (Amp) v tetracycline (Tet). S th t ca cc nucleotide trn vector c bt u vi v tr EcoRI n: T u tin trong chui GAATTC c quy c l nucleotide th nht. Cc s th t sau c tip tc quanh phn t vector theo hng t gen khng tetracycline ti gen khng ampicillin.

Hinh 9.3. Plasmid vector pBR322. Apr (hay Ampr) v Tetr: gen khng ampicillin v tetracycline, ori: trnh t khi u sao chp, v mt s v tr nhn bit cho cc RE.

Hnh 9.4. trnh by mt loi plasmid vector th h th ba l pUC19, y l loi vector to dng c trng, N mang vng to dng (multiple cloning sites) hay cn gi l vng a ni (polylinker), vng khi u sao chp (ori), v hai gen ch th (gen khng ampicillin v gen lacZ). Ampicillin l loi khng sinh git cht t bo vi khun, nhng nhng vi khun no cha vector pUC19 s khng li loi khng sinh ny. Gen lacZ m ha enzyme -galactosidase, bnh thng enzyme ny ct lactose sn xut ra glucose v galactose. Enzyme ny cng ct X-gal to ra mt c cht mu xanh; khi X-gal c b sung vo mi trng, cc khun lc vi khun cha pUC19 s c mu xanh v d dng nhn bit. Vng polylinker ca vector pUC19 l tp hp mt s v tr nhn bit n ca cc enzyme hn ch cho php gn on DNA ngoi lai vo plasmid.

Hnh 9.4. Plasmid vector to dng c trng pUC19. Mang cc v tr ct hn ch n trong vng to dng, vng khi u sao chp (ori), v hai gen ch th (gen Apr v gen lacZ).

Plasmid c th c ct mt v tr xc nh bng enzyme hn ch. V th, cc on c to ra c th to vng bng cch kt hp cc u dnh b sung. Hn na, cc on c to ra bi mt enzyme c bit hot ng trn mt phn t DNA s c u tng ng (u dnh) vi on c to ra bi cng mt enzyme hot ng trn mt phn t DNA khc. V th, cc on t hai phn t DNA khc nhau, t hai c th khc nhau c th kt hp bng cc lin kt hydrogen thun nghch khi cc on ny c trn li. Nu s kt hp c gn li sau khi bt cp, th cc on c kt hp c nh bn vng, s kt hp ca cc on ny c thc hin nh enzyme DNA ligase (cn gi l polynucleotide ligase) lin kt cng ha tr cc phn t ti t hp bng cch to ra lin kt phosphodiester gia nhm 5-PO4 ca polynucleotide ny vi nhm 3OH ca polynucleotide khc.

Hnh 9.5. Phng thc c bn to dng gen trong vi khun E. coli Hnh 9.5 trnh by ton b phng thc sn xut DNA ti t hp (to dng gen). Plasmid c ct cc v tr xc nh bng enzyme hn ch. DNA ca mt genome ngoi lai c ct bi cng mt loi enzyme, mt s on trong c th c gen quan tm. Plasmid v cc on ca genome c phi trn v kt hp nh enzyme DNA ligase. Cc plasmid ti t hp c bin np vo vi khun bng ng nui cy plasmid v vi khun. 2. Bacteriophage vector Cc bacteriophage c nhiu u im khi c s dng lm vector to dng. Vector dc s dng rng ri nht l bacteriophage gy nhim vo t bo E. coli. Mt trong nhng u im chnh ca phage l c hiu qu chuyn DNA vo trong t bo vi khun cao. u im th hai l mt phn ba ca genome phage l khng cn thit cho s xm nhim v sinh sn ca n trong t bo vt ch, khng c nhng gen ny, mt tiu th vn chuyn thnh cng DNA ca n vo vi khun v sinh sn c. Cc gen khng cn thit ny, khong >15 kb, c th c thay th bng mt

on DNA ngoi lai khong 15-23 kb. u im th ba l DNA s khng b ng gi trong v tr khi n di t 40-50 kb; v th cc on DNA ngoi lai khng bao gi c chuyn vo t bo tr khi chng c chn vo trong genome phage , iu kin cn thit m bo on DNA ngoi lai s c ti bn sau khi n vo trong t bo vt ch. Cc gen cn thit ca genome phage c nh v trong mt cm. Cc chng ca phage , c gi l vector thay th, c bin i di truyn vi mt v tr RE duy nht cho EcoRI (chng phage th h mi EMBL 3 c ba v tr cho cc RE: EcoRI, BamHI v SalI) trn mt mt khc ca cc gen khng cn thit (Hnh 9.6). V th, c th loi b cc gen khng cn thit bng EcoRI. DNA ngoi lai c ct bng EcoRI s c u dnh b sung cho u ca cc on DNA ca phage cn thit (nhnh tri v phi), c th c kt ni bng DNA ligase. Genome ca phage c cc on si n ngn c gi l cc v tr cos cn cho s ng gi DNA trong u ca phage. Genome ti t hp ca phage sau c th c ng gi trong v protein v c b sung vo trong E. coli. Cc phage gy ra s xm nhim DNA ti t hp ca chng vo trong t bo vt ch, ni n s c ti bn. Ch cc on DNA c kch thc thch hp v mang cc gen cn thit c ng gi trong cc v ca phage, cung cp mt h thng chn lc t ng cho cc vector ti t hp.

Hnh 9.6. Bacteriophage l mt vector to dng hiu qu 3. Cosmid vector Cc vector phage ch c th mang cc on DNA c kch thc khong 15-23 kb. Tuy nhin, cc cosmid vector li mang c cc on DNA c kch thc ln hn nhiu, khong 45 kb.

Hnh 9.7. To dng trong cosmid. Hai v tr cos gn v tr ct hn ch ScaI v BamHI. DNA ngun b ct bi BamHI phn on c kch thc khong 45 kb. Phn t plasmid DNA c ct bi BamHI v ScaI. Hai mu DNA ny c trn vo nhau v c gn bng T4 DNA ligase. Sau khi gn xong, nhng phn t ny c ng gi trong phn u ca phage , v nhng phn t c th ly nhim s c hnh thnh sau khi to ui. Cosmid l cc plasmid nh mang cc v tr cos ca phage; chng c th c ng gi trong v virus v c chuyn vo vi khun nh s xm nhim ca virus. Do tt c cc gen virus, ngoi tr cc v tr cos, l khng c, nn cosmid c th mang cc on DNA ngoi lai ln hn hai ln cc on m phage vector c th mang. Cc

cosmid vector c cc thnh phn sau: (1) mt im khi u sao chp ca plasmid (ori); (2) mt s cc v tr ct hn ch n; (3) mt hoc hn cc gen ch th chn lc; v (4) cc v tr cos cho php ng gi DNA trong u ca phage. DNA ngoi lai c chn vo trong cosmid trong cng mt phng thc vi plasmid: cosmid v DNA ngoi lai u c ct bi cng mt RE to ra cc u b sung (u dnh), v chng c lin kt vi nhau bng DNA ligase. Cc cosmid ti t hp c hp nht trong v, v cc tiu th phage c s dng xm nhim vo t bo vi khun, cosmid s ti bn nh mt plasmid. Bng 9.2 so snh cc tnh cht ca cc vector plasmid, phage v cosmid.
Bng 9.2. So snh cc vector plasmid, phage v cosmid

4. Th vin cDNA Th vin cDNA (complementary DNA) l tp hp cc on DNA b sung (cDNA) c tng hp t mRNA ca mt b phn trong c th sinh vt. S dung th vin cDNA co hai u im sau: - Cac dong cDNA cha trinh t ma ha lin tuc cua mt gen. Nhiu gen eukaryote la gian oan, co cha nhiu oan intron. Sau khi ct mRNA tin thn (pre mRNA) va ni lai, cac oan intron a bi loai va mRNA hoan chnh (mature mRNA) co trinh t ma ha lin tuc c tao thanh. Do cDNA c tao thanh t khun mu mRNA hoan chnh nn cac dong cDNA co th tng hp protein cn thit vi s lng ln nh mong mun. - Nhiu protein c tng hp vi s lng ln do nhng t bao chuyn ha va nh vy trong cac t bao nay mRNA cua protein o se co ty l cao va th vin cDNA c tao ra t cac t bao nay se co nhiu cDNA ma ha cho cac protein tng ng. S di dao cDNA ca mt vai loai nao o lam giam nhe ang k vic xac inh ung dong mong mun t th vin gen. Phng phap tng hp gen t mRNA ngay cang c phat trin, no kt hp vi cac phng phap khac cua sinh hoc phn t cho phep ng dung trong nhiu linh vc. Xy dng mt th vin cDNA bao gm nm bc chnh sau: - Tinh sch mRNA t RNA tng s ca mt phn c th sinh vt.

- Tng hp si cDNA th nht t khun mu mRNA nh enzyme phin m ngc (reverse transcriptase). - Gy bin tinh (un si) th lai mRNA-cDNA ph hy si mRNA bng RNase H ca E. coli. Tng hp si cDNA th hai t khun mu si cDNA th nht nh enzyme DNA polymerase vi primer l vng cp tc ca n thu c phn t cDNA si i. - Ct vng cp tc bng enzyme nuclease S1 v dng enzyme Klenow sa cha hai u ca si i cDNA to ra u bng. - Gn cc on ni (linker) vo hai u ca cDNA si i trc khi to dng trong vector thch hp xy dng th vin cDNA. 5. Th vin genomic DNA Th vin genomic DNA la mt tp hp cac oan DNA cung ai din cho mt genome nguyn ven (hoc gn nguyn ven) cua ca th ma DNA c bt ngun t o. Cc th vin c dung chu yu hoc sang loc theo cac phng phap khac nhau nhm phn lp mt (hoc cac) trnh t nucleotide quan tm c bit, hoc xac inh vi tri va th t cua cac trnh t trong genome ma t o th vin c xy dng (physical mapping). Xy dng mt th vin genomic DNA bao gm bn bc chinh: Ct DNA, gn DNA vao vector, ong goi cac dong tai t hp trong v protein cua virus lam bc trung gian trc khi xm nhp vao t bao vt chu, va chuyn cu truc o vao t bao vt chu. Th vin genomic DNA c nhiu ng dng, chng hn lp bn vt l (physical mapping) ca DNA v xc nh cc gen gy bnh hoc cc chui DNA quan tm cho nhng phn tch khc. Sn xut ra cc dng mang cc on chn DNA khc nhau nhng gi ln nhau c nhiu thun li v th vin c th c s dng trong qu trnh chromosome walking. Chromosome walking thng c thc hin vi th vin ca cosmid, phage hoc YAC2. Cc th vin genomic DNA cng cn thit cho vic xc nh cc gen gy bnh bng cch to dng chc nng (functional cloning). Theo hng ny, thng tin v chc nng ca gen c khai thc phn lp gen mong mun t th vin. Mt oligonucleotide c trnh t da trn chui amino acid tng phn c dng nh l mt probe (mu d) phn lp dng cDNA bng cch sng lc th vin cDNA. Dng cDNA ny sau c th c dng sng lc th vin genome nhm phn lp cc dng genomic DNA v cho php kho st c im ca chui genomic hon chnh. IV. Biu hin gen ngoi lai trong vi khun

V mt l thuyt, k thut DNA ti t hp cho php a bt k mt gen no t mt sinh vt ny vo mt sinh vt khc. Vn quan trng l lm sao gen ngoi lai c th biu hin trong c th vt ch. biu hin tt c cc gen ngoi lai trong E. coli phi bt u bng vic gn on gen ngoi lai vo trong vector biu hin (thng l plasmid). Cac vector biu hin l vector c th mang cac gen ngoi lai mong mun cho php thc hin s phin ma cua cac ban sao c tao dong va s dich ma cac mRNA cua chung trong E. coli. Vector ny phi c cc cu trc cn thit sau: - Cc trnh t m ha gen ch th (marker) m bo duy tr vector trong t bo. - Mt promoter kim sot phin m (v d: lac, trp hoc tac) cho php sn xut mt lng ln mRNA t cc gen c to dng. - Cc trnh t kim sot dch m nh vng lin kt ribosome c b tr thch hp v codon khi u AUG. - Mt polylinker a gen ngoi lai vo trong mt hng chnh xc vi promoter. Ch khi c cu trc y nh th, cc vector biu hin mang gen ngoi lai mi c bin np vo chng E. coli thch hp. Nu on gen ngoi lai khng nm gia promoter v v tr kt thc phin m, th n s khng c phin m.

1. Cc protein nguyn th ti t hp Cc protein nguyn th (native protein) c th c sn xut trong E. coli bng cch s dng promoter mnh v mt vng lin kt ribosome (ribosome binding sitesRBS) hiu qu. biu hin gen prokaryote c RBS mnh ch cn cung cp mt promoter l . Trong khi , biu hin mt gen eukaryote (hoc mt gen prokaryote vi mt RBS yu) cn phi cung cp c promoter ln RBS.

1.1. Biu hin ca gen prokaryote-Promoter Bc u tin khi biu hin cc protein ca eukaryote trong vi khun l chn mt vector biu hin mang promoter mnh ca prokaryote. Cac promoter thch hp nht cho biu hin cua gen ngoai lai E. coli la nhng promoter c kha nng iu chinh manh. in hnh l promoter (lai) trp-lac. Promoter trp-lac cn gi l promoter tac (mt dng promoter lai gia promoter trp v promoter lac) c s dng thnh cng sn xut mt lng ln protein trong E. coli (Hnh 9.8). Promoter trp c iu chinh bi gen c ch trp va co th c cam ng bi s b sung cua 3-indolylacetic acid (3-IAA) vao mi trng hoc bng cach thiu tryptophan. Trong khi , promoter lac c iu chinh bi gen c ch lac va vi th co th c cam ng nh b sung nhn t cam ng isopropyl--D-thiogalactoside (IPTG) vo mi trng nui cy vi khun. Cui cung, promoter (lai) trp-lac mang oan

trp-35 c gn vi oan lac-10 va operator lac c iu chinh bi gen c ch lac (lac repressor).

1.2. Biu hin ca gen eukaryote-Promoter v vng lin kt ribosome co mc biu hin cao cua gen ngoi lai trong E. coli, khng nhng phai s dung cac promoter manh san xut mt lng ln mRNA ma con s dung cac RBS am bao chc chn mRNA c dich ma mt cach co hiu qua. Trong E. coli, RBS bao gm mt ma khi u AUG va mt trnh t nucleotide ngc hng t codon khi u. Trnh t nay c goi la chui Shine-Dalgarno (SD) giu A-G, b sung cho u 3 cua 16S rRNA cua E. coli. Lin kt cua ribosome vi mRNA c khi u bng cch bt cp gia chui SD trong mRNA va trnh t u 3 cua 16S rRNA.

Hnh 9.8. Vector pKK177-3. pKK177-3 l mt tac vector cha vng to dng gen ngoi lai cng hng vi promoter tac. Cng hng vi vng ny l rrnB mang gen 5S ca E. coli v hai nhn t kt thc phin m T1 v T2.

Hiu sut dich ma cua mRNA chiu s chi phi cua mt s yu t nh sau : - Mc b sung gia chui SD va u 3 cua 16S rRNA. - Khng gian va kha nng chui DNA nm gia chui SD va codon AUG.

- Nucleotide theo sau AUG anh hng s lin kt ribosome. a codon ATG vo trong gen c tao dong va duy tri RBS cua vi khun bng cach dung vector pAS1 (Hnh 9.9). Vector pAS1 mang promoter PL v RBS ca gen cII ca bacteriophage , codon khi u ATG c dung hp trc tip vi phn ma hoa u amino cua gen eukaryote mun biu hin. Cch lm ny co th hn ch vic ti u khoang cach gia chui SD ca vi khun va codon khi u ATG cua gen eukaryote co c s biu hin hiu qua cua gen. oan DNA c gn promoter va chui SD sau o c bin np vo cc chng E. coli thch hp. Sng lc th bin np thu cc khun lc c mc phin m cao.

Hnh 9.9. Vector pAS1. Vector pAS1 l mt plasmid di khong 5,8 kb mang promoter PL ca bacteriophage v v tr ct hn ch duy nht BamHI nh v codon khi u ATG ca gen cII ca bacteriophage . 2. Cc protein dung hp ti t hp 2.1. Protein dung hp Protein dung hp cn gi l protein lai c m ha bi mt gen lai (fusion gene) do s dung hp in vitro cc on gen khc nhau. V vy, protein dung hp s mang trnh t amino acid ca hai protein khc bit. Cc protein dung hp c xy dng cho

cc mc ch khc nhau. S dung hp gen c thc hin bng cch gn phn m ha ca gen c to dng gn u tn cng 3 ca gen lacZ. Protein dung hp c nhng u im chnh sau: - Protein dung hp thng c sn xut vi hm lng ln do s khi u phin m v dch m c iu khin bi cc trnh t tiu chun ca E. coli. - Dung hp cc trnh t ngoi lai vi cc gen ca E. coli thng cho kt qu cc sn phm n nh hn cc protein ngoi lai nguyn th. 2.2. Vector biu hin cc gen dung hp vi gen lacZ Mt s h thng vector c pht trin biu hin cc gen dung hp vi gen lacZ, in hnh l cc vector h pUR (Hnh 9.10). Chn vector v v tr ct hn ch thch hp ngi ta c th tin hnh dung hp cho hu ht gen c to dng. 2.3. Pht hin cc protein dung hp Gn vector plasmid (v d: pUR) thch hp vi on DNA ngoi lai to ra mt s dung hp trong khung c. Bin np cc vector ny vo E. coli. Kim tra cc khun lc ring bit mang on DNA ngoi lai mong mun bng cch tch chit DNA ca vector plasmid, sau ct bng enzyme hn ch thch hp v in di kim tra trn agarose gel. Sng lc cc khun lc sn xut protein dung hp. 2.4. Tch chit cc protein dung hp sn xut khng th Protein dung hp c th c tch chit theo mt s cch: dng dch chit urea, sc k i lc aminophenylthiogalactoside, in di sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) hoc phi hp gia cc cch ny. K thut n gin nht l SDS-PAGE, nhum gel v pht hin bng protein mi, sau ct bng ny ra khi gel, ng kh ri nghin thnh bt mn. Bt ny s c dng tim vo th sn xut khng th.

