PGS. TS. Nguyn Hong Lc (Ch bin) TS. Trn Th L - ThS.

H Th Minh Thi

Gio trnh

Sinh hc phn t

Nh xut bn i hc Hu
Nm 2007

NH XUT BN I HC HU a ch: 01 in Bin Ph, Hu - in thoi: 054.834486 Chu trch nhim xut bn: Gim c: Nguyn Xun Khot Tng bin tp: Hong Hu Ha

Ngi phn bin: PGS. TS. Nng Vn Hi

Bin tp ni dung: TS. Nguyn Th Mai Dung

Bin tp k thut-m thut: Hong Tu

Trnh by ba: Nguyn Hong Lc

Ch bn vi tnh: Nguyn Hong Lc

SINH HC PHN T
In 500 bn kh 1624 cm, ti Cng ty In Thng k v Sn xut Bao b Hu, 36 Phm Hng Thi, Hu. S ng k KHXB 151-2007/CXB/02-03/HH. Quyt nh xut bn s: 08/Q-HH-NXB, cp ngy 12/4/2007. In xong v np lu chiu thng 4 nm 2007.

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

Cc tc gi

Chng 1

I. Nucleic acid

. (RNA). 1. Deoxyribonucleic acid

5 .

5 m.

Sinh hc phn t

Lin kt hydrogen

(a) Cu trc ca DNA

RNA Hnh 1.1. Chui xon kp ca DNA

mch .

20 A
o

100 A
o

300 A 1.3).
Sinh hc phn t
6

100 A

ng knh 100 A 300 A . 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.
u 5 O O Phosphate P O CH2 O H H O O O P O CH2 O H H O O HOCH2 OH H O H H NH2 H H O H H O O O N N O H P O CH2 O H H O u 3 H O H H H H H CH 3 H O N N O H Thymine (DNA) N N O Cytosine H O H H H N N N O N H Guanine NH2 H N N NH2 N N H Adenine

Deoxyribose

O
H HO OH

P O CH2 O

Ribose (RNA) O H

H Uracil (RNA)

Hnh 1.2. Cu trc cc nucleotide in hnh

Sinh hc phn t

DNA xon kp

Nhn ca 8 phn t histone DNA

2 nm

11 nm

Histone H1

30 nm

300 nm

700 nm 1400 nm

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.

: 10-15% genome (h gen) . -

Sinh hc phn t

). 5S RNA. . . .

, . 2. Ribonucleic acid sau: . ,s -protein. . .

Sinh hc phn t

. : (mRNA)

2:
DNA Phin m RNA Dch m Protein

E. coli 1,2 kb. (tRNA)

: . 2.3. RNA ribosome (rRNA) n. .

Sinh hc phn t

10

E. coli 5S.

exon. Ribosome l nhng phn t cn thit cho s t

. Ngi ta cng thy ribosome trong ty th, c s tng hp mt s protein ty th.

E. coli

(%)

(S)1
23

(kDa)
1,2 10 3 0 ,55 10 3 3,6 101

nucleotide
3700 1700 120

rRNA

80

16 5

tRNA mRNA

15 5

2,5 101

75

2.3.1. Ribosome ca prokaryote T bo c nghin cu v ribosome nhiu nht l E. coli. Ribosome (70S) ca E. coli gm hai tiu n v: tiu n v nh (30S) v tiu n v
S (Svedberg): n v o vn tc lng. H s lng ca mt tiu n v ph thuc khng nhng vo khi lng ca tiu n v m cn ph thuc vo hnh dng v rn ca n, iu ny gii thch ti sao s kt hp ca hai tiu n v 50S v 30S li to ra mt ribosome 70S.
1

Sinh hc phn t

11

ln (50S). Cn c vo h s lng, ngi ta phn bit ba loi rRNA: 23S rRNA, 16S rRNA v 5S rRNA. - Tiu n v 30S cha: 1 phn t 16S rRNA (c 1540 nu) v 21 ribosomal protein khc nhau. - Tiu n v 50S cha: 1 phn t 5S rRNA (c 120 nu), 1 phn t 23S rRNA (c 2900 nu) v 34 ribosomal protein. Hai tiu n v nh v ln khi kt hp vi nhau s to ra mt rnh ch tip gip ca chng cho mRNA i qua. 2.3.2. Ribosome ca eukaryote Ribosome ca eukaryote (80S) ln hn ribosome ca prokaryote cng bao gm hai tiu n v: tiu n v nh (40S) v tiu n v ln (60S). - Tiu n v 40S cha: 1 phn t 18S rRNA (c 1900 nu) v 33 ribosomal protein. - Tiu n v 60S cha: 3 phn t rRNA (5S; 5,8S v 28S) v 49 ribosomal protein. T RNA polymerase. CTP. 5 Th . . . : ATP, :

. E. coli .

