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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 53). 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 mm.
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
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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 1.3. 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:
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- 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:
E. coli, kich thc trung binh cua mt phn t mRNA khoang 1,2 kb.
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.
II. Protein
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Cng thc tng quat cua L-a-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 Ca. Vi vy, ngi ta goi la nhom a-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-a-amino acid. Mc du protein rt a dang nhng hu ht chung u c cu tao t 20 L-a-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):
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- 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.
nhau tao thanh mt dimer. Chng hn: hemoglobin c tao nn t hai chui a vi mi chui co 141 gc amino acid va hai chui b 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.
Enzyme
Protein iu khin
Protein vn chuyn
Protein d tr
Protein vn ng v co rt
Protein cu trc
Protein bo v
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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
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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.
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.
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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
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.
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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.
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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.
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 li 1 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.
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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.
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.
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.
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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.
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 (half-completion). y l tch s ca C0t1/2 v c gi l C0t1/2. Gi tr ny t l nghch vi hng s tc . Do C 0t1/2 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 Phn ng lai ph thuc vo C0t
Tc phn ng Phn ng theo phng trnh bc hai dC = kC 2 dt C: nng ca DNA si n thi im t K: hng s tc ti lin kt Tin phn ng Thng s ti hn l C0t1/2 Ly tch phn phng trnh tc Khi phn ng hon thnh mt na gia cc gii hn: nng ban u thi im t=1/2 ca DNA=C0 thi im t=0; nng C 1 1 = = duy tr si n=C sau thi gian t C0 2 1 + kC 0 t1 / 2 C 1 1 = V th C 0 t1 / 2 = C 0 1 + kC 0 t k
S hi tnh ca DNA thng c dng ng cong C 0t, 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
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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 3,3109 bp, trong khi genome ca nhng loi lng c di khong 3,1 109 bp hoc thc vt c th ln n 10 11 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 8,6108 bp, ln gp su ln kch thc genome ca rui gim khong 1,410 8bp. Ngoi ra, trong cc loi lng c kch thc genome ca chng cng thay i kh ln t 10 9-1011 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.
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.
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.
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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 l 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.
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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 10-10. 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-X n-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 T n. 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:
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- 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
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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 vng 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 Ri-plasmid 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.
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 TDNA. 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 T-DNA 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.
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 - HMLa (cho MATa chuyn thnh MATa) - HMRa (cho MATa 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.
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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 1,2-dimyristoyglycerol c gai trong mng plasma. Gc glycolipid l yu t quyt nh phn ng lai (cross-reacting)
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(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.
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.
Chng 3
I. nh ngha gen
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...
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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.
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).
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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.
4.2.2. 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.
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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.
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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).
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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
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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.
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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 thi allele khc nhau.
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...
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.
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.
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Chng 4
TI BN DNA
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.
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).
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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 4.7 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 l. 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).
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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.
3. Kt thc
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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.
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 a2, , 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).
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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 53 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.
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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:
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.
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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).
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.
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.
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.
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 promoterpolymerase. 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:
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.
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 G-C nn chng d dng b ph v bi hiu qu ca cu trc kp tc, v v th RNA d dng c gii phng.
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.
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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.
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Hnh 5.4. Cc tc nhn hot ha, phc hp trung gian v cc thnh phn bin i nucleosome trong phin m
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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.
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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.
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- 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.
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.
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.
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.
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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.
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 fMettRNAifMet 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
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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).
1.2.3. 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'-AUG-3' 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, MettRNAiMet 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).
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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 EF-G-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.
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.
V. Cc nhn t c ch dch m
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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.
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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.
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.
- 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.
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.
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- 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.
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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. N-glycosylase 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.
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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.
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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.
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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.
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: 5-CTGTN8ACAG-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.
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.
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 f 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.
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.
Chng 8
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.
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.
1. Mc cht nhim sc
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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 pro-insulin thnh insulin.
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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 -35. C hai trnh t phi hp nhau cho php RNA polymerase gn vo v khi s dch m, trnh t -35 to iu kin u tin cho vic gn vo.
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
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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.
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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.
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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.
- 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).
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- 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.
5.1. 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.
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.
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 .
Chng 9
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.
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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):
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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).
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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:
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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 (GTCGAC) 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:
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.
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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-PO 4 ca polynucleotide ny vi nhm 3-OH ca polynucleotide khc.
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2. Bacteriophage l vector
Cc bacteriophage c nhiu u im khi c s dng lm vector to dng. Vector dc s dng rng ri nht l bacteriophage l gy nhim vo t bo E. coli. Mt trong nhng u im chnh ca phage l 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 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 l 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 l 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 l, 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 l c nh v trong mt cm. Cc chng ca phage l, c gi l vector thay th, c bin i di truyn vi mt v tr RE duy nht cho EcoRI (chng phage l 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 l cn thit (nhnh tri v phi), c th c kt ni bng DNA ligase. Genome ca phage l 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 l sau c th c ng gi trong v protein v c b sung vo trong E. coli. Cc phage l 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.
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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 l, 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 l 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 l 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.
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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.
1. Cc protein nguyn th ti t hp
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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 sites-RBS) 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.
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 l, codon khi u ATG c dung hp trc
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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 l v v tr ct hn ch duy nht BamHI nh v codon khi u ATG ca gen cII ca bacteriophage l. 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.
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 b-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
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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 nguyn-khng th
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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.
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 bit cua protein quan tm. Do s thoai bin cua ma di truyn 3, chui amino acid xut c th
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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.
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 32P (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 109 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.
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.
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.
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.
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.
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