Hnh 9.10. Cc vector h pUR. y l cc vector dung hp vi gen lacZ, c cc v tr to dng BamHI, SalI, PstI, XbaI, HindIII v ClaI u 3 ca gen lacZ. S chn vo ca on DNA ngoi lai (cDNA) trong cc v tr to dng thch hp cho php sn xut protein dung hp ca -galactosidase hot ng vi chui peptide c m ha bi DNA ngoi lai. 3. Xc nh mc biu hin ca gen c to dng Ni chung, c ba cch thng c dng xc nh mc biu hin protein ngoi lai ca gen c to dng: - in di polyacrylamide gel c SDS xc nh protein c kch thc thch hp c sn xut mc cao trong cc t bo mang vector biu hin. Thng thng, protein quan tm c th quan st bng cch nhum gel vi Coomassie Brilliant Blue hoc bng AgNO3. Nu khng thy c bng protein mi khi dng cc thuc nhum ny, th nh du s trao i cht vi 100 Ci ca [35S]Met hoc [35S]Cys trn 1 mL

dch nui cy trong 5 pht. Sau , s dng k thut SDS-PAGE hoc phng x t ghi c th pht hin c protein quan tm. - Phn tch Western blot bng cch dng cc khng th lin kt c hiu vi protein quan tm c thm tch ln mng nitrocellulose sau khi thc hin in di SDS-polyacrylamide gel. - Nu mc biu hin thp th nn t gen lacZ cng hng vi gen c biu hin. Nh vy, nu s phin m hoc dch m hn ch biu hin th nhng thay i trong h thng biu hin c th c kim sot bng nhng thay i trong hot tnh ca -galactosidase. Phn tch Western blot - K thut SDS-PAGE L k thut in di trn polyacrylamide gel vi s c mt ca SDS cho php phn tch cc phn t protein c khi lng khc nhau. SDS c in tch m rt ln v c kh nng lin kt vi mch peptide. Nh vy, s lng SDS tng tc vi protein t l vi kch thc phn t protein v in tch ca SDS bm vo c th lm bt c phn t protein no cng chuyn ng trong in trng t cc m sang cc dng. Do , bng phng php in di, c th phn tch ring bit cc phn t protein c khi lng phn t khc nhau. - Phn ng lai khng nguyn-khng th Phn ng khng nguyn-khng th c tnh c hiu rt cao. V vy, c th p dng phn ng ny pht hin s c mt v tinh sch protein. Khng th (antibody) c sn xut khi a khng nguyn vo ng vt th nghim (th, chut) v c tinh sch t mu ng vt sau khi gy nhim. Nhng khng th to ra bng cch ny l nhng khng th a dng (polyclonal antibodies-do cc t bo lympho khc nhau tit ra), do chng c kh nng nhn bit mt s khng nguyn. Ngc li, khng th n dng (monoclonal antibodies) ch tng tc vi mt khng nguyn nht nh. Khng th c nh du bng enzyme (hoc bng cht pht hunh quang) pht hin protein c hiu (thng c thm tch/chuyn thm ln mng nitrocellulose sau khi chy in di SDS, v c nh ) thng qua k thut Western blot (hoc immunoblot) (Hnh 9.11). Sau khi protein trn mng lai gn vi khng th th nht c hiu v tip n l khng th th hai c nh du enzyme (alkaline phosphatase, horse-radish peroxidase) th phc hp ny s c lin kt vi c cht to mu. S hin din ca protein ngoi lai (sn phm dch m ca gen ngoi lai c chuyn vo t bo vt ch) s c pht hin nh s xut hin mu ca phn ng lai. S phn b ca protein c hiu trong t bo v t chc m cng c th pht hin bng k thut lai in situ (in situ hybridization) vi nguyn tc tng t Western

blot. Ngoi ra, khng th c s dng tinh sch protein c hiu bng kt ta min dch hoc sc k i lc (affinity chromatography). Khng th nh du c dng nh lng khng nguyn trong k thut xt nghim hp th min dch lin kt enzyme (enzyme-linked immunosorbent assay) gi tt l ELISA (Hnh 9.11). V. Phng php pht hin dng vi khun c DNA ti t hp 1. Lai khun lc v vt tan DNA c to dng trong plasmid hoc YAC sn xut ra cc khun lc khi cc nui cy bin np c dn mng trn a agar cha mi trng sinh trng v nui cy di nhng iu kin thch hp. Cc bacteriophage sinh tan t bo b chng xm nhim v sn xut ra cc plaque (vt tan) c dng hnh trn chu vi khong 2-3 mm c mu sng trn thm vi khun (bacterial lawn) ca lp agar nh (trong trng hp ny ngi ta chun b a agar c hai lp: lp agar nh c nng agar thp bacteriophage d sinh tan t bo vi khun, v lp agar y c nng agar cao hn). Cc vector, nh m t trn, thng cha gen ch th cho php chn lc nhng t bo vt ch mang vector (th bin np). Cc ch th ny thng l gen khng khng sinh v cc t bo bin np sinh trng trn mi trng cha khng sinh tng ng.

Hnh 9.11. K thut Western blot v ELISA da trn phn ng lin kt khng nguynkhng th

Ngoi ra, cc vector cn cha cc gen ch th b sung phn bit cc t bo bin np cha on chn ca DNA ngoi lai vi cc t bo cha cc vector t ti to li vng. V d: gen lacZ m ha enzyme -galactosidase. Mt dng (clone) cha cc chui DNA quan tm c bit c th c xc nh bi lai khun lc hoc vt tan. Mt lng nh khun lc bin np hoc vt tan c chuyn ln mng nitrocellulose hoc nylon bng cch ph (overlay) mng ny ln trn a agar. DNA c bin tnh v c nh trn mng bng un nng (baking) hoc chiu tia cc tm (UV-crosslinking), v sau c lai trong m cha probe nh du ng v phng x c trnh t b sung mt phn ca chui c xc nh. V d: C th mt oligonucleotide tng hp nhn to, c ngun gc t genomic DNA tng phn, cDNA hoc trnh t protein hoc sn phm PCR. Trong mt vi trng hp, probe c th c thit k da trn trnh t bt ngun t gen tng ng ca cc loi khc v thng c lai vi cng lc thp. Cc probe tha c ra khi mng vi phim X-quang. Theo hng ca phim (sau khi ra) so snh vi a agar gc, sau s

c kh nng gn kt cc khun lc thc t vi cc khun lc lai dng tnh tng ng trn phim X-quang. 2. Sng lc th vin gen bng PCR PCR (polymerase chain reaction) cng c th c ng dng sng lc cc th vin genomic DNA hoc cDNA c xy dng trong cc plasmid vector hoc bacteriophage vector. u im chnh ca sng lc bng PCR so vi sng lc da trn lai truyn thng l t tn thi gian hn. Sng lc bng PCR c th c m bo trong 3-4 gi trong khi n c th mt mt vi ngy trc khi pht hin bng k thut lai phn t (molecular hybridization). K thut sng lc bng PCR th hin ch th v kch thc ca cc on chn DNA c to dng hn l trnh t ca on chn, tuy nhin cc primer PCR c trng cho on chn DNA ngoi lai cng c th c s dng. iu ny cho php kho st chnh xc hn c im ca cc dng t th vin cDNA v genomic DNA. Nguyn tc ca PCR PCR l mt k thut c s dng ph bin trong cng ngh sinh hc hin i v ng gp rt ln cho nhng tin b v sinh hc phn t, nh du mt bc tin v cung quan trng tng ng vi vic khm ph ra cc enzyme hn ch v k thut Southern blot. K thut PCR da trn c s phan ng m rng primer nh enzyme Taq polymerase khuch ai in vitro cac nucleic acid c trng. PCR cho phep khuch ai theo ham mu ln n hang triu ln cac oan DNA co chiu dai khong t 200-3.000 bp. oan DNA c khuch ai (DNA ich) c nhn din nh cp primer c trng (oligonucleotide) thng co chiu dai khoang 20 nucleotide. Taq polymerase l mt loi enzyme DNA polymerase chu nhit (c vi khun chu nhit cao Thermus aquaticus) c dng tng hp cc on DNA mi trong mi trng co 4 loi deoxyribonucleotide (dATP, dCTP, dGTP v dTTP) v hai primer, trn c s khun mu ca mt on DNA nht nh a bit hoc cha bit trinh t. Cc on DNA mi hnh thnh li c s dng lm khun mu. Sau nhiu chu ky, s lng on DNA ni trn c nhn ln gp nhiu ln, nh vy c th s lng tch ra, phn tch trnh t hoc to dng... Primer bn trai tac ng trn si DNA 3-5 c goi la primer thun (forward primer-ky hiu la F). Primer bn phai tac ng trn si DNA 5-3 c goi la primer ngc (reverse primer-ky hiu la R). Nguyn tc ca PCR c trnh by trong hnh 9.12. Theo , t chu ky th hai Taq polymerase bt u to ra cc on DNA c chiu di xc nh. Cc primer thng l mt oligonucleotide tng hp (synthetic oligonucleotide) c khong 10-20 nucleotide hoc di hn. Nu bit trnh t ca on gen cn khuch i th c th tng hp nhn to cc primer tng ng thc hin PCR v tch chng ra bng k thut in di.

PCR thng tin hnh khoang 25-35 chu ky, qua t 10-6 g DNA ban u c th khuch i (amplification) ln ti trn 1 g (khong 2 kb). Mi chu ky PCR bao gm ba giai on c nhit khc nhau: - Gy bin tnh (denaturation) 90-95oC. Trong giai on bin tnh phn t DNA khun mu dng xon kp c tch thnh hai si n (single strands). - Gn primer (annealing) 40-65oC. Trong giai on ny cc primer gn vo cc v tr c trnh t tng ng DNA khun mu. Cc lin kt ion tao thanh mt on nh (cc primer lp rap chnh xc) v trn cc on nho DNA si i (khun mu v primer) enzyme Taq polymerase c th bt u qu trnh sao chp khun mu. - Ko di phn t (extension) 70-72oC. y l khong nhit ti thch cho Taq polymerase tin hnh tng hp DNA bt u t cc v tr c primer theo chiu 53.

Hnh 9.12. S phn ng chui DNA polymerase (PCR)

3. Sng lc cc th vin cDNA bng cc probe khc nhau Sang loc th vin cDNA bng cch lai nucleic acid la phng phap c s dung ph bin va ang tin cy nht tim kim cac dong quan tm. Sang loc bng phng phap lai nucleic acid cho phep phn tich ng thi nhiu dong va nhanh, khng oi hoi cac dong cDNA phai hoan chinh va san phm c tng hp trong t bao vt chu phai co hoat tinh sinh hoc hoc khang nguyn. Hn na, c s ly thuyt cua ky thut lai nucleic acid c hiu bit y u. iu nay cho phep phat trin mt s lng ln cac ky thut khac nhau co th iu chinh cac probe ca nucleic acid co cac chiu dai va c im khac nhau.

- Cac probe tng ng. Cac probe tng ng cha it nht mt phn cua chui nucleic acid chinh xac cua dong cDNA quan tm. Chung c dung trong nhiu trng hp khac nhau, vi du: khi mt dong b phn cua cDNA hin co c s dung phn lp mt dong hoan chinh t th vin cDNA. Thng thng, oan bt ngun t mt u hoc u kia cua dong hin co c phn lp, anh du phong xa in vitro va dung thm do th vin. Lai vi cac probe tng ng thng c tin hanh di cac iu kin nghim ngt. - Cac probe tng ng tng phn. Cac probe tng ng tng phn c dung phat hin cac dong cDNA co quan h ho hang, nhng khng ging ht nhau hoan toan, vi cac trnh t ca probe. - Cac probe oligonucleotide nhn tao. Cac probe oligonucleotide nhn tao la cac vung dNTP cua trinh t xac inh c tng hp in vitro. Trinh t cua cac probe nay c suy lun, bng cach dung ma di truyn, t cac vung ngn cua chui amino acid a 3 bit cua protein quan tm. Do s thoai bin cua ma di truyn , chui amino acid xut c th khng c c trng chnh xc bi cc oligonucleotide n c d oan cho trinh t. Mc du, trong rt nhiu trng hp, trinh t cac amino acid ging nhau co th c c trng bi nhiu oligonucleotide khac nhau. 4. Phn tich genomic DNA bng phng phap lai Southern Vic phat hin va xac inh cac chui nucleic acid c trng la cng vic thng xuyn trong nghin cu sinh hoc phn t. Nguyn tc cua ky thut nay la da vao s lai phn t, di cac iu kin thich hp hai chui nucleic acid n tao thanh mt phn t lai. Phan ng lai phu thuc rt nhiu vao mc tng ng cua hai chui nucleotide. Vic tao thanh phn t si i nh th xay ra chu yu thng qua lin kt hydrogen gia cac base guanosine vi cytosine, va adenosine vi thymidine. Thanh phn va s phn b cua cac base khng ging ro rt vi cac phn t nucleic acid cho kt qua cac tinh cht lai khac nhau, vi th lin kt hydrogen (hoc lai phn t gia cac base tng ng) khi c ghep cp thich hp a cung cp mt cng cu co gia tri xac inh cac chui lin quan hoc ng nht vi chui nucleic acid quan tm (probe). Trong s cac ky thut khac nhau s dung lai phn t phn tich nucleic acid, thi ky thut c s dung ph bin nht la lai mu do nucleic acid c anh du vi nucleic acid ich c c inh trn mt vt rn, c trng la mang nitrocellulose hoc nylon. Trinh t cua ky thut lai Southern blot bao gm cac bc sau: phn tch cac oan ct han ch cua DNA h gen bng in di agarose gel, chuyn DNA t agarose gel ln mang lai, lai cac mu do c anh du ng vi phong xa vi cac nucleic acid c c inh trn mang lai (Hinh 9.13).

Hinh 9.13. S cua ky thut lai Southern blot. Cc mu DNA ch v genomic DNA (A) v (B) trong v d ny c ct bng EcoRI v c phn on bng in di trn agarose gel. DNA ca plasmid mang on chn DNA dng lm mu d (C) cng c ct bng EcoRI v in di nh l mt i chng dng tnh.

Cc th nghim lai trn mng thng c tin hnh bng cch nh du probe bng P (mc d cc ng v phng x khc nh 35S cng c th c s dng). Phn ng lai ca Southern i hi hot tnh c hiu ca probe ti thiu phi l 10 9 dpm/g, mc d hot tnh 108 dpm/g c th c xem nh l chp nhn c trong cc ng dng cn cng lc thp. Cc phng php khng dng ng v phng x (v d: digoxigenin-dUTP) ngy cng c s dng ph bin hn mc d nhy ca chng l khng ln. Nhng cc k thut khng dng ng v phng x an ton hn cho nghin cu vin, to ra cc probe c th c bo qun trong thi gian di trc khi dng v kt qu pht hin cc tn hiu lai nhanh hn.
32

VI. Cc ng dng ca cng ngh DNA ti t hp Cng ngh DNA ti t hp hin nay c rt nhiu ng dng trong i sng. Cc ng dng ny bao gm sn xut dc phm v cc loi ha cht khc, cc vi khun c bit, cc cy trng nng nghip quan trng, v cc vt nui c bin i di truyn... K thut ny cng c s dng rng ri trong cc xt nghim y khoa v (trong mt vi trng hp) ang c dng kim tra cc khim khuyt di truyn ngi. Hng trm cng ty hin nay ang c bit pht trin cc sn phm thng qua bin i di truyn cc c th sng. Di y l cc ng dng chnh ca cng ngh DNA ti t hp. 1. ng dng trong dc phm

Sn phm thng mi u tin c pht trin bng cng ngh DNA ti t hp l cc dc phm dng trong iu tr cc bnh v cc ri lon ngi. Nm 1979, T hp Eli Lilly bt u sn xut thng mi insulin ngi bng cng ngh DNA ti t hp. Trc thi im ny, tt c insulin dng trong iu tr bnh tiu ng c tch chit t ty ca cc vt nui cho tht. Mc d ngun insulin ny c tc dng tt cho nhiu ngi mc bnh tiu ng, nhng n vn khng phi l insulin ngi, v mt s ngi tri qua cc phn ng ph vi protein ngoi lai. Gen insulin ngi sau c chn vo plasmid v chuyn vo vi khun sn xut insulin ngi. Cc dc phm c sn xut thng qua cng ngh DNA ti t hp bao gm: hormone sinh trng ngi (cho tr em mc bnh ci), nhn t to t huyt (cha bnh kh ng mu), hot t plasminogen m (dng ha tan cc huyt khi trong bnh nhn b nhi mu c tim), interferon, interleukin, DNA vaccine

2. Cc vi khun c bit Cc vi khun c vai tr quan trng trong cc qu trnh cng nghip, bao gm sn xut ethanol t cc nguyn liu thc vt, lc khong t qung, x l nc thi v cc loi cht thi khc. Cc vi khun ang s dng trong cc qu trnh ny c bin i di truyn bng cng ngh DNA ti t hp sao cho chng c th hot ng hiu qu hn. Cc chng vi khun mi hu ch thu c t k thut hin i ny ang c pht trin phn hy cc ha cht c v cc cht gy nhim, tng cng thu hi du, tng nitrogen cho cy trng, v c ch sinh trng ca cc vi khun v vi nm gy bnh.