Sinh hc phn t

12

II. Protein
(monome chung:
COOH H2N CH R L- -amino acid

amino acid, qu (NH2 L D-

.
2

. ca chng. Nhng amino acid trung tnh c mt nhm amine v mt nhm carboxyl.

protein.
L- -

20 L:

Sinh hc phn t

13

- Amino acid t . threonine. (-S-S-). . - Iminoacid. Proline. . . Bao

. Bao . Bao . Bao

. Bao

. . Bao

2O.

. nh sau (Hnh 1.4):

Sinh hc phn t

14

).

1. L

L phin

(a) Cu trc s cp (bc 1)

Xon (b) Cu trc th cp (bc 2) (c) Cu trc bc 3

(d) Cu trc bc 4

Hnh 1.4. Cc mc t chc ca phn t protein

2. L

Sinh hc phn t

15

( , cun xun

. chui polypeptide. 3. L ,l

. 4. L

Sinh hc phn t

16

.
Bng 1.2. Cc chc nng sinh hc ca protein v mt s v d Cc nhm chc nng Enzyme V d Ribonuclease Trypsin Phosphofructokinase Alcohol dehydrogenase Catalase Malic enzyme Insulin Somatotropin Thyrotropin lac repressor NF1 (nuclear factor 1) Catabolite activator protein (CAP) AP1

Protein iu khin

Sinh hc phn t

17

Protein vn chuyn

Hemoglobin Serum albumin Glucose transporter Ovalbumin Casein Zein Phaseolin Ferritin Actin Myosin Tubulin Dynelin Kinesin -Keratin Collagen Elastin Fibroin Proteoglycans Grb 2 crk shc stat IRS-1 Immunoglobulins Thrombin Fibrinogen Antifreeze proteins Snake and bee venom proteins Diphtheria toxin Ricin Monellin Resilin Glue proteins

Protein d tr

Protein vn ng v co rt

Protein cu trc

Protein cu trc tm thi (scaffold protein)

Protein bo v

Protein l/ngoi lai (exotic protein)

Sinh hc phn t

18

1016 .

, tu

disulfite ).

albumin.

1.5).

Sinh hc phn t

19

.
(a) (b)

Bn ngoi Hemoglobin (Hb) 4O2 Hb(O2)4 Glucose Vn chuyn glucose (mt protein mng)

Bn trong

Phi

Tun hon tnh mch Tun hon ng mch

Mng t bo

Tim

M 4O2

Hemoglobin (Hb)

Hb(O2)4

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. Sinh hc phn t
20

).

. Chng hn: -

. Fibroin ( .

: ( -glutamyl-cysteinyl)n-glycine

Sinh hc phn t

21

109

nguyn. Trong c

. . 0oC. 2.8. Protein l/ngoi lai . .

III. Lipid

Sinh hc phn t

22

i.
a NH2 H2N HOOC HOOC NH2 NH2 COOH
V tr kt hp vi khng nguyn

V tr kt hp vi cytophage

COOH NH2 NH2

b H2N NH2

Khng nguyn

Khng th

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.

1.7)

Sinh hc phn t

23

1.3.

CH3(CH2)10 COOH CH3(CH2)14 COOH

Lauric Palmitic

CH3(CH2)7CH=CH (CH2)7 COOH CH3(CH2)4 CH= CH- CH2 CH= CH (CH2)7 COOH

Oleic Linoleic

Oligosaccharide Protein xuyn mng

Glycoprotein

Glycolipid

Protein ngoi bin

Hai lp phospholipid

Lp tch ri

Li k nc

Phospholipid

Protein a nc Protein xuyn mng Protein ngoi bin

Gc acid bo k nc u a nc
Glycoprotein

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

Phn t l

ng

IV. Polysaccharide

a C1 .
Sinh hc phn t
24

nucleic

nucleic acid hay

Ti liu tham kho/c thm

1. H Hunh Thy Dng. 1998. Sinh hc phn t. NXB Gio dc, H Ni. 2. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 3. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 4. Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J. 2004. Molecular Cell Biology. 5th ed. WH Freeman and Company, New York, USA. 5. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Loscik R. 2004. Molecular Biology of the Gene. The Benjamin Cummings/Cold Spring Habor Laboratory Press, San Francisco, CA, USA. 6. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company Inc. New York, USA. Sinh hc phn t
25