3. Cc sn phm nng nghip Cng ngh DNA ti t hp cng c mt nh hng quan trng n sn xut nng nghip, ni m hin nay n c dng to ra cc cy trng v vt nui mang cc c im c gi tr. Trong nhiu nm, cc nh bnh hc thc vt tha nhn rng thc vt b nhim bnh virus th nh sau s khng li s nhim trng cc chng mang c tnh mnh. V vy, cc nh di truyn hc to ra tnh khng virus trong cy trng bng cch chuyn cc gen v protein ca virus vo t bo thc vt. Cy b (squash) bin i di truyn c tn l Freedom II, mang cc gen ca virus 2 gy bnh khm da hu (watermelon mosaic virus 2) v virus gy bnh khm mu vng cy zucchini (zucchini yellow mosaic virus) chng s nhim trng virus. Mt hng khc c bin i di truyn l tnh khng su (pest) thc vt lm gim s ph thuc vo cc thuc tr su ha hc. c t protein t vi khun Bacillus thuringiensis git mt cch chn lc cc u trng ca cc su b cn trng nht nh nhng li khng gy c cho s sng ca cc ng vt hoang d, ngi v

cc loi cn trng khc. Gen c t c phn lp t vi khun, lin kt vi promoter hot ng sau c chuyn vo trong ng, c chua, khoai ty v bng. Gen ny sn xut c t i vi cn trng trong cy, v su bm khi n cy b cht. Cng ngh DNA ti t hp cng cho php pht trin tnh khng cht dit c thc vt. Mt vn quan trng trong nng nghip l iu khin cc c di cnh tranh nc, nh sng v cht dinh dng vi cy trng. Mc d cht dit c c tc dng git c di, nhng chng cng c th gy nguy him cho cy trng. Cc gen cung cp tnh khng cho nhiu loi cht c di c chuyn vo c chua, u tng, bng, cy ci du v cc cy trng thng mi quan trng khc. Khi ng rung c canh tc cc cy trng ny c phun thuc dit c, th c di b git nhng cc cy trng bin i di truyn li khng b nh hng. Nm 1999, c hn 21 triu ha cy u tng bin i di truyn v 11 triu ha cy ng chuyn gen c trng trn th gii. Cng ngh DNA ti t hp cng c ng dng cho cc vt nui. V d: gen sn xut hormone sinh trng c phn lp t gia sc v c to dng trong E. coli, cc vi khun ny sn xut mt lng ln hormone sinh trng ca b, yu t lin quan n tng sn xut sa. Cc ng vt chuyn gen c pht trin mang gen m ha cho cc sn phm dc liu. V d: mt gen ngi m ha cho nhn t VIII (hnh thnh t huyt) c lin kt vi vng iu ha ca gen sn xut -lactoglobulin cu, mt protein to sa. Gen dung hp c tim vo phi cu, to ra mt con cu chuyn gen c kh nng sn xut trong sa ca n nhn t ng mu ca ngi. Mt phng thc tng t c dng chuyn gen 1-antitrysin, mt protein dng iu tr bnh nhn kh thng di truyn (hereditary emphysema), vo trong cu. Cu ci mang gen ny sn xut rt nhiu 1-antitrysin khong 15g trong 1 lt sa ca chng.

4. Thuc oligonucleotide Mt ng dng cng ngh DNA ti t hp trong thi gian gn y l pht trin cc thuc oligonucleotide c trnh t phn t DNA hoc RNA nhn to ngn c th c dng iu tr bnh. Cc antisense oligonucleotide b sung cho cc RNA khng mong mun, nh l RNA ca virus. Khi c b sung vo t bo, th cc antisense DNA ny lin kt vi mRNA ca virus v c ch s dch m ca chng. Cc DNA oligonucleotide si n lin kt cht ch vi cc trnh t DNA khc, to thnh mt phn t triplex DNA. S to thnh triplex DNA cn tr lin kt ca RNA polymerase v cc protein khc cn cho s phin m. Cc oligonucleotide khc l cc ribozyme, cc phn t RNA m chc nng nh l cc enzyme. Nhng phc hp ny lin kt vi cc phn t mRNA c hiu v ct chng thnh tng on, ph hy kh nng m ha protein ca chng. Mt s thuc oligonucleotide sn sng th nghim trong iu tr bnh AIDS v ung th.

5. Liu php gen i vi loi bnh di truyn, bnh do t bin gen ngi ta phi cn n s can thip ca liu php gen, mt hng cha bnh gn lin vi cc k thut tin tin trong lnh vc cng ngh sinh hc hin i nh cc vi thao tc gen, sa i thay th gen... Liu php gen (gene therapy) thc cht l phng php cha bnh bng gen. C nhiu khi nim khc nhau v liu php gen, nhng cch hiu chung nht l tp hp cc bin php s dng cc gen cn thit (cn gi l gen tr liu) nhm mc ch cha bnh cho con ngi. Trong , gen tr liu c th l: - Cc gen hot ng bnh thng (gen lnh) c th a vo t bo thay th gen hng, gen mt chc nng, khi phc hot ng bnh thng ca t bo v s sng ca c th. - L nhng gen c kh nng m ha mt protein c hiu, khi a vo t bo sng c th to nn cc loi protein c hiu. Cc loi protein c hiu c th c ch hot ng ca mt gen khc trong t bo, km hm kh nng phn chia ca t bo hoc gy cht cc t bo b bnh. - Nhng gen khi a vo t bo hot ng ng thi vi cc gen bnh (gen b t bin trong t bo) lm hn ch tc ng ca gen bnh hoc h tr, b p cho cc gen b hng. - Gen tr liu cn l cc gen bt hot c a vo t bo thay th cho mt gen lnh no , nhm hn ch sn phm khng cn thit ca gen lnh hoc to ra cho t bo mt trng thi mi, c tc dng chng li bnh tt. - Gen tr liu c th l nhng on oligonucleotide c tc dng km hm hot ng ca gen b hng, b t bin trong t bo. 6. Chn on bnh can thip sm Bng k thut chc i, kt hp ng thi phn tch mu b m bng enzyme hn ch, ngi ta c th chn on sm trc khi sinh (v enzyme hn ch c kh nng phn bit c gen t bin vi gen bnh thng). Cc on DNA ct ra, c phn tch qua phng php in di, em lai vi cc mu d DNA hoc RNA nh du bng ng v phng x 32P hoc hunh quang. nh phng x t ghi hoc tn hiu hunh quang cho ta thy cc on DNA c lai vi cc mu d. Cc on ny c tch ra nghin cu v xc nh t bin hay bnh thng bng enzyme hn ch, v mt s t bin di truyn c nh hng n cc v tr dnh cho enzyme hn ch. ng dng cc k thut sinh hc phn t trong lnh vc ny c nhiu thun li hn cc phng php kinh in. Th nht, c th rt ngn c thi gian nh ng dng k thut PCR, chng hn chn on bnh HIV-1, dng phng php PCR ch

mt mt ngy so vi 3-4 tun nui cy ca phng php truyn thng. Th hai, i vi cc tc nhn gy bnh kh nui cy hay khng th nui cy th k thut sinh hc phn t l gii php duy nht, chng hn Chlamydia, Brucella, vim gan B,... Th ba, vic to dng mt gen dng lm probe n gin hn vic sn xut khng th c hiu, hn na vi mt probe ngi ta c th pht hin tt c cc kiu huyt thanh (serotype) ca tc nhn gy bnh, iu m ch mt khng th khng th lm c. Cui cng, nu trc kia phi cn n nhiu k thut (quan st di knh hin vi, nui cy trn mt lot mi trng, min dch hc...) th hng chn on bng sinh hc phn t ch cn mt k thut (lai phn t hoc PCR). i vi tc nhn l virus, cc k thut lai phn t v PCR cho php chn on nhiu nhm nh papillomavirus, HBV, HIV. c bit phng php lai ti ch cn cho php xc nh trc tip virus trn lt ct sinh thit. i vi cc tc nhn vi khun, c nhiu b kit chn on sinh hc phn t c thng mi ha nh: Mycobacterium tuberculosis, Mycobacterium pneumoniae, Salmonella, Streptococcus pneumoniae, Haemophilus influenzae... i vi cc tc nhn k sinh trng, ngi ta cng thnh cng trong chn on Plasmodium, Schistosoma, Trypanosoma, Toxoplasma, Leishmania bng lai phn t. Phng php PCR cn cho php pht hin tc nhn ngay trong vt trung gian truyn bnh. 7. DNA fingerprinting S dng cc trnh t DNA nhn dng mt ngi, c gi l DNA fingerprinting, l mt cng c rt mnh kho st ti phm v cc ng dng php l khc. Trong mt ng dng c trng, DNA fingerprinting c th c dng xc nhn mt s nghi ng hin din hin trng gy n. Mt mu DNA ca mu, tc, tinh dch hoc m ca c th c thu thp t hin trng. Nu mu l rt nh, phng php PCR c th c dng khuch i cho DNA, sn sng cho vic kim tra. Cc mu DNA b sung c thu thp t mt hoc nhiu nghi can khc. Mi mt mu c ct vi mt hoc nhiu RE, v kt qu cc on DNA c phn tch bng in di. Cc on trn gel c bin tnh v chuyn ln mng nitrocellulose bng thm tch Southern (blot). Mt hoc nhiu probe (c nh du phng x) sau c lai vi mng nitrocellulose v c pht hin bng phng x t ghi. Kiu ca bng c to ra bi DNA t mu c thu thp hin trng sau c so snh vi cc kiu c to ra bi DNA t nhng nghi can. Cc probe c dng trong DNA fingerprinting pht hin cc vng thay i cao ca genome, v th c hi DNA t hai ngi sn xut chnh xc cng mt kiu bng l rt thp. Khi mt s probe c dng trong phn tch, th xc sut m hai ngi c cng mt tp hp cc kiu l gn nh khng c (tr khi h l cp sinh i ging ht nhau).

Mu khp nhau t hin trng v nghi can c th cung cp bng chng rng nghi can c mt ti hin trng v n. Probe c dng ph bin nht trong DNA fingerprinting b sung cho cc trnh t lp li ngn c tm thy rt nhiu trong genome ngi. Con ngi khc nhau rt nhiu v s lng bn sao ca cc trnh t lp li ny; v vy, cc a hnh ny c gi l s lng bin thin ca cc on lp li tun t (VNTRs). DNA fingerprinting cng c s dng cung cp thng tin v mi quan h huyt thng v cc ngun c th sng khc. V d: DNA fingerprinting c dng xc nh mt s mu vi khun gy bnh than (anthrax) gi bng bu in n nhng ngi khc trong nm 2001 tt c l t mt ngun mu. 8. Lp bn gen Mt ng gp c ngha ca cng ngh DNA ti t hp l cung cp mt lng ln cc marker (ch th) di truyn c dng trong lp bn gen. Mt nhm trong s cc marker c dng trong lp bn gen l restriction fragment length polymorphism (RFLP). RFLP l nhng bin i a hnh trong cc kiu ca cc on DNA c to ra khi phn t DNA c ct cng mt enzyme hn ch. Nu DNA t hai ngi c ct bi cng mt enzyme v kiu ca cc on DNA khc nhau c to ra, th hai ngi ny phi c s khc nhau v cc trnh t DNA ca h. Nhng s khc nhau ny c di truyn v c th c dng trong lp bn , tng t vi phng thc m trong s khc nhau v allele c dng lp bn cc gen thng thng. Theo truyn thng, bn gen c da vo vic s dng cc sai khc di truyn to ra nhng sai khc kiu hnh c th quan st d dng. ng tic l do hu ht cc tnh trng b nh hng bi a gen v mi trng, nn s lng cc tnh trng c c s di truyn n gin thch hp cho vic s dng bn b gii hn. RFLP cung cp mt s lng ln cc marker di truyn c th c dng lp bn gen ca sinh vt.

Ti liu tham kho/c thm

1. Dale JW and Von Schanzt M. 2002. From Gene to Genome. John Wiley & Sons, Ltd. West Sussex, UK. 2. Erlich HA. 1989. PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, USA. 3. Glick BR and Pasternak JJ. 2003. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 3rd ed. ASM Press, USA. 4. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK.

5. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory, USA. 6. Primrose SB, Twyman R and Old RW. 2001. Principles of Gene Manipulation. 6th ed. Blackwell Science, Oxford, UK. 7. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook. Humana Press Inc. New Jersey, USA. 8. Surzycki S. 2000. Basic Techniques in Molecular Biology. Springer-Verlag, Berlin, Heidelberg, Germany. 9. Walker JM and Rapley R. 2000. Molecular Biology and Biotechnology. Chapman & Hall Limited, London, UK.

Cht ty lm bin i b mt t bo gii phng cc thnh phn t bo ra mi trng bn ngoi.

1 2

YAC (yeast artificial chromosome): Nhim sc th nhn to ca nm men. S thoi bin (suy bin) ca m di truyn (degeneracy): Bnh thng c th c 64 t hp b ba m ha khc nhau ca 4 nucleotide trong khi ch c 20 amino acid ph bin v mt amino acid li c th c nhiu b ba m ha.
3

Ph lc

Mt s thut ng c bn
Adenine (A). Mt trong bn nitrogen base co trong thanh phn cua DNA va RNA, bt cp vi thymine (T) trn DNA va vi uracil (U) trn RNA. Adenine cung co trong thanh phn cua mt s coenzyme. Adenosine diphosphate (ADP). Mt ribonucleoside 5-diphosphate c cu to t adenine, ng ribose (5C) v hai gc phosphate. ADP c tc dng nhn phosphate trong chu trnh nng lng ca t bo. Adenosine triphosphate (ATP). Mt ribonucleoside 5-triphosphate c cu to t adenine, ng ribose (5C) v ba gc phosphate. ATP l phn t cha nng lng ha hc chnh ca t bo, chu yu c tp hp trong ty th (mitochondria) va lp th (chloroplast). Cc gc phosphate ca ATP c mang cc lin kt khi b thy phn s phng thch mt nng lng t do ln. Nng lng ca qua trinh h hp hoc quang hp c s dung tao thanh ATP t ADP. Sau o, ATP c bin i ngc tr lai thanh ADP nhiu vung khac nhau cua t bao, nng lng phong thch ra c dung iu khin cac phan ng ha sinh ni bao. i khi cng xay ra s thy phn tip ADP thanh nhng AMP (adenosine monophosphate) phng thch nng lng nhiu hn.

Allele. Mt trong nhiu dng khc nhau ca mt gen chim mt locus xc nh trn nhim sc th. Cac allele khac nhau cua mt gen c trinh t cac nucleotide khc nhau, cung lin quan n mt tinh trang cua t bao hoc c th. V du: trong thi nghim lai u Ha Lan cua Mendel, mau vang va mau xanh cua hat u la hai allele cua gen quy inh mu sc hat u. Ca th c goi la ng hp t v mt gen khi hai allele o ging nhau, va goi la di hp t khi hai allele o khac nhau. trang thai di hp t allele ny (allele tri) thng ln t hiu qua cua allele kia (allele ln). Allele quy inh trang thai binh thng la tri, trong khi cac allele t bin thng la ln. Amber codon. La mt trong ba codon (m b ba UAG) kt thuc mt trnh t ca mRNA trong sinh tng hp protein. Amino acid. L mt phn t nh mang mt gc amine (-NH3) v mt gc carboxyl (-COOH) lin kt vi cng mt nguyn t carbon. Amino acid l n v cu trc c s ca chui polypeptide. C 20 amino acid khc nhau trn cc chui polypeptide co trong t nhin. Trinh t sp xp cua cac amino acid trn chui polypeptide quyt inh cu truc va chc nng cua polypeptide va protein ma no tao thanh. Aminoacyl-tRNA (aminoacyl-tRNA). Mt ester gia aminoacyl vi tRNA tng ng ca amino acid . AMP vng (cyclic AMP, cAMP). L mt phn t AMP m trong gc phosphate c lin kt vi v tr 3 v c 5 ca ng ribose. cAMP l thng tin th hai trong t bo. S to thnh cAMP (xc tc bi enzyme adenylate cyclase) c kch thch bi mt s hormone. Ampicillin (Amp). Cht khang sinh ban tng hp c dung trong mi trng chon lc chon cac t bao mang t bin khuyt dng hoc chon dong t bao (ti t hp) mang oan DNA c to dong. Antisense. Trnh t khng m ha ca mt phn t DNA si i. Chui cy (chui ngc chiu) b sung vi mRNA v lm khun mu cho s tng hp mRNA. Antisense molecule. La mt phn t gn c hiu ln DNA hoc mRNA lam dng qua trinh phin ma hoc dch m. Thng phn t nay co ngun gc t DNA va RNA. t ch (epistasis). Tc dng tng h gia cc gen khi mt gen cn tr s biu hin kiu hnh ca mt gen khng alelle vi n. ATP-ase. Enzyme thy phn ATP thnh ADP v phosphate, thng phi hp vi s bin i nng lng. ATP-synthetase. Enzyme tng hp ATP t ADP v phosphate trong qu trnh phosphoryl oxy ha (oxidative phosphorylation) mng trong ca ty th. Baculovirus. Loai virus c bit gy nhim cac t bao cn trung va tao ra cac th vi ln trong cac t bao bi nhim.