Chng 2

Cu trc genome
Genome (h gen, b gen) l thut ng c dng vi cc ngha khc nhau nh sau: - Nguyn liu di truyn ca mt c th: 1) nhim sc th trong t bo vi khun (hoc mt trong mi loi nhim sc th nu hn mt loi c mt, v d: cc nhim sc th ln hoc b ca Vibrio cholerae), 2) DNA hoc RNA trong mt virion, 3) nhim sc th cng vi mi plasmid c kt hp (v d: nhim sc th v hai plasmid nh trong vi khun Buchnera). - Tt c cc gen (khc nhau) trong t bo hoc virion. - B nhim sc th n bi hoc genome n bi trong t bo. Chui genome hon chnh (ngha l trnh t hon chnh ca cc nucleotide trong genome) c cng b cho mt s loi vi khun. Cc trnh t khc cng c cng b, v d genome ca cy cc di (Arabidopsis thaliana) v genome ngi. Genome cha ton b thng tin di truyn v cc chng trnh cn thit cho c th hot ng. cc sinh vt nhn tht (eukaryote), 99% genome nm trong nhn t bo v phn cn li nm trong mt s c quan t nh ty th v lp th. a s genome vi khun v phn genome cha trong cc c quan t thng c kch thc nh v dng vng khp kn. Ngc li, phn genome trong nhn thng rt ln v phn b trn cc nhim sc th dng thng. D n genome l d n xc nh cu trc di truyn chnh xc ca mt genome c th sng, ngha l trnh t DNA ca tt c cc gen ca n. D n genome ca mt s sinh vt m hnh (model organisms) c hon thnh nh sau: - Cc genome vi khun. Cc trnh t hon chnh ca genome Escherichia coli c xc nh theo phng thc t hp/tp hp (consortium) ca cc phng th nghim. Nm 1995, hai trnh t genome hon chnh ca vi khun Haemophilus influenzae v Mycoplasma genitalium cng c hon thnh. Loi M. genitalium c mt genome n
Sinh hc phn t
26

gin (khong 580.067 base), do n da vo vt ch vn hnh nhiu b my trao i cht ca mnh. Loi H. influenzae l mt vi khun c trng hn, v c genome khong 1.830.121 base vi 1.749 gen. - Chui genome hon chnh ca nm men Saccharomyces cerevisiae c hon chnh trong nm 1996, nh mt consortium ca cc phng th nghim. Genome ca chng di 12.146.000 base. - Cc d n genome ng vt nh: chut, cu, ln, giun trn (Caenorhabditis elegans), rui gim (Drosophila melanogaster), hoc thc vt nh: la nc, la m, ng, to, cc di, m ni bt nht trong s l d n genome ngi cng c thc hin. Ngy 12. 2. 2001 genome ngi c cng b vi khong 30.000 gen, t hn nhiu so vi d kin trc y (hng trm ngn gen), v ch gp hai ln giun trn hoc rui gim. Ngi ta xc nh h gen ngi ging 98% so vi tinh tinh v c n 99% l ging nhau gia cc dn tc, cc c th. Do , vn hnh thnh v pht trin nhn cch, ch s thng minh... phi ch yu trn c s x hi v s rn luyn ca tng ngi pht trin tim nng sinh hc ca bn thn. Trnh t genome ca nhng sinh vt m hnh rt c ngha trong nhng nghin cu ca mt chuyn ngnh khoa hc mi l genome hc (genomics). Da vo y, cc nh sinh hc phn t c th phn tch cu trc, hot ng v chc nng ca cc gen, lm sng t c vai tr ca DNA lp li, DNA khng cha m di truyn, DNA nm gia cc gen... iu c bit c ngha l khi so snh cc genome vi nhau, c th hiu c hot ng ca genome trong cc c th sng, mi quan h gia chng, s a dng sinh hc v mc tin ha. Kt qu bc u so snh genome gia cc loi sinh vt vi nhau cho thy c ba c im ni bt: 1) cc gen phn b trong genome khng theo qui lut, 2) kch thc ca genome thay i khng t l thun (tng quan) vi tnh phc tp ca loi, 3) s lng nhim sc th cng rt khc nhau ngay gia nhng loi rt gn nhau.