Bn ct hn ch (restriction map). Trnh t cc v tr nhn bit (recognition sites) ca tt c cc enzyme hn ch (restriction enzyme hay restriction endonuclease, RE) trn mt phn t DNA. Ban di truyn (genetic map). S v trinh t sp xp cua cac cu t di truyn va khoang cach tng i gia chung trn nhim sc th ( eukaryote) hoc trn si DNA ( prokaryote). Nu cac cu t o la cac gen trn nhim sc th va khoang cach gia chung c xac inh bng tn s trao i cheo thi o la ban nhim sc th hay ban lin kt gen. Nu cac cu t la cac im bi t bin bn trong gen va khoang cach gia chung cung c xac inh bng tn s trao i cheo gia cac im noi trn thi o la ban gen. Nu cac cu t di truyn la cac im co th bi ct bi enzyme han ch trn phn t DNA th gi l bn ct hn ch (restriction map). Xem b n ct hn ch. Baz ng ng (analog base). Cht ha hoc co cu truc phn t rt ging cac base binh thng cua DNA. Chung co th thay th cac nitrogen base binh thng trong DNA va hoat ng nh mt tac nhn t bin. Trong ln sao chep tip theo cua DNA, base ng ng co th bt cp sai vi mt base binh thng, tao nn t bin im. V d: base ng ng cua adenine (A) la 2-aminopurine (AP) co th gn vao DNA vi tri ca adenine; trong ln sao chep tip o co th bt cp vi cytosine (C), trong ln sao chep tip theo na C kt cp vi guanine (G). Nh vy a din ra s thay th cp A-T bng cp G-C. Baz nit (nitrogen base). Loai phn t cu tao nn nucleic acid (DNA v RNA). Cac nitrogen base co trong nucleic acid l adenine, guanine, cytosine v thymine (trong phn t DNA) hoc uracil (trong phn t RNA). Trinh t sp xp cua chung doc theo phn t nucleic acid tao nn thng tin di truyn ca c th sinh vt. Bt cp b sung (complementary base pairing). S kt hp thnh tng i gia cc nitrogen base nm trn hai mch n ca chui xon kp DNA-DNA, DNARNA hoc RNA-RNA thng qua cc mi lin kt hydrogen. S bt cp mang tnh c hiu: guanine bt cp vi cytosine, cn adenine bt cp vi thymine trn DNA hoc uracil trn RNA. Bin i hu dch m (post-translational modification). S thay i cc lin kt ha tr xy ra trong chui polypeptide, sau khi chui polypeptide tch khi ribosome v trc khi tr thnh protein hot ng thc s. Bin i mRNA (mRNA processing). Qu trnh to thnh mRNA hon chnh (mature mRNA) hot ng chc nng t sn phm phin m s cp l mRNA tin thn cha hon chnh (pre-mRNA). Trong qu trnh ny cc on intron b loi b v cc on exon c ni li vi nhau. Bin np (transformation). L qu trnh truyn DNA ngoi lai vo mt t bo nhn, chng hn sphaeroplast hoc protoplast, v c th hp nht trong nhim sc th nh s ti t hp tng ng hoc c bin i trong mt n v sao chp t tr

(autonomous replicon). S bin np c th xut hin trong cc iu kin t nhin mt s vi khun (v d: Bacillus, Haemophilus, Neisseria v Streptococcus), nhng nhiu vi khun (v d: E. coli) v cc c th sinh vt eukaryote s bin np ch c th xut hin nhng t bo thm c DNA bng cc phng php nhn to nh: ha bin np, in bin np... Bin tnh (denaturation). L hin tng chuyn t dng mch kp sang dng mch n ca DNA v RNA thng do nhit gy nn. Bin tnh ca protein l hin tng chuyn t cu hnh hot ng thnh dng khng hot ng. Biu hin ca gen (gene expression). L cc qu trnh phin m (transcription) v dch m (translation) ca mt gen to ra sn phm protein ca n. B khung (backbone). Thut ng c dung chi chui xon cua khung ng phosphate trong phn t DNA mach kep. Cm ng (induction). Lin quan n kh nng tng hp mt s enzyme (sn phm ca gen) vi khun ch khi c mt c cht (substrate) ca cc enzyme ny trong mi trng. Khi s dng cho khi nim iu ha biu hin ca gen, t ny c ngha l s khi ng qu trnh phin m do tng tc gia mt cht cm ng (inducer) vi protein iu ha. V d: trng hp operon lac E. coli, lactose ong vai tro cht cam ng co tac dung m operon cac gen trong operon hoat ng (phin ma va dich ma). CAP (catabolite gene activator protein). Protein hot ha gen d ha. Mt protein iu ha c bit, kim sot s khi u phin m cc gen sn xut cc enzyme cn thit cho mt t bo, c th s dng nhng thc n khc khi khng c mt ca glucose. Cp baz (base pair, bp). L lin kt A-T hoc C-G trn mt phn t DNA mch kp, v l n v o chiu di ca mt phn t DNA. Cu trc bc mt (primary structure). Bao gm trnh t cc amino acid v nhng cu ni disulfide trong chui hoc gia cc chui polypeptide. trng hp nucleic acid, l trnh t ca cc nucleotide trn mt chui DNA hoc RNA c ni vi nhau nh lin kt phosphodiester. Cu trc bc hai (secondary structure). L s ni lin nhng amino acid t u n (u tn cng N) ti u kia (u tn cng C) ca mt phn t protein. Cng nh vy, trn mt trnh t nucleic acid l s ni lin nhng nucleotide t u n (u 5) ti u kia (u 3) ca mt chui DNA. Cu trc bc ba (tertiary structure). Cu trc ba chiu dng cun xon ca phn t protein hoc chui polynucleotide. Cu trc bc bn (quaternary structure). Cu trc ba chiu ca mt protein c nhiu tiu n v (subunit), theo kiu m nhng tiu n v kt hp khp vi nhau. Cu disulfide (disulfide bridge). Lin kt ng ha tr to thnh gia hai chui polypeptide qua trung gian ca mt gc cystine.

Chc ba sao chp (replication fork). Cu trc hnh ch Y hnh thnh khi phn t DNA mch kp dn xon l ra hai si n lm khun mu cho tng hp DNA (s ti bn). Cht cm ng (inducer). Mt hp cht ha hc hoc mt tac nhn cua mi trng co tac dung xuc tin qua trinh phin ma operon cua vi khun hoc cc phn t c tc dng san xut ra s lng ln cac enzyme trong trao i cht. Cht d nhim sc (heterochromatin). Bao gm cc vng ca h gen thng xuyn trng thi c c v khng biu hin di truyn. Trng thi ny c th vnh vin hay tm thi. Cht nhim sc (chromatin). Phc hp DNA v protein trong nhn giai on ngh ca qu trnh phn bo. Trong giai on ny khng th phn bit tng nhim th ring l. Tn gi ny bt ngun t phn ng vi cc mu nhum c trng cho DNA ca phc hp. Cht nhim sc tht (euchromatin). Cht nhim sc cha nhng gen c phin m. Cht c ch (repressor). Sn phm protein ca mt gen c ch nm trong thanh phn operon, co tac dung bam vao vung chi huy cua operon lm ong operon lai, do qua trinh phin ma khng bt u c va tt ca cac gen cu truc (gen m ha) trong operon u ngng hoat ng. Chromosome walking. K thut ny dng lp bn nhim sc th t tp hp cc on DNA ct hn ch chng ln nhau (overlapping). Bt u t mt th vin trong cha cc on DNA ni trn c to dng. Mt on DNA mang mt gen bit c la chn v s dng nh mt probe nhn dng (v d: bng cch lai khun lc) cc on khc, l cc on chng ln nhau cha cng mt gen. Sau , trnh t nucleotide ca cc on ny s c phn tch v nh vy c th xc nh c ton b cc on ca nhim sc th. T , bn ca mt vng c bit s c xy dng dn dn. Chu trnh sinh tan (lylic cycle). Mt kiu chu trnh sng ca thc khun th (bacteriophage) khi n xm nhim vi khun, iu khin cc hot ng sinh sn v sinh trng bng cc gen ca n v sinh ra cc bacteriophage th h con, chui ra khoi t bao vi khun sau khi pha v t bao o. Chu trinh tim tan (lysogenic cycle). L hin tng h gen ca bacteriophage hin din trng thi n nh v khng sinh tan trong t bo vt ch sng ca n. Cc t bo vt ch c th tip tc sinh trng v phn chia, v s sao chp ca h gen bacteriophage (prophage) c phi hp vi nhim sc th ca vt ch sao cho khi t bo phn chia th prophage cng c chuyn vo trong c hai t bo con. Prophage c duy tr bng cch hoc hp nht trong nhim sc th vt ch (v d: bacteriophage , bacteriophage 105) hoc nh l mt plasmid bn ngoi nhim sc

th (v d: bacteriophage P1 v bacteriophage F116). T bo vt ch c th hoc khng th biu hin ra mt kiu hnh bin i. Chui contig (contiguous sequence). Mt oan trnh t dai c hinh thanh t mt s cac oan phn t ngn chng ln nhau (overlapping). Chui kham (concatemer). Phn t DNA bao gm nhiu oan ca bit ni vi nhau thng qua cac u dinh. Chui m ha (coding sequence). on phn t DNA mang m di truyn xc nh phin m thnh mRNA v sau dch m thnh chui polypeptide. Chuyn gen (transgenic). Qu trnh chuyn mt oan DNA ngoi lai (foreign DNA) bng cc k thut khc nhau (Agrobacterium, vi tim, bn gen, xung in...) vo mt c th vt ch (vi sinh vt, ng vt hoc thc vt). Cistron. Thut ng c, l mt n v ca DNA tng ng vi mt gen ma ha mt chui polypeptide hoc mt phn t RNA. Cordycepin. La 3 deoxyadenosine, mt cht ngn can phan ng polyadenine ha (polyadenylation) cua RNA. Corepressor. La mt phn t nho kich hoat s kim ham gen trong khi giai ma bng cach gn cht vi mt protein iu ha. Cosmid. Vector lai (hybrid vector) c cu thanh t cac oan trinh t ca plasmid va cac v tr cos (u dinh) cua bacteriophage . Cytosine (C). Base pyrimidine c trong DNA. Trn DNA mch kp cytosine bt cp vi mt base purine l guanine (G). D-loop. Vung DNA ty th ni co mt oan ngn RNA bt cp vao thay th phn mch n DNA nguyn thy y. Deoxyribonucleotide triphosphate (dNTP). Tin cht a c triphosphoryl ha (nng lng cao) cn thit cho qua trinh tng hp DNA. N c k hiu cho mt trong bn nitrogen base (A, G, T hoc C). Deoxyribonuclease (DNase). Loai enzyme nuclease thy phn (phn hy) DNA si i hoc DNA si n. Deoxyribonucleic acid (DNA). DNA la ai phn t sinh hoc co cu truc xon i, tn tai chu yu trong nhn t bao, trn cac nhim sc th, mang thng tin di truyn cua sinh vt. Phn t DNA gm hai chui polynucleotide, chui no xon quanh chui kia tao nn chui xon kep. Trong cac nucleotide, theo chiu doc cac gc phosphate ni xen ke vi cac phn t ng deoxyribose tao nn b khung bn ngoai cua chui xon kep, theo chiu ngang mi phn t ng u kt hp vi mt trong bn nitrogen base: adenine, guanine, cytosine hoc thymine. DNA khng trc tip th hin chc nng sinh hoc ma gian tip qua protein do no ma ha. DNA tao RNA, RNA tao protein. RNA cung la acid nhn (nucleic acid). No co

thanh phn cu tao kha ging DNA, ngoai tr gc thymine (T) trong DNA c thay th bi gc uracil (U), va RNA dang si n ch khng phai dang xon kp nh DNA. Qua trinh oc ma di truyn cha trong DNA tng hp protein goi la s phin m (transcription) tao ra RNA mang thng tin di truyn l mRNA (messenger RNA). mRNA kt hp vi mt c quan t trong t bao la ribosome tao ra protein trong qua trinh dch ma (translation). Qua trinh trn c gi la qua trinh sinh hoc cn ban. Nm 1962, Watson (M) v Crick (Anh) chia s Gii Nobel vi Wilkins (Anh) v pht minh ra cu trc khng gian ca DNA v ngha ca n trong vic truyn thng tin di truyn. iu ng tic l Franklin, ngi c nhng ng gp ng k cho pht minh ny mt trc . Theo qui nh th Gii Nobel khng dc php tng cho ngi mt. Deoxyribose. Phn t ng co trong thanh phn cua DNA. Di polymer (heteropolymer). Mt polymer bao gm cac loai monomer khac nhau. Phn ln protein va nucleic acid la di polymer. Di tng ng (heterologous). Cc trinh t gen khng ging nhau hon ton m ch ging nhau mt mc no . Dich chuyn im t (nick translation). Phng phap anh du DNA bng 32 phong xa [- P]dCTP nh enzyme DNA polymerase I ca E. coli. Dich ma (translation). S tng hp protein trn khun mRNA. Qu trnh chuyn thng tin di truyn trong trnh t base ca mRNA sang trnh t amino acid ca chui polypeptide trong t bo cn gi l qu trnh sinh tng hp protein. Dich ma gi oan lai (hybrid-arrested translation). Ky thut m trong s dch m ca mt phn t mRNA c bit (trong h thng dch m in vitro) b ngn chn do s hnh thnh on lai ca mRNA vi mt phn t RNA (antisense RNA) hoc DNA (v d: cDNA) b sung. Dch m ngc (reverse translation). L k thut phn lp cc gen nh kh nng ca chng trong vic lai vi mt on m oligonucleotide no , on ny c chun b bng cch d on on m nucleic acid t nhng on m ha ca protein bit trc. Dideoxyribonucleotide triphosphate (ddNTP). Mt ng phn cua dNTP dung kt thuc mt chui DNA trong cac thi nghim xac inh trnh t gen (sequencing). Dimer. La mt hn hp c tao ra gia hai phn t ng nht nhng khi lng phn t thi gp i so vi phn t nguyn thy. DNA b sung (complementary DNA, cDNA). DNA c tng hp trn khun mu mRNA nh qu trnh phin ma ngc (reverse transcription). Do vy, no co trinh t sp xp cac nucleotide b sung vi trinh t cac nucleotide trn mRNA. V du: trn mRNA trinh t o la UUGAAG thi trn cac DNA b sung se co trinh t tng ng l AACTTC. DNA b sung c tng hp t nhin trong chu trinh sng cua virus mang

vt cht di truyn la RNA. V d: HIV, virus cum va cac retrovirus noi chung. DNA b sung c tng hp nhn to trong cac phong thi nghim xy dng th vin cDNA (cDNA library). DNA cun ngc (foldback DNA). DNA co cac on xui ngc ging nhau (palindrome) hoc cac on lp lai ao ngc (inverted repeats) nn co th nhanh chong gn kt tr lai sau khi chui xon kep DNA bin tinh. DNA khun mu (template DNA). Si DNA ma trinh t cac nucleotide cua no c dung lam khun mu tng hp nn si DNA mi trong qua trinh ti bn (sao chp) hoc khuch i DNA (PCR) hoc tng hp nn si RNA mi trong qua trinh phin ma. DNA polymerase. Enzyme tng hp ban sao DNA trn khun mu DNA bng cch xuc tac phan ng gn tng nucleotide ring bit vao u chui DNA ang c tng hp. Nm 1959, hai nh khoa hc ngi M l Kornberg v Ochoa c nhn Gii Nobel v nhng nghin cu lm sng t c ch c bn ca qu trnh sao chp DNA lin quan n DNA polymerase I. DNA replicase. Phc hp enzyme v protein c hiu, cn thit cho s ti bn DNA. DNA siu xon (supercoiled DNA). DNA xon li trn bn thn n, thng l kt qu ca s gp khc, m xon hoc xon li ca chui xon kp DNA. DNA v tinh (satellite DNA). L nhng on DNA mang cc trnh t lp li ni tip c thnh phn khc vi tr s trung bnh ca DNA h gen. DNA v tinh tao thanh bng theo gradient ty trong v d dang phn bit vi bng cua phn ln DNA h gen do DNA v tinh co ty trong nho hn. Ban sao cua DNA v tinh lp lai hang triu ln trong h gen, tp trung chu yu vung tm ng va hai u cua nhim sc th. Dng (clone). Tp hp cc t bo hoc phn t ging ht nhau cng bt ngun t mt t bo hay phn t ban u. Dong phi (germ line). Cac t bao co chc nng sinh san, t o sinh ra trng va tinh bao. Dot blot. L ky thut trong o cac vt tron nho (hoc cac im) co cha nucleic acid c t ln mang nitrocellulose hoc nylon lai vi on mi DNA c nh du ng v phng x. a cistron (polycistronic). Phn t RNA ma ha nhiu chc nng. Nhiu operon vi khun c biu hin thng qua cac mRNA a cistron. a hinh dai cac oan ct han ch (restriction fragment length polymorphism, RFLP). Tnh a hnh chiu di cc on ct hn ch ch cc sai bit di truyn v tr nhn bit ca cc enzyme hn ch (chng hn nh do s thay i mt nucleotide) dn n s sai bit trong chiu di ca cc on hnh thnh t

phn ng ct hn ch DNA vi cng mt enzyme. RFLP thng c dng thit lp bn di truyn vi mt s marker di truyn bit trc. i phn t (macromolecule). Mt phn t c khi lng khong t vi nghn ti nhiu triu. anh du ui (end labelling). B sung phn t phong xa vao ui cua mt polynucleotide nh enzyme T4 polynucleotide kinase. u bng (blunt end). Cac u cua DNA si i khng co cac u li 3 hoc 5 (protruding ends). u dinh (cohesive ends hoc sticky ends). Cac u cua phn t DNA co cac trinh t b sung ngn co th dinh kt lai ni hai phn t DNA vi nhau. Cac u dinh thng do cac enzyme han ch tao ra. u tn cng C (C terminus). Gc carboxyl (COOH) t do v tr tn cng cua mt phn t protein hoc chui polypeptide. u tn cng N (N terminus). Gc amine (NH2) v tr tn cng ca mt phn t protein hoc chui polypeptide. Tt c cc polypeptide u c tng hp bt u t u ny, n gc carboxyl tn cng. im t (nick). im t gy mt si n trn DNA si i. im nng (hot spot). V tr ca gen c xu hng b t bin nhiu hn hn so vi cc v tr khc trn phn t DNA. in di trn gel (gel electrophoresis). K thut dng phn tch cc phn t nucleic acid hoc protein da vo s dch chuyn ca chng trn gi th dng gel (agarose hoc polyacrylamide) di nh hng ca in trng. S dch chuyn ca cc phn t ny ph thuc vo in tch, cu hnh, kch thc v khi lng phn t ca nucleic acid hoc protein cng nh dung mi v nng ca cht dng lm gi th. iu ha hot ng gen (gene operation regulation). iu khin s hot ng ca cc gen trong qu trnh pht trin c th, m bo cho cc gen hot ng thi im cn thit, v do vy qu trnh pht trin c th din ra bnh thng. M hnh operon ca Monod v Jacob l c ch iu ha hot ng gen n gin nht. nh m (capping). Gn nucleotide guanine (G) c methyl ha (ci m) vo u 5 ca phn t mRNA tin thn (pre-mRNA) sinh vt eukaryote, ci m sau c gi li phn t mRNA hon chnh (mature mRNA). on duy nht (unique sequence). Cc on DNA ch c mt hoc mt vi bn sao trong h gen. oan Klenow (Klenow fragment). Cn gi l oan ln cua DNA polymerase I. y l mt oan cua DNA polymerase I (khi lng phn t 76.000) ca E. coli bi mt hoat tinh exonuclease 53.