I. Thnh phn v c im ca genome

Genome cha mi thng tin di truyn c trng cho tng loi, thm ch cho tng c th trong loi. Genome c th bao gm cc phn t DNA
Sinh hc phn t
27

hoc RNA. i vi sinh vt bc cao, kch thc genome thay i t 109 bp (ng vt c v) n 1011 bp (thc vt). Khc vi t bo tin nhn (prokaryote), cc gen trong genome ca eukaryote thng tn ti nhiu bn sao v thng b gin on bi cc on m m khng mang thng tin di truyn (cc intron). V vy, mt trong nhng vn ang c quan tm l cn phi bit s lng cc gen khc nhau c mt trong genome cng nh s lng cc gen hot ng trong tng loi m, tng giai on pht trin v t l cc gen so vi kch thc genome... 1. Genome ca c quan t Hu ht genome ca c quan t, nhng khng phi lun lun, c dng phn t DNA mch vng n ca mt chui duy nht. Genome ca c quan t m ha cho mt s, khng phi tt c, cc protein c tm thy trong c quan t. Do c nhiu c quan t trong mt t bo, cho nn c nhiu genome ca c quan t trn mt t bo. Mc d bn thn genome ca c quan t l duy nht. Nhng n cu to gm mt chui lp li1 lin quan vi mi chui khng lp li2 ca nhn. V nguyn tc, cc gen c quan t c phin m v dch m bi cc c quan t. 1.1. Genome ca ty th DNA ty th (mitochondrial DNA-mtDNA) l mt genome c lp, thng l mch vng, c nh v trong ty th. - DNA ty th ca t bo ng vt m ha c trng cho 13 protein, 2 rRNA v 22 tRNA. - DNA ty th ca nm men S. cerevisiae di hn mtDNA ca t bo ng vt nm ln do s c mt ca cc on intron di. Cc genome ty th c kch thc tng s rt khc nhau, cc t bo ng vt c kch thc genome nh (khong 16,5 kb ng vt c v) (Hnh 2.1). C khong mt vi trm ty th trn mt t bo. Mi ty th c

DNA lp li m t cc chui hin din hn mt bn sao trong mi genome. DNA khng lp li cha cc chui duy nht: ch c mt bn sao trong genome n bi.
1 2

Sinh hc phn t

28

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.
Cyt b D-loop

ND5

ND6 12S rRNA 16S rRNA 16,6 kb

ND4 ND4L ND3 CO3 ATPase 8 ATPase 6

ND1

ND2 CO1

CO2

Cc gen tRNA Cc vng m ha Hng ca gen, 5 n 3 CO: cytochrome oxidase ND: NADH dehydrogenase

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

Sinh hc phn t

29

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

Ty th m ha cho cc RNA v protein Loi Kch thc (kb) Nm Sinh vt nguyn sinh Thc vt ng vt 19-100 6-100 186-366 16-17 Cc gen m ha protein 8-14 3-62 27-34 13 Cc gen m ha RNA 10-28 2-29 21-30 4-24

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.

Sinh hc phn t

30

- DNA lp th di t 120-190 kb. Cc genome ca lp th c phn tch trnh t cho thy c khong 87-183 gen. Bng 2.2 m t cc chc nng c m ha bi genome lp th cy trng.
Bng 2.2. Genome ca lp th cc cy trng m ha cho 4 rRNA, 30 tRNA v khong 60 protein

Cc lp th c hn 100 gen Cc gen - M ha RNA 16S rRNA 23S rRNA 4,5S rRNA 5S rRNA tRNA - Biu hin gen Cc r-protein RNA polymerase Khc - Cc chc nng ca lp th Rubisco v thylakoids NADH dehydrogenase

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

Sinh hc phn t

31

ca nhn nm men S. cerevisiae; 2) nhng intron trong cc gen m ha protein tng t vi cc intron ca cc gen ty th. Vai tr ca lp th l thc hin qu trnh quang hp. Do , nhiu gen ca n m ha cho cc protein ca cc phc hp nh v trong cc mng thylakoid. Mt vi phc hp protein ca lp th ging cc phc hp protein ca ty th: c mt s tiu n v c m ha bi genome ca c quan t v mt s khc c m ha bi genome ca nhn. Nhng cc phc hp cn li c m ha hon ton bi genome lp th. 2. ng hc ca phn ng lai DNA Bn cht chung ca eukaryotic genome c phn nh qua ng hc ca s ti lin kt cc DNA (DNA reassociation kinetics) b bin tnh. S ti lin kt gia cc chui DNA b sung xy ra nh bt cp base, ngc li vi qu trnh bin tnh (denaturation) m nh chng c tch ri (Hnh 2.2) thc hin s ti bn hoc phin m. ng hc ca phn ng ti lin kt phn nh s khc nhau ca cc chui hin din, v th phn ng ny c th c dng nh lng cc gen v cc sn phm RNA ca chng.
DNA si i Bin tnh

DNA si n

Hi tnh

DNA c hi tnh

Hnh 2.2. DNA c th bin tnh v hi tnh

Sinh hc phn t

32

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 C0 t1/2 v c gi l C0t1/2. Gi tr ny t l nghch vi hng s tc . Do C0t1/2 l tch s ca nng v thi gian yu cu cho mt na ng, nn mt gi tr C0t1/2 ln hn dn n mt phn ng chm hn.
Bng 2.3. Mt phn ng ti lin kt ca DNA c m t bi C0t1/2