on lp li o ngc (inverted repeats). Hai oan DNA ng nht, trn hai hng ngc nhau cung mt phn t. S lp lai nh vy se tao ra hin tng palindrome. on lp lai cng chiu (direct repeats). La nhng chui m di truyn ng nht ai din cho mt hoc hai ban sao theo cung mt hng cua mt phn t DNA. on Okazaki (Okazaki fragment). Cc on DNA si n tng i ngn ln lt c tng hp trong qu trnh sao chp (ti bn) DNA theo kiu gin on dc theo mt trong hai si khun, trong khi dc theo si khun kia th mch DNA mi c tng hp lin tc. Sau , cc on Okazaki c ni lin vi nhau bng cc mi lin kt ng ha tr nh enzyme ligase. oan xp (nested fragments). Mt lot cc on nucleic acid c chiu di ch khc nhau mt hoc mt vi nucleotide. oan xui ngc ging nhau (palindrome). on DNA mch kp c trnh t sp xp cc base trn hai mch n ging ht nhau nu cng c c theo mt chiu (chng hn 53). V d: cc on nhn bit ca enzyme hn ch. ng du (replica plating). Phng php chuyn nguyn mu cc khun lc hoc vt tan t mt a thch gc sang a thch mi bng cch dng mng nylon (v d: mng Hybond-N+) va kht p ln mt thch ca a gc dnh ly cc t bo trong cc khun lc (colony) hoc vt tan (plaque) ca a gc, ri a mng ny p ln mt thch mi. n cistron (monocistron). Mi mRNA monocistron ch m ha cho mt protein eukaryote. n vi phin ma (transcriptional unit). oan DNA ma ha cho phn t RNA, bt u t im khi u phin ma n im kt thc phin ma, n c th di hn mt gen. n v sao chp (replicon). on DNA bt u t im khi u sao chp ko di v hai pha ti hai im kt thc sao chp. n v ti t hp (recon). on DNA ca gen c chiu di ngn s trao i cho khng th din ra bn trong n c na. Hin nay, c bit l mt cp nucleotide. c tnh (virulence). Kh nng ca cc bacteriophage gy tan t bao vi khun chu khi chung xm nhim vao. i m (anticodon). L mt on gm ba nucleotide trn tRNA bt cp vi cm m (codon) trn mRNA theo nguyn tc b sung. ng polymer (homopolymer). Mt polymer chi bao gm mt loai monomer, chng han nh polyphenylalanine (protein) hoc polyadenine (nucleic acid). t bin (mutation). Bin i trong trinh t cac base cua DNA. Co th c gy nn bi chn oan, mt oan hoc co cac base bi sa i.

t bin dch khung (frameshift mutation). t bin lm mt hoc thm mt cp base trong gen dn ti hu qu lm dch khung dc bnh thng ca mRNA i mt nucleotide. Do vy, lm thay i ton b thng tin di truyn on gen bt u t im xy ra t bin n cui gen. t bin ng hon (transition mutation). Kiu t bin thay th mt cp base purine-pyrimidine ny bng mt cp base purine-pyrimidine khc ti mt im trn DNA. V d: thay cp GC bng cp AT hoc ngc li. t bin gen (gene mutation). Cn gi l t bin im, xy ra do nhng bin i ca tng nucleotide nh: mt, thm hay thay i v tr ca nucleotide trn phn t DNA. L nguyn nhn ca s hnh thnh protein khng bnh thng. Phn ln t bin l c hi nhng chng vn thng c gi li trong qun th v thng l ln v do c th c duy tr trong kiu gen m khng nh hng n sc sng ca sinh vt. Tc t bin t nhin thp, nhng tn s t bin c th tng nh cc tc nhn nh phng x ion ha v cc ha cht gy t bin gy ra. t bin khuyt dng (auxotrophic mutation). Cn gi l t bin ha sinh. t bin lm mt kh nng tng hp mt cht cn thit cho s sinh trng ca t bo. t bin l r (leaky mutation). t bin ch lm gim hot tnh enzyme m khng lm mt hn hot tnh . t bin ngc (reverse mutation hay reversion). Cn gi l hi bin. t bin xy ra theo chiu t dng t bin tr v dng bnh thng, c tc dng phc hi trnh t nucleotide ban u. t bin nhm ngha (missense mutation). t bin gen khi mt cp base trong b ba (codon) b thay th bi mt base khc, dn n s thay th amino acid v tr tng ng trn chui polypeptide bng mt amino acid khc. t bin c ch (suppressor mutation). t bin xy ra mt v tr trn DNA c tc dng loi b hon ton hoc tng phn hu qu ca t bin ban u xy ra mt v tr khc. t bin v ngha (nonsense mutation). t bin gen khi mt base trn DNA b thay th bng mt base khc lm cho b ba tng ng trn mRNA t ch xc nh mt amino acid chuyn thnh b ba v ngha (khng xc nh amino acid no), mang tn hiu chm dt qu trnh dch m. V d : amber codon (t bin amber, amber mutation) hay ochre codon (t bin ochre, ochre mutation). Kt qu l chui polypeptide ch c tng hp n b ba xy ra t bin v ngha th dng li. ui polyA (polyA tail). on trnh t di 50-200 nucleotide adenine c b sung vo u 3 ca hu ht cc mRNA eukaryote sau khi phin m. E. coli (Escherichia coli). Vi khun thng c trong rut non ca ng vt c xng sng. E. coli c coi nh sinh vt mu cho vic nghin cu hot ng ca t

bo. y l vi khun Gram m c kch thc genome khong 4106 base-pair. Cc qu trnh biu hin gen (phin m v dch m) i i vi nhau, sinh ra si mRNA c tng hp mi v c s dng ngay cho qu trnh dch m. Khng c hin tng bin i sau dch m (post-translation). V th, E. coli c xem l mt trong nhng t bo vt ch n gin nht. Rt nhiu th nghim to dng gen ang c thc hin hng ngy ti cc phng th nghim u s dng E. coli lm vt ch vi nhiu chng khc nhau v mt di truyn v cho nhng ng dng c bit. Endopeptidase. Enzyme xc tc ct lin kt peptide trong chui polypeptide. Endonuclease. L enzyme nuclease ct bn trong phn t nucleic acid, ngc vi exonuclease la enzyme phn giai DNA t mt u hoc ca hai u. Nuclease thy phn nhng lin kt phosphodiester gia cc nucleotide ca mt phn t nucleic acid. Cc nuclease c th c hiu i vi DNA (deoxyribonuclease) hoc c hiu i vi RNA (ribonuclease). Enzyme. Cht xc tc sinh hc, l cc phn t sinh hoc co ban cht protein ong vai tro cht xuc tac cho cac phan ng bin i ha sinh. Enzyme ng phn ha (isomerase). Enzyme xc tc cho s chuyn ha mt hp cht thnh mt ng phn v tr. Enzyme gn DNA (DNA ligase). Enzyme dung ni cac phn t DNA vi nhau bng cach tao ra mi lin kt phosphodiester gia nhom 5-phosphate va nhom 3 hydroxyl trong qua trinh ti bn hoc sa cha DNA. Enzyme han ch (restriction enzyme, RE). Loai endonuclease co kha nng ct DNA tai nhng oan trinh t nht nh m chung nhn bit. Enzyme hn ch c pht hin vo nm 1970, chng tn ti trong t bo vi khun, c tc dng ct DNA ngoi lai (v d: DNA ca phage) ti nhng im xc nh, tiu dit DNA ny. Cho n nay hn 900 enzyme hn ch c tm thy. Cc enzyme hn ch c s dng rng ri trong cc phng th nghim thao tc gen nh nhng chic ko ct DNA ti nhng im c hiu. V tr ct ph thuc vo loi enzyme hn ch c la chn. Nm 1978, Arber (Thy S), Nathans (M) v Smith (M) c nhn Gii Nobel nh pht minh ra enzyme hn ch v nhng ng dng ca chng gii quyt nhiu vn quan trng ca di truyn hc phn t. Cc enzyme ny l nhng chic ko phn t c th ct DNA thnh nhng on xc nh, m ra mt thi k pht trin mi ca sinh hc hin i-Thi k thao tc gen. Enzyme phin ma ngc (reverse transcriptase). DNA polymerase phu thuc RNA (RNA-dependent DNA polymerase) co trong cac RNA virus (retrovirus) c dung tng hp cDNA trong iu kin in vitro. Enzyme ton phn (holoenzyme). Enzyme hot ng gm tt c cc tiu n v cn thit, cc nhm ngoi v nhng cofactor.

Episome. Nhn t di truyn c trong vi khun, c kh nng t sao chp trong t bo cht, ng thi li c th xen vo nhim sc th chnh ca vi khun v sao chp ng thi vi nhim sc th. Exon. Cc on DNA trong gen c chc nng phin m. Exon tn ti c sinh vt prokaryote v eukaryote. Ring sinh vt eukaryote cc exon nm xen k vi cc on intron. Cc intron chim ti 90% tng s DNA ca t bo eukaryote v khng c chc nng phin m. Eukaryote. Sinh vt c t bo mang nhn in hnh (nhn tht) ngha l nhn c bao bc bi mng nhn v tham gia vo hai c ch phn bo quan trng l nguyn phn v gim phn. Exonuclease. Loi enzyme nuclease ch tc ng vo u tn cng ca phn t nucleic acid, ct ra tng nucleotide mt theo thi gian. Chng c th chuyn ha theo u 5 hoc 3 ca si DNA. Ex vivo. Thut ng dng ch cc th nghim thc hin trn t bo nui cy, cc t bo ny sau s c a vo mt c th sng. -galactosidase. Enzyme c m ha bi gen lacZ. Enzyme ny thy phn lactose thnh glucose v galactose. Gy t bin nh hng bng oligonucleotide (oligonucleotide-directed mutagenesis). Cn gi l gy t bin nh hng im (site-directed mutagenesis), l qu trnh to ra mt bin i xc nh trn DNA bng cch s dng mt oligonucleotide tng hp (primer) c mt vi bin i trong trnh t nucleotide. Gel chm (gel retardation). Phng php xc nh im bm ca protein trn cc on DNA, da vo di chuyn chm ca chng so vi DNA khng b protein bm trong cc th nghim in di trn gel. Gen (gene). L n v di truyn, yu t quyt nh mt tnh trng c th. Thng tin di truyn ca cc gen c m ha trong DNA quyt nh tnh bin d ca loi v ca c th. DNA l mt chui bao gm cc n v nucleotide, c bn loi nucleotide mang bn nitrogen base khc nhau l adenine (A), guanine (G), cytosine (C), v thymine (T). Trnh t cc nucleotide ca mt gen xc nh mt polypeptide hoc mt RNA. Gen c kh nng b t bin. Cc gen ch yu nm dc theo nhim sc th trong nhn t bo. Mi gen chim mt v tr xc nh trn nhim sc th gi l locus. Gen c th tn ti nhiu dng gi l cc allele. Cc gen biu hin thng qua cc phn t do chng sinh ra l RNA (trong qu trnh phin m) v protein (trong qu trnh dch m). Gen cu trc (structural gene) hay gen m ha (coding gene). L trnh t m ha sn phm protein. Gen ch th (reporter gene). L mt gen m ha m sn phm ca n c trc nghim mt cch d dng (v d chloramphenicol acetyltranferase), n c th c gn

vi bt c mt promoter no sao cho s biu hin ca gen ny c dng th nghim chc nng ca promoter. Gen iu ha (regulatory gene). Mt gen m sn phm protein ca n tham gia vo s iu ha biu hin ca mt gen khc. V d: gen m ha mt protein km hm. Gen gy cht (death gene). Gen lm cht c th mt giai on no trong qu trnh pht trin ca chng. Gen nhy (transposon). L mt on DNA nh c cu trc c bit nn c kh nng di chuyn t mt v tr ny n mt v tr khc trn phn t DNA hay t mt nhim sc th ny sang mt nhim sc th khc trong t bo lng bi. Gen nhy ch chuyn n nhng v tr xc nh trong h gen. Trong nhiu trng hp gen nhy c th gy t bin ti v tr n di chuyn n. Gen nhy c th dng lm phng tin gy t bin nh hng. Thng cac gen nay mang tnh khng khng sinh giup vi khun thich nghi nhanh vi thuc khang sinh. Nm 1983, McClintock (M) nhn Gii Nobel nh khm ph ra cc nhn t di truyn vn ng (gen nhy) trn i tng cy ng. Gen c ch (repressor gene). C hai chc nng: (1) L mt gen iu ha (regulator) m sn phm ca n l mt protein c tc dng c ch hot ng phin m ca mt operon. (2) L mt gen phc hi (suppressor) kiu hnh ban u ca t bin trong mt gen khc. Ghp i lch (mismatch). S ghp i khng ng vi quy lut b sung gia cc nucleotide thuc hai si n DNA trong mch kp. Ghp exon hay splicing (RNA). Qu trnh ct b nhng intron v ni cc exon ca sn phm phin m ban u (tin thn mRNA) to thnh mRNA hon chnh (mature mRNA). Qu trnh bin i ny xy ra trong nhn t bo. Gi thuyt mt gen-mt enzyme (one gene-one enzyme hypothesis). Gi thuyt da trn cc cng trnh ca Beadle v Tatum v di truyn hc ha sinh, theo mi gen kim sot s tng hp mt enzyme. Gim phn (meiosis). S phn bo tri qua hai giai on ca mt t bo lng bi (2n) dn n s hnh thnh cc giao t n bi (1n) hoc cc bo t hu tnh mang mt na vt cht di truyn ca t bo lng bi ban u. Gim phn I (meiosis I). Ln phn chia th nht ca gim phn lm gim s lng nhim sc th i mt na v to ra cc t hp nhim sc th mi. Ln phn chia ny gm bn giai on: pha u, pha gia, pha sau v pha cui. Gim phn II (meiosis II). Ln phn chia th hai ca gim phn dn n s phn tch cc nhim sc t v hon thnh qu trnh gim phn ni chung. Gc ti bn (origin, ori). Trnh t nucleotide hoc v tr trn DNA m bt u s ti bn (sao chp).

Guanine (G). Mt trong nm nitrogen base c trong thnh phn ca DNA v RNA, bt cp vi cytosine (C) trong chui xon kp nh ba lin kt hydrogen. Helicase. Enzyme xc tc vic tho xon ca chui xon kp DNA trong qu trnh sao chp E. coli. H gen hay b gen (genome). L tp hp cc gen c trong mt t bo n bi eukaryote, trong mt t bo prokaryote hoc trong mt virus. H gen cha toan b DNA cua c th, vi du: h gen ngi cha DNA dai khoang 1,6 m nhng chi rng khoang 5 phn ti mm. ngi, s DNA noi trn c chia lam 46 phn co dai ngn khac nhau goi la nhim sc th (chromosome), la tp hp DNA dang nen cht n kich thc ng kinh chi con 3-4 phn triu met. Tuy nho nh vy, nhng nhim sc th ngi co n 3 t gc nucleotide. S sp xp c thu cua chung quyt inh ban cht sinh hoc cua c th. Hiu ng glucose (glucose effect). Hin tng kh hot tnh ca operon cm ng vi khun khi c mt glucose k c khi hin din cht cm ng operon. Histone. Nhm protein kim nm trong phc hp vi DNA cc nhim sc th eukaryote v ng vai tr quan trng trong vic xc nh cu trc ca nhim sc th. Hon chuyn gen (gene conversion). S hon chuyn gen la s thay i mt mch n cua chui xon kp DNA giup no b sung bng mt mch n khac bt c vi tri nao ma no a bi mt cp ni base. Hot tnh c hiu (specific activity). L hot phng x trn mt n v nguyn liu, chng hn: mt mu d (probe) nh du phng x c th c hot tnh c hiu 106 ln m/pht trn microgram. Hot tnh c hiu cng c dng xc nh hot ca enzyme. Hi tnh (renaturation). S ti bt cp ca hai mch n DNA b sung ca mt phn t DNA mch kp. Hp CAAT (CAAT box). on DNA nm ngc hng cch im bt u phin m khong 75 nucleotide hin din sinh vt eukaryote. y l mt trong nhng on tng cng s bm ca RNA polymerase. Hp Goldberg-Hogness (Golderg-Hogness box) hay hp Hogness (Hogness box) hay hp TATA (TATA box). on DNA c trong cc promoter eukaryote tng t hp Pribnow c trong cc sinh vt prokaryote, nm cch v tr bt u phin m khong 30 nucleotide, c coi l operator sinh vt eukaryote, c trnh t ph bin ca si bn l TATAAAT. Hp Pribnow (Pribnow box). Mt phn ca promoter trong cc h gen prokaryote, nm cch pha trn im khi u phin m 10 nucleotide. Trnh t ca si tng ng vi hp Pribnow l TATAAT.

In du DNA (DNA fingerprinting) hay in du di truyn (genetic fingerprinting). L phng php dng cc mu d phng x hoc dng k thut PCR nhn dng cc bng DNA c cc on lp li vi tn s cao. Bn mu hnh cc bng DNA l duy nht i vi mi c th, v do vy c th dng xc nh c trng c th hoc quan h huyt thng. In du chn DNA (DNA footprinting). Phng php nhn dng cc vng DNA m cc protein iu ha bm vo. Intron. Nhng on DNA nh sinh vt eukaryote khng mang thng tin m ha amino acid, phn b ri rc dc theo phn t DNA. Sau khi thng tin t DNA c phin m sang mRNA th cc intron trn mRNA b ct b, cc on mRNA cn li gm ton cc exon c ni li vi nhau v chuyn n ribosome dng lm khun mu cho qu trnh dch m. Intron khng thy c sinh vt prokaryote. In vitro v in vivo. La thut ng m ta thi nghim trong ng nghim (in vitro) va trong c th (in vivo). Cung vi s pht trin ng dung cua may tinh, hin nay cac nha khoa hoc con tin hanh thi nghim m phong bng computer. Qua trinh nay goi la thi nghim in silico. Isozymes hay isoenzymes. Nhm cc enzyme cng xc tc mt phn ng nhng khc nhau v cu trc s cp hoc tc in di, thut ng allozymes cung co y nghia tng t. Ko di on mi (primer extension). S tng hp mt bn sao nucleic acid bt u t on mi. c s dng nh du phng x on DNA lm mu d hoc khuch i mt on DNA bng k thut PCR. Khong cch bn (map distance). S trao i cho trung bnh xy ra gia hai locus gen trn nhim sc th hoc trn si DNA. Khuch i (amplification). S sn xut nhiu bn sao ca mt trnh t DNA. Khung c m (open reading frame, ORF). L mt trnh t m ha chui polypeptide c bt u vi m khi u (initiator codon) v kt thc bng mt m dng (stop codon). Mt khung c m b ngn chn nu mt stop codon c nh v gn vi m khi u. Mc d v l thuyt ma di truyn c xy dng da trn b ba nucleotide do s co ba khung c co th co trn mi si DNA, tuy nhin trong thc t khung c chnh xc c xc nh bi mt im bt u c nh. Khuyt on (deletion, deficiency). t bin nhim sc th dn n lm mt mt on vt cht di truyn v thng tin di truyn cha trong n ri khi nhim sc th. Kiu gen (genotype). Cu trc di truyn ca c th. Hin trng ca c th (kiu hnh) ph thuc vo quan h tri-ln gia cc allele trong kiu gen v vo mi tng tc gia kiu gen v mi trng.

Kiu hnh (phenotype). Ngoi hnh ca mt sinh vt, l kt qu tng tc gia kiu gen vi mi trng. Nhiu gen c trong kiu gen nhng khng biu hin ra kiu hnh v chng b che khut bi cc allele tri. Nhng sinh vt c cng kiu gen c th c cc kiu hnh rt khc nhau trong nhng mi trng khc nhau. Kiu hnh ca c th ng hp t v allele ln gi l kiu hnh ln. Kiu hoang di (wild type). Dng thng thy nht ca mt gen trong qun th hoang di. Allele kiu hoang di c k hiu bng mt ch in hoa hoc thm du cng sau ch vit thng, v d: A hay a+. Allele kiu hoang di thng l tri v cho kiu hnh bnh thng. Kiu nhn (karyotype). B nhim sc th c trng ca mt loi vi s lng, kch thc v hnh thi xc nh ca cc nhim sc th. Mi loi c c trng bi kiu nhn ring ca n. Cc loi khc nhau c kiu nhn khc nhau. Kilobase (kb). 1000 base (hoc cp base), c dng nh n v o hoc xc nh chiu di ca cc phn t DNA hoc RNA. Kinase. Cc enzyme xc tc phn ng phosphoryl ha mt phn t nhn nh ATP. Lai (hybridization). Ghp cc phn t nucleic acid tch bit li vi nhau thng qua cc mi lin kt hydrogen gia cc base b sung. Lai khun lc (colony hybridization). Ky thut lai in situ xac inh vi khun mang vector khm (chimeric vector) ma DNA cua vector nay tng ng vi mt oan ma ha c bit nao o. Lai phn t (molecular hybridization). Qu trnh trong hai mch nucleic acid b sung (A-T, G-C) bt cp hnh thnh nn mt mch kp. y l mt k thut hu ch pht hin mt trnh t nucleotide chuyn bit. Lai ti ch (in situ hybridization). Qu trnh bt cp gia mu d (l mt trnh t DNA si n hay RNA) vi DNA ca t bo c c nh trn lam knh. Lp bn hn ch (restriction mapping). K thut dng xc nh v tr cc im ct hn ch trn phn t DNA. Lin kt peptide (peptide bond). Mi lin kt ng ha tr trong chui polypeptide, ni nhm -carboxyl ca mt amino acid vi nhm -amine ca mt amino acid k tip. Lin kt phosphodiester (phosphodiester bond). Mt phn t cha hai alcol, ester ha vi mt phn t phosphoric acid lm cu ni gia chng vi nhau. V d: chui polynucleotide c ni bi lin kt 53 phosphodiester gia hai nucleotide cnh nhau. Linker. Mt oligonucleotide tng hp c hai u bng, cha mt hoc nhiu vi tri ct han ch cho php ni cDNA si i vi cac plasmid vector hoc bacteriophage

vector. cDNA si i trc c x ly vi DNA polymerase cua bacteriophage T4 hoc DNA polymerase I cua E. coli to u bng. Cc linker sau khi gn vi hai u bng ca on cDNA nh DNA ligase s c ct hn ch to ra u so le tng ng vi hai u ca vector. Phn ng gn gia on cDNA c mang linker hai u vi vector cng c xc tc nh DNA ligase. Locus. V tr m gen nh v trn nhim sc th. LTR (long-terminated repeat). La mt oan ma c lp lai trc tip ca hai u cua mt retroviral DNA. Lysosome. Mt bo quan c mng bao bc trong t bo cht ca nhng t bo eukaryote. Lysosome cha nhiu enzyme thy phn. L thuyt trung tm (central dogma). Thuyt cho rng thng tin c truyn theo mt chiu t DNA n RNA v n protein. Mapping and sequencing gene. Lp ban gen va xac inh trnh t cc nucleotide. M di truyn (codon). Nhm ba nucleotide nm k nhau (b ba) trn phn t mRNA xc nh mt amino acid trn chui polypeptide, hoc l tn hiu kt thc vic tng hp polypeptide. M thoi bin (degenerate codon). M di truyn m mt amino acid c quy nh bi mt s b ba nitrogen base, ch khng phi ch bi mt b ba. Thoi bin l c im vn c ca m di truyn tn ti ph bin sinh gii. Ma v nghia (nonsense codon) hay m dng (stop codon). L codon ma o qua trinh dich ma dng lai (ni kt thuc cua chui polypeptide). Co tt ca ba codon v nghia vi cac tn goi la amber (UAG), ocher (UAA) va opal (UGA) Maturation. Qu trnh trong cc mRNA va c phin m tri qua mt s bin i ha hc tr thnh mRNA hon chnh sn sng lm khun mu cho vic tng hp protein. My m nhp nhy (scintillation counter). My dng xc nh hot tnh phng x trong mt mu th nghim. Mu d DNA (DNA probe). Mt on RNA hay DNA chuyn bit c nh du bng ng v phng x hay bng ha cht (cht pht hunh quang hoc enzyme), dng nh v mt trnh t nucleic acid nht nh thng qua k thut lai phn t (xem Northern blot, Southern blot...) Monomer. L cc phn t n v nh, c th lin kt vi cc phn t n v ging n hnh thnh nhng phn t ln hn (polymer). V d: cc nucleotide l cc monomer ca nucleic acid v cc amino acid l monomer ca protein. M hnh bo ton. M hnh sao chp DNA m theo hai si DNA b m vn c gi nguyn v lm khun tng hp chui xon kp mi (chui con).

Mi (primer). Mt trnh t DNA hay RNA ngn, bt cp vi mt mch ca DNA khun mu v c mang mt u 3-OH t do gip DNA polymerase bt u tng hp mt chui DNA mi. M chp (cap). Mt nucleotide bin i, 7-methyl guanine, gn vo u 5 ca base u tin mi chui mRNA ngay khi bt u phin m. M cn thit cho s hon chnh, s n nh v s dch m ca mRNA. Ngoi t bo (exocytosis). Hin tng phng thch vt cht ni bo ra ngoi t bo. Nguyn dng (prototroph). Cc t bo c th mc trn mi trng khng cn b sung thm cht dinh dng. Nguyn phn (mitosis). Ti bn cc nhim sc th trong t bo sinh dng eukaryote. S phn chia nhng t bo eukaryote ko theo s phn chia nhn gi l mitosis. Qu trnh phn chia t bo qua nm giai on (prophase, prometaphase, metaphase, anaphase v telophase), sau mt t bo c phn chia v to ra hai t bo c tnh cht di truyn ging vi t bo m. Nhn t (housekeeping factor). L mt nhn t khi u c bn cht enzyme ca sinh vt prokaryote, n l mt phn ca RNA polymerase gn vi cc v tr promoter trn DNA trong qu trnh phin m. Cc nhn t khc nhau c hot ha trong phn ng vi cc iu kin mi trng khc nhau. Mi phn t RNA polymerase c chnh xc mt nhn t tiu n v. Vi khun E. coli c by nhn t , trong khi cc loi vi khun khc c s lng cc nhn t rt khc nhau. Nhn t gii tnh (fertility factor, F). L nhn t di truyn vi khun xc nh gii tnh c hoc ci ca chng. E. coli, nhn t hu th k hiu l F xc nh gii tnh c. Khi trong t bo khng c nhn t F th l t bo ci, k hiu l F . Khi nhn t F nh vo nhim sc th th l t bo siu c k hiu l Hfr. Nhn t ko di (elongation factor, EF). Protein c hiu tham gia vo vic a cc phn t aminoacyl-tRNA n ribosome cho amino acid trong phc h phn t ni trn c th gn vo chui polypeptide ang c tng hp. Nhn t kt thc (termination factor). Yu t protein trong cytosol cn thit cho vic gii phng chui polypeptide hon thnh trn ribosome (cng gi l release factors-RF). Nhn t khi u (initiation factor). prokaryote vit tt l IF, cn eukaryote l eIF. L cc protein kt hp vi tiu n v ca ribosome vo giai on khi ng ca qu trnh dch m. Nhn t Rho (Rho factor). Mt protein c vai tr kt thc s tng hp mt s mRNA. Nhim sc th (chromosome). Mt thnh phn ca t bo, bao gm cc protein v mt phn t DNA c nht. DNA cha nhiu gen, c chc nng d tr v truyn

thng tin di truyn. S lng nhim sc th trong mt t bo c th thay i t 1 ( vi khun) cho ti trn 100, nhng khng thay i mt loi nht nh (v d: 46 i vi ngi). Nhim sc th thng (autosome). Tt c nhim sc th tr nhng nhim sc th gii tnh hoc nhim sc th ca ty th. Nhim sc t (chromatide). Hai chui song song v ging nhau, ni vi nhau bi phn tm ca mt nhim sc th dng n c nhn i, thc th ny tn ti cho n khi bt u k sau (anaphase). Northern blot. K thut chuyn v c nh RNA t formaldehyde agarose gel (sau khi c phn on bng in di) ln mng lai bng nylon hoc nitrocellulose lai vi mu d (probe) c nh du ng v phng x [-32P]dCTP hoc digoxigenin-dUTP. Tn hiu lai sau c pht hin trn phim X-quang (trng hp [-32P]dCTP) hoc trn mng lai (trng hp digoxigenin-dUTP). Ni thc bo (endocytosis). S xm nhp ca cht ngoi t bo vo trong t bo. Nuclease S1. Enzyme thy phn DNA si n. Nucleic acids. Nhng polynucleotide sinh hc thin nhin, trong nhng n v nucleotide c kt hp vi nhau bi nhng lin kt phosphodieste thnh trnh t DNA hoc RNA ring bit. Nucleolar organizer. La vung mang gen ma ha cho rRNA cua mt nhim sc th. Nucleolus. La mt vung c bit cua nhn, c tao ra do s giai ma cua nhng gen rRNA. Nucleoside. Mt hp cht gm mt base purine hoc pyrimidine kt hp ng ha tr vi mt phn t ng pentose. Nucleosome. n v cu trc ca nhim sc cht, gm mt on DNA c kch thc khong 200 bp qun bao quanh mt li tp hp 8 protein histone. Nucleotide. Mt nucleoside phosphoryl ha vi mt trong nhng hydroxyl ca pentose. Phn t ng vai tr cu trc c s ca DNA v RNA, gm ba phn: ng pentose (ribose trong RNA, deoxyribose trong DNA), nitrogen base v gc phosphate. Nucleolytic. Phan ng thy phn mt cu ni phosphodiester trong mt nucleic acid. Nuclease Bal 31. Mt loi enzyme exonuclease thy phn c hai si ca phn t DNA cng mt lc. Oligo. Tip u ng c ngha l mt t, v d: oligonucleotide (polynucleotide) c mt t nucleotide hoc oligopeptide (polypeptide) c mt t peptide.

Oligo(dT)-cellulose. Mt on ngn gm cc gc deoxythymidine lin kt vi c cht cellulose, c s dng tinh sch mRNA eukaryote. Oligomer. Thut ng chung ch mt on ngn cc monomer. Oligonucleotide. Mt on ngn cc monomer l nucleotide, thng t 20-30 nucleotide. Operon. Nhm cc gen vi khun chu s iu khin chung ca mt gen iu ha. Cu trc operon bao gm: 1) Nhm gen cu trc m hot ng ca chng chu s iu khin chung, 2) Gen iu ha sn sinh ra protein iu ha, 3) Vng ch huy (operator) v trnh t khi ng (promoter) chu tc ng ca protein iu ha. Protein iu ha c tc dng bm vo vng ch huy lm cho n b ng, do vy nhm gen cu trc ngng hot ng. Khi trong mi trng xut hin c cht ca cc gen cu trc th c cht ny bt hot ha protein iu ha khin n khng bm vo vng ch huy c na, vng ch huy c m v nhm gen cu trc li hot ng. Operon chu c ch (repressible operons). Cc operon sinh tng hp chui amino acid b c ch khi mt cht (v d: chui amino acid ) c a vo mi trng. Orche codon. Chui kt thuc bng codon UAA. Opal codon. Chui kt thuc bng codon UGA. n ha (temperate). Trng thi tim tan ca cc bacteriophage t bo vt ch. -peptide. Mt phn ca protein -galactosidase, c m ha bi on gen lacZ. Periplasm. Phn ngoi bo tng nm gia lp mng ngoi v mng trong ca vi khun. Peroxisome. Bo quan c trong t bo cht eukaryote cha enzyme xc tc cho s to thnh v phn hy peroxide (H2O2). Phage. Vit tt ca bacteriophage (thc khun th), l loi virus xm nhim v sinh sn bn trong vi khun. Phage thng c v bc protein, phc hp bao gm phn u c hnh a din cha nucleic acid (DNA hoc RNA) v ui m qua nucleic acid xm nhp vo vi khun ch. Sau qu trnh nhn ln ca nucleic acid ca phage, t bo vi khun ch thng b tan bin. Loi phage lun lun lm tan t bo vi khun khi chng xm nhim vi khun gi l phage c. V d: phage T4. Ngc li, cn c phage n ha, khi xm nhim vi khun n gy nn phn ng tim tan, ngha l h gen ca phage gn vo nhim sc th vi khun v c sao chp cng vi nhim sc th . H gen ca phage trng thi gn nh vy vi nhim sc th vi khun gi l prophage. Phagemid. L mt loi plasmid vector c mang cc on trnh t ca phage.