Phn ng lai ph thuc vo C0t Tc phn ng Phn ng theo phng trnh bc hai

dC dt

kC 2

C l nng ca DNA si n thi im t. K l hng s tc ti lin kt. Tin phn ng Ly tch phn phng trnh tc gia cc gii hn: nng ban u ca DNA = C0 thi im t = 0; nng duy tr si n = C sau thi gian t

C C0

1 1 k .C 0 t

Thng s ti hn l C0t1/2 Khi phn ng hon thnh mt na thi im t =

C C0

1 2

1 1 k .C 0 t 1/2

V th C0t1/2 = C 0 t 1/2

1 k

Sinh hc phn t

33

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

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

3. Kch thc ca genome Khng phi tt c cc on DNA trong genome u tng ng vi cc gen (m ha cho protein hoc mt sn phm cn thit cho hot ng sng ca t bo). T nhng nm 1970, bng cc th nghim gy bo ha t bin ngi ta c th xc nh c s gen nm trn mt on nhim sc th. Ngy nay, nh cc k thut phn tch DNA v RNA hin i (Southern blot, Northern blot, microarray...) cc nh khoa hc c th xc nh s gen hot
Sinh hc phn t
34

ng trong mt t bo. V d: t bo nm men S. cerevisiae (sinh vt eukaryote bc thp) c khong 4.000 gen hot ng, cn t bo ng vt c v khong 10.000-15.000 gen. Nh vy, nu di trung bnh ca mt gen khong 10 kb th tng s chiu di cc gen hot ng trong mt t bo cng ch chim 1-2% genome. Hay ni cch khc, ch mt phn rt nh genome mang thng tin di truyn cn thit cho hot ng sng ca t bo. Vy phn genome cn li c vai tr g, v tnh phc tp ca loi c lin quan g vi kch thc genome hay khng? lm sng t vn trn, chng ta cn xem xt kch thc genome ca mt s loi gn nhau trong bc thang tin ha (c phc tp loi tng t nhau) cng nh genome ca nhng loi xa nhau (c tnh phc tp khc nhau). Chng hn: - Genome ca ngi c kch thc khong 3,3 109 bp, trong khi genome ca nhng loi lng c di khong 3,1 109 bp hoc thc vt c th ln n 1011 bp. Nh vy, c phi l cc loi lng c c tnh phc tp tng t c th chng ta? - Hay l ngay trong cng mt loi, chng ta cng nhn thy c s mu thun v kch thc genome? V d: rui nh (Musca domestica) c genome khong 8,6 108 bp, ln gp su ln kch thc genome ca rui gim khong 1,4 108bp. Ngoi ra, trong cc loi lng c kch thc genome ca chng cng thay i kh ln t 109-1011 bp. V sao ngay trong cng mt loi m kch thc genome li bin thin nhiu nh vy, c phi rui nh c cu to phc tp hn nhiu so vi rui gim? T nhng d liu trn, chng ta c th nhn nh rng tnh phc tp ca loi khng lin quan n kch thc ca genome. Tuy nhin, vai tr ca phn genome cn li (phn khng m ha) n nay vn cha c bit nhiu. 4. Tng s gen c bit mt s loi eukaryote C 6.000 gen nm men S. cerevisiae, 18.500 gen giun trn, 13.600 gen rui gim, 25.000 gen Arabidopsis, v c kh nng 30.000 gen chut v < 30.000 gen ngi. Nh chng ta bit, mi quan h gia kch thc genome v s lng gen khng cn na. Genome ca cc sinh vt eukaryote n bo
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c cng phm vi kch thc vi genome ca vi khun ln nht. Cc eukaryote bc cao c nhiu gen hn, nhng s lng khng tng quan vi kch thc genome. Hnh 2.4 cho thy genome ca loi nm men S. cerevisiae di 13.500 kb v loi nm men S. pombe l 12.500 kb, c khong 6.000 gen v 5.000 gen tng ng. Khung c m trung bnh khong 1,4 kb, v th khong 70% genome c chim gi bi cc vng m ha. S khc nhau ch yu gia chng l ch 5% gen ca S. cerevisiae c intron, so vi 43% ca S. pombe. Genome ca giun trn c khong 18.500 gen. Mc d genome ca rui gim ln hn genome ca giun trn, nhng chng li c s gen t hn. n nay, chng ta cha hiu ti sao rui gim-mt c th phc tp hn nhiu-ch c 70% s gen so vi giun trn. iu ny cho thy khng c mt mi quan h chnh xc gia s gen v tnh phc tp ca c quan.

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

Cy Arabidopsis c kch thc genome trung gian gia giun trn v rui gim, nhng li c s gen ln hn c hai (25.000). iu ny mt ln na cho thy khng c mt quan h r rng, v cng nhn mnh nt c bit
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ca thc vt, l c th c nhiu gen hn (do s nhn i ca ng b t tin truyn li) cc t bo ng vt. a s genome Arabidopsis c tm thy trong cc on c nhn i, gi rng c mt s nhn i xa xa trong genome (to ra mt dng t bi). Ch 35% cc gen ca Arabidopsis hin din nh cc bn sao n. Genome ca la ln hn Arabidopsis khong 4 ln, nhng s gen ch ln hn khong 50%, c kh nng khong 40.000 gen. DNA lp li chim khong 42-45% genome. Hn 80% gen tm thy trong Arabidopsis c hin din trong la. Trong s nhng gen chung ny, khong 8.000 c trong Arabidopsis v la nhng khng thy bt k genome ng vt hoc vi khun no (nhng gen c phn tch trnh t). C kh nng y l tp hp cc gen m ha cho cc chc nng c trng ca thc vt, chng hn nh quang hp.

II. Tnh phc tp ca genome

Kt qu nghin cu ng hc ca cc phn ng lai c tin hnh gia genomic DNA vi cDNA (complementary DNA-DNA b sung), gia DNA vi mRNA cho thy hu ht cc gen hot ng u nm trong thnh phn DNA khng lp li. Nh vy, thnh phn ny c ngha rt quan trng trong vic nh gi tnh phc tp ca genome. Hay ni cch khc, da vo thnh phn DNA khng lp li c th bit c kch thc genome cng nh mc tin ha ca loi. Nu nh kch thc genome ( trng thi n bi) c coi l mt thng s ng hc (k hiu C), th gi tr ny c trng cho tng loi v khng phi lun lun t l thun vi tnh phc tp ca loi. Ngc li, gi tr C phn nh cc c im sau: - S lng DNA m ha cho cc sn phm cn thit i vi hot ng sng ca c th rt nh so vi s lng DNA c trong genome. - C s bin i rt ln ca gi tr C gia mt s loi m tnh phc tp ca chng khng khc nhau nhiu. Genome vi khun c xem l ch cha cc on DNA khng lp li v cc gen thng tn ti bn sao n. Ngc li, genome ca eukaryote thng cha cc gen c hai hoc nhiu bn sao. Hn na, trnh t nucleotide ca cc bn sao ny c th khng ging nhau hon ton mc d sn phm protein m chng m ha c cng mt chc nng. Cc bn sao tng ng ca mt gen c xp chung vo mt nhm gi l mt h gen
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(gene family). Nh vy, ngoi cc gen c mt bn sao ging nh vi khun, genome ca eukaryote cn cha cc h gen. Hu ht cc gen m ha cho protein c phn lp u nm trong cc h gen khc nhau. Cc gen trong mt h thng hot ng theo thi gian v khng gian. iu c ngha, mi thnh vin trong h thng hot ng mt thi im nht nh trong qu trnh hnh thnh v pht trin c th hoc hot ng trong cc m chuyn bit. Khi mt thnh vin trong h b t bin (bt hot) th thnh vin khc c th hot ng thay th. Khi nim v gen c hnh thnh khi cc nh di truyn hc c in nghin cu biu hin cc tnh trng mi do gy t bin DNA ca genome vi khun hay thc khun th (bacteriophage). Mt gen c xem l mt on DNA m bt c t bin no xy ra trn u dn n xut hin tnh trng mi. iu ny d hiu i vi nhng genome ch cha cc gen c mt bn sao (nh genome ca prokaryote). Tuy nhin, genome ca eukaryote, khi c nhiu gen cng qui nh mt tnh trng hoc cc gen tng ng trong mt h c th hot ng h tr thay th nhau th t bin trn mt gen khng phi lc no cng quan st c mc phenotype. Mt khc, cc on DNA tng ng vi trnh t m ha (coding sequence, exon) cho mt protein thng b ngt qung bi cc on DNA khng cha thng tin di truyn (non-coding sequence, intervening sequence, intron). Cc intron c phin m cng vi cc exon sang phn t RNA gi l phn t tin thn mRNA (pre-mature mRNA hay pre-mRNA) nhng sau chng b loi b v cc exon c ni li vi nhau to thnh phn t mRNA hon chnh (mature mRNA hay mRNA) c dng cho qu trnh sinh tng hp protein. Qu trnh ct cc intron, ni exon khng tun theo mt trt t bt buc m bin ha a dng to ra cc phn t mRNA khc nhau t mt phn t premRNA (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