Phn ng chui polymerase (polymerase chain reaction, PCR). Phng php dng trong phng th nghim khuch i cc on DNA c bit ln hng triu ln trong vng vi gi thng qua 20-30 chu k nhit, mi chu k bao gm ba mc nhit : bin tnh 90-95oC, bt cp vi mi 40-65oC hoc hn v tng hp mch mi nh DNA polymerase chu nhit (Taq polymerase) 70-72oC. PCR c ng dng rng ri trong chn on y hc, phn tch s a dng sinh hc, chn ging v trong nhiu lnh vc khc. Nh khoa hc M (Tin s Mullis) ngi pht minh ra k thut PCR nhn gii Nobel nm 1993. Cng chia s Gii Nobel vi Mullis l Smith (Canada) do c nhng ng gp mang tnh nn tng cho vic gy t bin im nh hng, da trn cc oligonucleotide v vic pht trin chng trong cc nghin cu protein. Phan ng tp trung (condensation reaction). La phan ng trong o cu ni c hinh thanh vi s mt i mt phn t nc v thm vao mt amino acid trong qua trinh hnh thanh chui polypeptide. Phn ng thy phn (hydrolysis). Phn ng trong mt lin kt cng ha tr b ph v khi c s kt hp mt phn t nc. Pht sinh t bin (mutagenesis). Qu trnh to ra t bin trong DNA. Phn tch trnh t gen (gene sequencing). L k thut xc nh trnh t theo cu trc bc mt ca chui cc nucleotide trong mt phn t nucleic acid. Phn tch trnh t ca DNA c cc phng php ha hc ca Maxam-Gilbert v phng php enzyme ca Sanger. Trong nhng nm gn y, mt s phng php xc nh trnh t mi nh s h tr ca my tnh xut hin. Bn cnh k thut thng thng s dng cc polyacrylamide gel phn ly cc phn t DNA c di khc nhau, cc k thut mi lin quan n pht hin hunh quang ca cc nucleotide c nh du, phn tch trnh t DNA bng khi ph, in di mao dn hoc lai vi cc on oligonucleotide c tng hp nhn to cng ra i. Nm 1980, Sanger (Anh) v Gilbert (M) c trao gii Nobel do c nhng ng gp quan trn v phng php xc nh trnh t cc nucleotide trong phn t DNA. ng gp ny l mc lch s to ln trong sinh hc phn t, l nguyn l ca tt c cc my xc nh trnh t DNA t ng ang s dng hin nay trn khp th gii. Phn cui (telomere). on cui, phn cui ca mt nhim sc th thng ca eukaryote, bao gm nhng trnh t DNA ngn c lp li nhiu ln. Phn tm (centromere). Phn co tht c thy trn nhim sc th k gia, l ni hai nhim sc t nh vi nhau. Phin m (transcription). Qu trnh tng hp mRNA t khun mu DNA. Phin m ngc (reverse transcription). Qu trnh tng hp DNA t khun mu mRNA nh enzyme phin m ngc (reverse transcriptase).

Phng x t ghi (autoradiography). K thut pht hin cc phn t c nh du phng x thng qua hiu ng to nh ca cc phn t ny trn phim X-quang. Phosphoryl ha (phosphorylation). Phn ng to thnh mt dn xut phosphate ca mt phn t sinh hc di tc dng xc tc ca mt enzyme. Phosphatase kim (alkaline phosphatase). Enzyme loi b cc nhm 5phosphate t u ca cc phn t DNA v li cc nhm 5-hydroxyl. Phi t (ligand). Mt phn t hoc mt ion kt hp vi mt protein, v d: hormone c th kt hp vi th th (receptor) c hiu ca n. Plasmid. La DNA vi khun c cu trc mch vng kp, nm trong t bo cht v ngoi nhn, c kh nng sao chp c lp i vi nhim sc th ca t bo. Tn ti c sinh vt prokaryote v eukaryote. Ngy nay, cc plasmid thit k nhn to c s dng rng ri nh l cc vector dng trong cc k thut to dng v biu hin gen. Polynucleotide. Trnh t nhng nucleotide ni ng ha tr vi nhau, trong v tr 3 ca pentose ca mt trong nhng nucleotide c ni vi mt lin kt phosphodieste v tr 5 ca pentose ca nucleotide tip theo. Polypeptide. Mt chui di nhng amino acid ni vi nhau bi nhng lin kt peptide. Polysome (polyribosome). Cu trc gm nhiu ribosome kt hp vi mRNA trong qu trnh dch m. Nhng polysome t do trong t bo cht, tng hp nhng protein ni bo. Nhng polysome kt hp vi mng ngoi ca li ni sinh cht, tng hp nhng protein dng cho s tng hp mng hoc c bi tit ra ngoi. Primase (protein Dna G). L mt dng ca RNA polymerase v l sn phm ca gen Dna G. Trong vi khun, primase lin kt vi DNA helicase to thnh mt phc hp gi l primosome. Primase c hot ha bi DNA helicase nh n s tng hp mt RNA primer ngn xp x 111 nucleotide, enzyme DNA polymerase s ko di RNA primer bng cch b sung cc nucleotide mi. V th, primase l mt nhn t quan trng trong ti bn DNA bi v nu khng c n DNA polymerase khng th khi u s tng hp mt si DNA mi. Prokaryote. Sinh vt n bo khng c nhn t bo in hnh, DNA nm trong t bo cht khng c mng bao bc, khng c nguyn phn v gim phn; i din in hnh l vi khun. Prophage. Phage n ha xen vo nhim sc th ca vi khun tim tan. N sao chp ng thi vi nhim sc th ca t bo vi khun ch. Protein. Mt phn t ln gm mt hoc nhiu chui polypeptide, mi chui c mt trnh t amino acid v mt khi lng phn t c trng. Protein l hp cht quan trng bc nht i vi c th sng. V cu trc, protein l phn t mch di gm cc n v cu trc nh l cc amino acid ni vi nhau qua mi lin kt peptide. Khi

lng phn t ca protein t vi nghn n vi triu. C khong 20 loi amino acid. Cc loi protein phc tp hn c lin kt thm vi cc nhm b sung. Protein dung hp (fusion protein). L mt protein ti t hp lai c m ha bi mt gen lai (fusion gene) do s dung hp in vitro cc on gen khc nhau trn plasmid vector v sau bin np vo vi sinh vt ch (chng hn E. coli). V vy, protein dung hp s mang trnh t amino acid ca hai protein khc bit c tng hp t u N ca vector biu hin. Protein khng histone (non-histone). Protein acid lin kt vi DNA trong cc nhim sc th eukaryote. Cn gi l protein khng kim. Protein nguyn th (native protein). L mt protein ti t hp c m ha bi mt gen ngoi lai (foreign gene) trong vi sinh vt ch. Khc vi protein dung hp, protein nguyn th c tng hp t u N ca n ch khng phi t u N ca vector. Protein si (fibrous protein). Nhng protein khng tan, c chc nng bo v hoc cu trc, cha nhng chui polypeptide c cu trc bc hai. Purine. Mt hp cht d vng, kim, c nitrogen, l thnh phn ca nhng nucleotide v nucleic acid. Purine cha mt nhn pyrimidine kt hp vi mt nhn imidazol. Purmycin. Mt loi khng sinh c tc dng c ch s tng hp polypeptide bng cch gn vo chui polypeptide, cnh tranh vi aminoacyl-tRNA gn ln v tr ca ribosome, v nh vy to ra s kt thc sm tng hp chui polypeptide. Pyrimidine. Mt nitrogen base d vng c trong cc nucleotide v nucleic acid. Pyrophosphate. Phn t hnh thnh bi hai gc phosphate ni vi nhau bng lin kt anhydride. t Pyrophosphatase. Enzyme thy phn mt pyrophosphate hu c thnh hai phn orthophosphate.

Qu trnh phn tch v tinh sch u ra (downstream processing). Giai on tinh sach cc san phm ca mt qu trnh sinh hc (bioprocessing). Cc k thut thng c s dng trong bc ny la ly tm, siu loc hoc sc k. Quang ti hot ha (photoreactivation, light repair). Mt cch sa cha cc pyrimidine dimer. Cc dimer hi bin trc tip tr li dng ban u di tc dng ca nh sng bnh thng bc sng 320-370 nm. Quang hp (photosynthesis). Qu trnh s dng nng lng ca nh sng to thnh carbohydrate bt u t CO2 v mt tc nhn kh. Replisome. Mt phc hp a enzyme c tc dng khi ng s ti bn DNA chc ba ti bn.

Retroposon. Loi transposon hot ng di dng RNA; trc ht DNA c phin m thnh RNA, RNA ny sau c phin m ngc thnh DNA v gn xen vo mt v tr mi trn h gen. Retrovirus. L loi virus RNA cha enzyme reverse transcriptase v sinh sn di dng DNA mch kp. Chng c kh nng xm nhim t bo vt ch cao. Khi xm nhim n c kh nng gn h gen ca virus vi h gen ca t bo vt ch, l c s thit k cc vector liu php gen hiu qu. Ribonuclease. Enzyme xc tc c hiu vic phn hy RNA bng cch ct cc mi lin kt phosphodiester trn RNA. Ribonucleic acid (RNA). Thng l phn t a phn mch n gm cc n v cu trc c s l ribonucleotide. V mt ha hc RNA rt ging vi DNA. RNA l vt cht di truyn cua mt s virus va la cac phn t trung gian trong qua trinh tng hp protein ma thng tin v trinh t amino acid cua chung a c ma ha trong DNA. Ribonucleotide. n v cu trc c s ca RNA, gm ba thnh phn: ng ribose, nitrogen base v nhm phosphate. Ribosome. La c quan t khi kt hp vi mRNA tao ra b may tng hp protein. Trong t bao thng co hang nghin ribosome, ribosome cua moi t bao u gm mt tiu n vi nho va mt tiu n vi ln. Mi tiu n vi co mang nhiu protein va rRNA (trong rRNA l thnh phn ch yu chim khong 65%) co kich thc khac nhau. Ngi ta cng thy ribosome trong ty th, c s tng hp mt s protein ty th. Ribozyme. La oan RNA co hoat tinh xuc tac phan ng ha hoc ging nh enzyme. RNA b sung (complementary RNA). RNA sinh ra bng cach phin ma t khun mu si n DNA tng ng. RNA kch thc nh ca nhn (small nuclear RNA, snRNA). Ngoi mRNA, tRNA V rRNA, t bao eukaryote con cha nhng phn t RNA kch thc nho ca nhn (chim khong <1%) tham gia vao ghep ni cac exon. RNA ligase. Enzyme ni cac oan RNA vi nhau sau khi cac intron c ct ri khoi tin cht cua mRNA (pre-mRNA) cac sinh vt eukaryote, tao ra mRNA hon chnh sn sang tham gia vao qua trinh dich ma din ra trn ribosome. RNA polymerase. Con goi la RNA polymerase phu thuc DNA (DNA-dependent RNA polymerase), xuc tac vic tng hp RNA trn khun mu DNA trong qua trinh phin ma. RNA ribosome (ribosomal RNA, rRNA). La thanh phn c ban cua ribosome, ong vai tro xuc tac va cu truc trong tng hp protein. Ty theo h s lng rRNA c chia thanh nhiu loai: eukaryote co rRNA 28S; 18S; 5,8S va 5S; con cac rRNA E. coli co ba loai: 23S, 16S va 5S. rRNA chim nhiu nht trong bn loai RNA (khong 80% tng s RNA t bao), tip n la tRNA khong 16%, mRNA chi khong 2%. Ngoi

ra, t bao sinh vt eukaryote con cha nhng phn t RNA kch thc nho ca nhn (small nuclear, snRNA) chim khong <1% tham gia vao ghep ni cac exon. RNase. Enzyme thy phn RNA. RNA thng tin (messenger RNA, mRNA). Mt loi RNA c phin m t mt trnh t DNA. mRNA truyn thng tin di truyn t nhim sc th ti ribosome tng hp protein. Trong qua trinh o mt si cua chui xon kep DNA c dung lam khun mu, doc theo no cc nucleotide cua mRNA b sung c xp thanh hang, ni vi nhau tao nn mt polynucleotide ging ht si DNA khng lam khun mu ngoi tr thymine c thay bng uracil. Qua trinh nay goi la phin ma v phn t mRNA mang ma di truyn c dung iu khin s hinh thanh protein trn ribosome. RNA vn chuyn (transfer RNA, tRNA) . Loai RNA mang cac amino acid n ribosome va sp xp chung doc theo phn t mRNA a nm sn o. Tai y, cac amino acid ni vi nhau bng lin kt peptide tao thanh phn t protein. Mi amino acid co mt phn t RNA vn chuyn ring vi b ba c trng v nh vy cac amino acid c sp xp theo trt t ca cac nitrogen base trn mRNA trong qua trinh dich ma. 10 sequence. La oan ma ha co tac ng lin ng TATAATG tp trung khoang 10 bp trc im khi u cua mt gen cua vi khun. 35 sequence. La oan ma ha tp trung khoang 35 bp trc im khi u cua mt gen cua vi khun. Sn phm phin m s cp (primary transcript). Sn phm phin m ban u ca cc gen eukaryote cn c gi l mRNA tin thn (pre-mRNA), thng c kch thc rt ln. N phi c x l (splicing) to ra phn t mRNA hon chnh (mature mRNA) dng cho dch m. Sng lc (screening). K thut nhn dng mt dng DNA trong mt th vin h gen (genomic library) hoc th vin cDNA (cDNA library) bng mt phng php lai mu d c nh du [-32P]dCTP vi cc vt tan (trng hp dng bacteriophage lm vector to dng v cho xm nhim vo vi khun E. coli) hoc khun lc (dng plasmid lm vector to dng) ca cc th vin trn mng nylon hoc nitrocellulose. Tn hiu lai c pht hin bng phng x t ghi trn phim X-quang. Sinh hc phn t (molecular biology). Khoa hc nghin cu cc hin tng sng mc phn t. Lnh vc khoa hc tr tui ny l im gp nhau ca cc khoa hc kinh in nh di truyn hc, ha sinh hc, t bo hc, vt l hc, ha hc hu c v ha l. Theo cch hiu ph bin hin nay, sinh hc phn t l khoa hc nghin cu cc gen v hot ng ca chng mc phn t, bao gm phin m, dch m, sao chp, iu ha biu hin gen, ti t hp v chuyn gen...

Sinh tng hp protein (protein synthesis). Phn ng ha hc din ra trn ribosome to nn cc phn t protein t cc amino acid trn c s thng tin di truyn nhn c t trong nhn t bo thng qua mRNA. Somatotropin. y l loi hormone c bit, thng c tng hp trong no ng vt v ngi vi mt hm lng v cng thp. Somatostatin c vai tr iu ha hormone sinh trng v insulin i vo mu, kim tra s tng hp hai loi hormone ny. Hin nay, hormone nay a c san xut bng cng ngh DNA tai t hp cha bnh ln tre em do thiu somatotropin. Southern blot. K thut chuyn v c nh DNA bin tnh t agarose gel (sau khi c phn on bng in di) ln mng lai bng nylon hay nitrocellulose lai vi mu d (probe) c nh du ng v phng x [-32P]dCTP hoc digoxigenindUTP. Tn hiu lai sau c pht hin trn phim X-quang (trng hp [-32P]dCTP) hoc trn mng lai (trng hp digoxigenin-dUTP). S bn sao (copy number). (1) S cc phn t plasmid c trong mt t bo vi khun. (2) S lng cc bn sao ca mt gen trong h gen ca mt sinh vt. S phng x t ghi (autoradiogram). Hnh nh sinh ra trn phim X-quang do s pht x ca cc ht phng x. Si ch (leading strand). Chui DNA trong qu trnh ti bn c tng hp cng chiu vi hng di chuyn ca chc ba ti bn. Si c ngha (sense strand). Si n trong chui xon kp DNA c dng lm khun mu tng hp mRNA trong qu trnh phin m. Si n DNA th hai khng c dng lm khun mu phin m gi l si i ngha (antisense strand). Si th (lagging strand). Chui DNA trong qu trnh ti bn c tng hp ngc chiu vi hng di chuyn ca chc ba ti bn. Suppressor. C hai loi: (1) Extragenic-thng l mt gen m ha mt tRNA t bin c trn codon t bin, theo ngha ca codon u tin. (2) Intragenic-l t bin c tnh cht b p phc hi khung c m u tin sau khi c s chuyn dch khung. Sa cha bng ct b (excision repair, dark repair). Qu trnh sa cha cc pyrimidine dimer trn DNA (do tia t ngoi sinh ra) bng cch ct b cc dimer v tng hp on DNA mi thay th no di tc dng ca cc enzyme c hiu m khng cn n nh sng. Tc nhn t bin (mutation factor). Bt k tc nhn vt l hoc ha hc no lm tng ng k tn s t bin so vi tn s t bin t nhin th u c xem l tc nhn t bin. Cc tc nhn t bin thng dng l cc tia phng x nh tia X, tia gamma, tia t ngoi; cc ha cht nh cc ng ng ca cc nitrogen base, nitrogen acid, hydroxylamine, acridin...