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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 premRNA 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.
Trnh t m ha RNA

DNA
Promoter PHIN M M Exon Intron Exon Intron Exon AAAAAAA 3 Trnh t ch huy on ko di Hon chnh RNA: loi b intron Trnh t m ha protein ui Poly(A) Terminator

Pre-mRNA 5

mRNA

5 DCH M

AAAAAAAA 3

Polypeptide

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

Ngy nay, theo quan im ca sinh hc phn t, mt gen c xem l mt on DNA m ha cho mt sn phm cn thit i vi hot ng sng
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ca t bo. R rng rng khng phi ch c DNA m ha cho protein m c cc DNA m ha cho rRNA, tRNA v cc loi RNA khc tham gia vo nhng phng thc kim sot hot ng ca genome cng c xc nh l gen.

III. Thay i trt t ca cc on DNA trong genome-Transposon

1. S thay i trt t ca cc on DNA trong genome Nh bit kch thc v cu trc genome ca cc loi rt khc nhau. Nguyn nhn ca s a dng ny l do: 1) trao i cho gia cc cp nhim sc th tng ng xy ra trong phn bo gim nhim dn n s a dng trong loi; 2) trong genome trt t cc on DNA cng nh cu trc cc gen c sp xp li, c trng cho tng c th. Chnh cc yu t di truyn c kh nng di chuyn gia cc v tr trong mt genome hoc gia cc genome khc nhau gp phn lm a dng di truyn gia cc c th trong loi. Cc yu t di truyn c kh nng di chuyn c xp vo ba nhm chnh ty thuc vo tnh c lp ca chng: - Nhm th nht gm cc yu t c kh nng di chuyn gia cc v tr khc nhau trong genome. - Nhm th hai gm cc yu t c kh nng ghp vo v tch ra khi genome tn ti c lp trong t bo (cc episome nh plasmid F, bacteriophage). - Nhm th ba ch di chuyn di s kim sot ca t bo nhng giai on sinh trng pht trin nht nh sp xp khi ng mt s gen c bit (hnh thnh cassette hot ng nm men S. cerevisiae, k sinh trng n bo Trypanosome). S di chuyn ca cc yu t nhm th hai v th ba lin quan ti ti t hp tng ng hoc ti t hp cc v tr c hiu. Mc d khng c kh nng tn ti c lp bn ngoi genome, nhm th nht t kim sot s di chuyn ca chng trong genome v khng i hi s tng ng gia chng vi v tr ghp vo. Do , c th ni chng di chuyn mt cch t do trong genome v c gi tn chung l cc yu t di chuyn (transposable elements, transposons). Trong khi thc khun th c xem l episome,

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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.
DNA ca genome Retroelement Bn sao mi ca retroelement

(1) Phin m (2) Dch m (5) Xen on ca retroelement DNA

RNA (3) Phin m ngc RNA thnh DNA

Reverse transcriptase (4) Tng hp si DNA th hai

Hnh 2.6. Retroelement

( . sinh vt eukaryote, cc transposon cn c gi l yu t kim sot (controlling elements).

. cy ng, ng
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. 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 10-510-7 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

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oligonucleotide phn b hai u transposon. y l v tr enzyme nhn bit v ct ni transposon v tr i v n. Cc transposon n gin nht vi khun c gi l IS (insertion sequences). Chng c th nm trn chromosome hoc trn cc plasmid. Transposon vi khun khng gi mt chc nng no trong t bo. Trnh t nucleotide mt u IS thng lp li nhng ngc chiu so vi u kia (inverted repeat). V d nh GGTAT-Xn-ATACC. Do , khi si i IS tch thnh hai si n th mi si ny c kh nng hnh thnh lin kt b sung ti hai u ca IS to cu trc thn-quai/cn-thng lng (stem-loop). Transposon thng m ha cho cc enzyme transposase lm nhim v nhn bit chui nucleotide lp li ngc chiu (inverted repeat) ct transposon v di chuyn. Khi mt IS c ghp vo v tr bt k ca genome th mt on ngn DNA ti v tr ny c nhn i, tc l hai u ca IS c chn bi cc nucleotide ging nhau, sp xp theo cng mt chiu. V vy, on ngn DNA ny c gi l lp li xui chiu (direct repeat). Chiu di ca chng thng khong 9 bp (Hnh 2.7). Da vo s c mt ca cc on cng chiu v ngc chiu c th xc nh c v tr transposon ghp vo hoc chuyn i. Ngoi cc IS, vi khun cn c cc on DNA c kh nng di chuyn vi kch thc di hn, gi l Tn. Cc Tn thng phn b trn plasmid (phn t DNA dng vng, kch thc thng khng ln) v c kh nng chn vo bt k v tr no trong genome. Chng thng mang thng tin di truyn m ha cho cc protein khng khng sinh. Gia IS v Tn c mi quan h v trnh t cc nucleotide. Cc Tn thng c gii hn hai u bi mt loi IS no . Qu trnh di chuyn ca mt transposon t v tr c (v tr cho, donor) sang v tr mi (v tr nhn, recipient) xy ra theo hai c ch khc nhau: - C ch sao y bn chnh (transposon c mt c hai v tr). Theo c ch ny, phin bn sau khi c sao chp t v tr cho s c ghp vo v tr nhn. Nh vy, mi ln di chuyn th s lng bn sao c tng ln. Qu trnh ny lin quan n hai loi enzyme: transposase (tc ng vo hai u bn gc transposon) v resolvase (tc ng ln bn sao). - C ch tch ra khi v tr c di chuyn n v tr mi. Theo c ch ny, mt transposon c th tch ra khi v tr c v ghp vo v tr mi. Nh vy, s lng transposon khng thay i. Kiu di chuyn ny ch cn
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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.