Ti bn (replication). S sao chp vt cht di truyn trong chu trnh phn bo hoc s tng hp DNA ca phage khi phage sinh sn trong t bo vi khun. Ti bn DNA kiu bn bo ton (semiconservative DNA replication). Qu trnh sao chp DNA m hai si cha m dn xon v c dng lm khun tng hp hai si mi. Khi sao chp xong th mi chui xon kp con gm mt si c v mt si mi. Ti bn DNA kiu gin on (discontinuos DNA replication). S tng hp DNA thnh nhng on ngn, sau ni li vi nhau to thnh chui polynucleotide di. Ti bn DNA kiu na gin on (semidiscontinuos DNA replication). S tng hp DNA lin tc trn mt si khun mu trong hai si ca DNA mch kp; cn trn si khun mu kia, DNA c tng hp thnh nhng on ngn gi l on Okazaki, sau tt c chng c ni li vi nhau nh ligase to thnh chui polynucleotide di. Ti bn hai hng (bidirectional replication). S ti bn theo hai hng ngc chiu nhau. Ti t hp (recombination). Qu trnh m trong nhim sc th hay phn t DNA t ra ri cc phn t c ni li theo mt t hp mi. Qu trnh ny c th xy ra trong t bo sng (qua s trao i cho trong phn bo gim nhim) hay trong ng nghim nh cc enzyme ct v ni DNA. To dng (cloning). Cn gi l nhn dng, tch dng hay dng ha, l s sn sinh nhiu bn sao ca mt phn t DNA, thng l phn t DNA ti t hp trong plasmid vector, bng cch sao chp phn t trong mt vt ch thch hp chng hn vi khun E. coli. Tn s lp li (repetition frequency). La s ln sao chp cua mt oan ma ha nao o co trong h gen n bi, tng ng vi 1 trong trng hp nonrepetitive DNA va ln hn 2 trong trng hp repetitive DNA. Terminal transferase. Enzyme b sung cc gc nucleotide vo u 3 ca oligonucleotide hoc polynucleotide. T bao bach huyt (B lymphocyte). La nhng t bao co chc nng tng hp cac khang th. T bo Hfr (high frequency recombination cell). T bo gii tnh c E. coli, c mang nhn t F gn lin vi nhim sc th vi khun. Khi nhn t F thc y s tip hp ca t bo Hfr vi t bo ci (F ) th cc gen ca vi khun c truyn sang t bo ci vi tn s cao. T bo mm phi (embryonic stem cell). T bo phi cha bit ha, a th chut, c th c nui cy trong mt thi gian di m vn gi c tnh a th,

ngha l kh nng bit ha theo nhiu hng pht trin thnh cc t bo khc nhau nh: t bo c tim, t bo thn kinh, t bo gan... Th bin np (transformant). T bo hoc sinh vt nhn c gen ca mt sinh vt khc trong qu trnh bin np v biu hin chc nng ca gen ra kiu hnh. Th a hinh (polymorphism). M ta s co mt ng thi cua qun th trong h gen biu hin bin di co tinh cht allele, co th quan sat trn cc allele tao ra cac kiu hinh khac nhau, hoc s thay i cua DNA anh hng n kiu ct hn ch (restriction patterns). Th t bin (mutant). Sinh vt (hoc gen) mang t bin di truyn. Th khm (mosaic). Phi hoc c th c cc t bo mang cc h gen khng ging nhau. Th nhn (nucleosome). Cu trc c s ca cc nhim sc th eukaryote gm DNA bao quanh mt li histone. Th nhn c dng ht, quan st thy di knh hin vi in t. Th ti t hp (recombinant). Cc c th hoc t bo mang cc t hp gen khc vi cha m ca chng do cc qu trnh ti t hp di truyn sinh ra. Thng tin di truyn (genetic information). Thng tin c lu tr trong cc phn t DNA ca sinh vt dng trnh t sp xp ca bn nucleotide k hiu l A, T, C v G ng vai tr nh nhng ch ci ca ngn ng di truyn. Trong ngn ng ny, mi t ch c ba ch ci gi l mt b ba. Ngha ca mi t l mt amino acid c mt trn phn t protein tng ng. Mi cu ca ngn ng di truyn l mt gen cha ng thng tin di truyn m nhim mt chc nng trn vn. Mi chc nng l mt c tnh sinh l, hnh thi hay cu trc ca s sng. Do c ch sao chp theo kiu na bo ton ca DNA m thng tin di truyn c truyn chnh xc t th h n sang th h kia hu nh khng thay i. Th th (receptor). Protein mng, mt phn nm trong mng t bo, c kh nng gn mt phi t (ligand) mt ngoi ca mng kt qu l gy ra mt bin i hot ng trong t bo cht. Thuc th nghim (orphan drug). La thuc c phap lut M (va mt s nc khac) cho phep dng th trong mt vai nm ( M la 7 nm) cha nhng cn bnh him gp. Hormone phat trin ngi va erythropoetin c san xut nh cng ngh DNA tai t hp thuc dang thuc th nghim noi trn. Th vin cDNA (cDNA library). Tp hp cc dng DNA c to ra t mRNA ca mt t bo hoc mt m c th trong bacteriophage vector, i din cho thng tin di truyn m cc t bo biu hin. Th vin h gen (genomic library). Tp hp tt c cc on DNA c to ra t phn ng ct hn ch genome trong bacteriophage vector, i din c cho ton b cho thng tin di truyn ca mt h gen.

Thymine (T). Base loi pyrimidine c trong DNA nhng khng c trong RNA. Trong mch kp DNA thymine bt cp vi adenine (A). Tnh trng (trait). Mt c im hoc tnh cht bn ngoi ca cc c th sinh vt. Tnh trng a gen (polygenic trait). Tnh trng c xc nh bi mi tng tc gia nhiu gen, chng hn mu mt ngi. Topoisomerases. Nhng enzyme xc tc s thay i nhng dng siu xon m hoc dng ca chui DNA xon kp vng. Trao i cht (metabolism). Cn gi l qu trnh chuyn ha, l ton b nhng thay i trong t bo sng ca nhng phn t hu c nh xc tc bi nhng enzyme. S chuyn ha bao gm c qu trnh tng hp ln thoi ha. Trnh t dn u (leader sequence). Mt trong ba phn ch yu ca mt phn t mRNA. Trnh t ny nm u 5 ca mRNA v mang thng tin ribosome v cc protein c hiu nhn bit bt u qu trnh tng hp polypeptide, trnh t dn u khng c dch m thnh trnh t cc amino acid. Trnh t iu ha (regulatory sequence). Mt trnh t ca DNA tham gia vo qu trnh iu ha ca gen. V d: trnh t promoter hoc operator. Trnh t chn (insertion sequence, IS). C vi khun, kch thc nh v tng ng vi transposon, ch mang gen cn cho s gn chn ca chnh n. Trnh t ch huy (operator). on DNA ngn nm u operon, k st promoter ni m protein c ch c th bm vo, c tc dng m hoc ng operon cho cc gen cu trc trong operon hot ng hoc ngng hot ng. Trnh t khi ng (promoter). Trnh t nucleotide c hiu nm trong thnh phn operon, c chc nng iu ha hot ng ca operon, ni RNA polymerase bm vo bt u qu trnh phin m. Trnh t c trng ca promoter c khong 20-200 nitrogen base. Trnh t lp li (repetitive sequence). Cc trnh t base c nhiu bn sao cng mt lc trn phn t DNA. C bn loi trnh t lp li: trnh t lp li n (c mt bn sao), trnh t lp li va (c t 1 n 10 bn sao), trnh t lp li trung bnh (c t 10 n vi trm bn sao) v trnh t lp li cao (c t vi trm n vi triu bn sao). Trnh t lp li ngc chiu (inverted repeat sequence). Mt on ngn ca DNA c lp li, thng cc u ca mt on di hn, c hng ngc chiu. Trnh t lp li xui chiu (direct repeat sequence). on DNA gm mt dy cc trnh t nucleotide c lp li theo cng mt chiu. Trnh t ph bin (consensus sequence). on trnh t thy c trong hu ht cc mu ca mt yu t di truyn c th v c bn vng cao, v d hp CAAT.

Trnh t Shine-Dalgarno (Shine Dalgarno sequence, SD). Cn gi l vng lin kt ribosome (RBS), l mt phn ca trnh t nucleotide u 5 ca mt mRNA prokaryote c th kt hp b sung cp base vi u 3 ca 16S rRNA, dng lm tn hiu cho s khi u dch m. Trnh t tng cng (enhancer). Trnh t nucleotide dng cis lm tng cng phin m ca promoter trong gen eukaryote. N c th nm cch promoter hng ngn cp base v hot ng theo c hai hng bt k v tr no so vi promoter. Tng ng (homologous). (1) Ni v cc nhim sc th cp i trong cc sinh vt lng bi. (2) Dng m t cc trnh t DNA ging ht nhau mt cch tuyt i; tuy nhin, phn trm tng ng gia cc on lin quan i khi ch tng i. Ty th (mitochondria). Bo quan c mng bao bc, nm trong t bo cht ca cc sinh vt eukaryote. Ty th cha nhng enzyme cn thit cho chu trnh citric acid, vn chuyn electron v qu trnh phosphoryl oxy ha. gn mi (annealing). Dung chi s bt cp cua khun mu DNA si n vi primer (mi) tng hp nn mt si DNA b sung bng cch s dng cc dNTP c trong mi trng ko di primer nh s xc tc ca enzyme Taq DNA polymerase (trong khuch i PCR) hoc DNA polymerase I (trong tng hp cDNA). c ch amber (amber suppresser). La cac gen t bin (ma ha cho tRNA) ma nhng anticodons cua no a c kich hoat co th nhn UAG codon cung nh cac codon trc o. Vt ch (host). T bo dng nhn cc phn t DNA ln nhiu ln. Vector. L cc phn t DNA c s dung trong tao dong v biu hin gen, va nhn ban chung trong t bo vt ch (E. coli hoc nm men). C ba nhm vector chnh gm: (1) Nhm plasmid, (2) Nhm phage/phagemid, v (3) Nhm nhim sc th nhn to (artificial chromosome: BAC, YAC). Y tng v vector chuyn gen bt ngun t cac plasmid cua vi khun. Vector chuyn gen la cc phn t DNA co kha nng t tai sinh, tn tai c lp trong t bao va mang c cac gen cn chuyn. Vector biu hin (expression vector). L vector c th mang cac gen ngoi lai mong mun cho php thc hin s phin ma cua cac ban sao c tao dong va s dich ma cac mRNA cua chung trong t bo vt ch, v d: E. coli. biu hin cc gen ngoi lai trong E. coli phi bt u bng vic gn n vo trong vector biu hin (thng l plasmid). Vector ny phi c cc cu trc cn thit sau: (1) Cc trnh t m ha gen ch th chn lc (selectable marker) m bo duy tr vector trong t bo. (2) Mt promoter kim sot phin m cho php sn xut mt lng ln mRNA t cc gen c to dng. (3) Cc trnh t kim sot dch m nh vng lin kt ribosome c b tr thch hp v codon khi u ATG. (4) Mt polylinker a gen ngoi lai vo trong mt hng chnh xc vi promoter.

Vector to dng (cloning vector). Phn t DNA mch kp c kh nng t sao chp trong t bo vt ch. C th gn vo phn t ny mt on hoc mt vi on DNA khc ngun to nn phn t DNA ti t hp dng nhn dng. Vector thay th (replacement vector hay substitution vector). L mt bacteriophage vector m cc im to dng sp xp thnh cc cp, do vy phn ca h gen nm gia cc im ny c th c thay th bng on DNA chn. Vt tan (plaque). Vng trn trong sut xut hin trn thm c ca cc vi khun mc trn mi trng thch c, do s tan v lp li nhiu chu k ca cc t bo vi khun b bacteriophage xm nhim v sinh tan. V tr cos (cos site). Vung c tao ra khi cac u dinh cua DNA phage ni lai vi nhau. Virus. Phc hp cha nucleic acid (DNA hoc RNA) nm trong mt v bc protein, c kh nng gy nhim v ti bn bn trong t bo vt ch c hiu, to ra nhiu virus, lan truyn t t bo ny sang t bo khc. Virus l dng sng khng c cu trc t bo, c kh nng xm nhp vo cc t bo sng xc nh v ch sinh sn bn trong cc t bo . Ging nh tt c c sinh vt khc, virus c b my di truyn ca ring mnh, m ha vic tng hp cc ht virus t cc cht c trong t bo vt ch. Nh vy, virus l nhng vt k sinh ni bo. Virus phn b khp ni trong t nhin, xm nhp vo tt c cc nhm sinh vt. Ngi ta bit khong 500 loi virus xm nhp ng vt mu nng, 300 loi xm nhp thc vt bc cao. Mt s khi u ung th ng vt v ngi c th do virus. Virus tn ti hai dng: dng ngh hay ngoi bo v dng sinh sn hay ni bo. Kch thc ca cc ht virus t 15-350 nm, chiu di ca mt s loi virus c th t ti 2000 nm. Phn ln virus ch nhn thy c qua knh hin vi in t. Cht mang thng tin di truyn ca virus l nucleic acid: DNA hoc RNA. V vy, c th phn virus thnh hai loi: loi mang DNA v loi mang RNA. Vi tim (microinjection). K thut a DNA vo nhn hoc vo t bo cht ca t bo bng kim mao dn v bm p lc. y la ky thut s dung ph bin trong cng ngh t bao ng vt (animal cell biotechnology), n i hi thit b vi thao tc (micromanipulator) cc nhy, k nng thc hnh v s kin nhn cao ca k thut vin. VNTR (variable number of tandem repeats). Cn gi l tiu v tinh, ch cc vng ca h gen c trng bi s lp li ca cng mt trnh t DNA. S lng ln lp li thay i theo c th nn mang tnh a allele rt cao. Vng kp tc (hairpin loop). Vng chui n b sung to np gp cha cc cp base to thnh xon kp, c dung lam mi (primer) cho qua trinh tng hp si cDNA th hai. Vn gen (gene pool). Ton b thng tin di truyn c trong tt c cc gen ca mt qun th ti mt thi im xc nh.

Vng cng hng (downtream). cp n v tr ca mt on trnh t no nm pha u 3 ca gen hoc mt on gen quan tm. Vng a ni (polylinker hay polycloning sites). Mt trnh t DNA mch kp c tng hp nhn to c mang mt lot cc v tr nhn bit ca cc enzyme hn ch. Trnh t ny c gn vo cc vector dng trong k thut to dng gen (nh vng to dng). Vng lin kt ribosome (ribosome binding sites, RBS). Vung trn phn t mRNA giup cho no bam vao ribosome trong qua trinh dich ma (xem trnh t Shine Dalgarno). Vng nhn (nucleoid). Mt vng trong t bo vi khun tp trung vt cht di truyn. Vng ngc hng (upstream region). V tr ca mt trnh t nucleotide no nm pha u 5 ca phn t DNA so vi gen quan tm. Vng siu bin (hypervariable region, HVR). Vng trong h gen bao gm mt s lng bin ng cc trnh t lp li v c dng xc nh c trng c th (xem in du di truyn). Vng to dng (multiple cloning sites, MCS). oan DNA ngn trong mt vector th h th ba (v d: cc nhm pUC, pGEM, pBluescript) co cha cac vi tri nhn bit cho mt s enzyme ct hn ch thng dng, c thit k chn on DNA ngoi lai vo y (xem vng a ni). Western blot. K thut phn tch protein da trn nguyn l lin kt khng nguyn-khng th pht hin protein c hiu c bn cht khng nguyn (thng c thm tch ln mng nitrocellulose sau khi chy in di SDS-PAGE, v c nh ). Sau khi protein trn mng lai gn vi khng th th nht c hiu v tip n l khng th th hai c nh du enzyme (alkaline phosphatase hoc horse-radish peroxidase) th phc hp ny s c lin kt vi c cht to mu. S hin din ca protein ngoi lai (sn phm dch m ca gen ngoi lai c chuyn vo t bo vt ch) s c pht hin nh s xut hin mu ca phn ng lai. Xon alpha (helix ). Cu trc xon ca chui polypeptide, thng l quay phi, vi s lng ti a lin kt trong chui, mt trong nhng cu trc bc hai ph bin ca protein c tm thy keratin. Xon kp (double helix). Cu trc ba chiu t nhin ca hai chui DNA b sung, i song v xon vi nhau. YAC (Yeast artificial chromosome). Nhim sc th nhn to ca nm men, c dng lm vector to dng nhng on DNA c kch thc rt ln trong nm men. Yu t ngoi nhim sc th (extrachromosomal element). Phn t DNA khng phi l thnh phn ca nhim sc th t bo vt ch.

Yu t tc ng cis (cis-acting element). on trnh t DNA ch biu hin hiu qu trn chnh phn t DNA m n tc ng. V d: hp CAAT l mt phn t tc ng cis i vi qu trnh phin m cc sinh vt eukaryote. Yu t tc ng trans (trans-acting element). Yu t di truyn c th biu hin hiu qu m khng cn nm trn cng mt phn t vi on trnh t ch. Thng yu t m ha cho mt sn phm protein (c th l mt enzyme hay mt protein iu ha) v sn phm ny c th khuch tn n im tc ng.

Ti liu tham kho/c thm

1. Ban T in-NXB Khoa hc v K thut. 2002. T in Bch khoa Sinh hc. NXB Khoa hc v K thut, H Ni. 2. Alberts B, Johnson A, Lewis J, Raff M, Roberts K and Walter P. 2002. Molecular Biology of the Cell. 4th ed. Garland Science Publishing, Inc. New York, USA. 3. Karp G. 2002. Cell and Molecular Biology. John Wiley & Sons, Inc. New York, USA. 4. Lawrence E. 1995. Hendersons Dictionary of Biological Terms. 7th ed. Longman Group Ltd. Singapore. 5. Singleton P and Sainsbury D. 2001. Dictionary of Microbiology and Molecular Biology. 3rd ed. John Wiley & Sons, Ltd. UK. 6. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Losick R. 2004. Molecular Biology of the Gene. Pearson Education, Inc./Benjamin Cummings Publishing, San Francisco, USA.