123456789 123456789
Transposase gene

987654321 987654321

ATGCA TACGT DNA ch V tr ch DNA ch

ATGCA123456789 TACGT123456789 Transposon Lp li Lp li xui chiu ngc chiu

987654321ATGCA 987654321TACGT Lp li Lp li ngc chiu xui chiu Chn lc ch ngu nhin cc im nng AAAN20TTT cc im nng NGCTNAGCN cc im nng ngu nhin

IS1 IS2 IS4 IS5 IS10R IS50R IS903

9 bp 5 bp 11-13 bp 4 bp 9 bp 9 bp 9 bp

23 bp 41 bp 18 bp 16 bp 22 bp 9 bp 18 bp

Chiu di tng s 768 bp 1327 bp 1428 bp 1195 bp 1329 bp 1531 bp 1057 bp

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.

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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 A. rhizogenes thc vt. Tuy nhin, sau bnh c duy tr li khng ph thuc s tn ti ca vi khun. l do mt s gen ca vi khun c chuyn vo genome cy ch v hot ng gy bnh. Cc gen vi khun c kh nng di chuyn v hot ng trong t bo thc vt nm trn Ti-plasmid (tumor inducing plasmid) ca A. tumefaciens hoc trn Ri-plasmid (hairy-root inducing plasmid) ca A. rhizogenes. Cng nh cc khi u ng vt, cc t bo thc vt c DNA vi khun ghp vo genome b chuyn sang trng thi mi, s pht trin v bit ha ca chng hon ton khc vi cc t bo bnh thng. l do hot ng ca cc gen vi khun trong genome ca thc vt. Bnh thng, nhng gen ny c mt trong genome vi khun nhng chng ch c hot ng (m) sau khi hp nht vo genome thc vt v chu s kim sot ca t bo cy ch. Qu trnh ny c tnh cht c hiu vt ch, tc l mt loi vi khun ch c kh nng gy khi u trn mt s loi cy ch ny m khng tng tc c vi cc loi cy khc. Vic to khi u hay thc hin qu trnh chuyn gen t vi khun sang genome thc vt dn n bin i trng thi sinh l ca t bo thc vt i hi cc iu kin sau: - Phi c hot ng ca cc gen trn ba 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.

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1. Ti-plasmid v Ri-plasmid -

. 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 Agrobacterium A. tumefaciens A. rhizogenes . 2. T-DNA , cytokinin v opine (trng hp Ti-plasmid) hoc auxin v opine (trng h (right border-RB) v bin tri (left border-LB), mi bin c chiu di 25 bp. 3. Vng vir vir vir virA, virE2, virB, virD, virD2, vir -

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. 4. Qu trnh chuyn T-DNA vo t bo thc vt Qu trnh ct on T-DNA ra khi plasmid v vn chuyn n vo t bo thc vt trc ht ph thuc vo sn phm ca cc gen vir. Vi khun xm nhim ti bt k v tr tn thng no trn thn cy. Cy c vt thng do s hng ngu nhin ca mng t bo thc vt hoc do vi khun tit ra hn hp nhng cht c m ha bi cc gen vir. Hot ng ca cc gen ny c hot ha bi hp cht phenolic ca cy (v d nh acetosyringone, catechol, cc dn xut ca chalcone...). Ngoi ra, cc monosaccharide nh glucose, arabinose c mt trong mi trng cng lm tng kh nng gy cm ng nhm gen vir. Protein VirA ng vai tr quan trng trong vic quyt nh loi cy ch b nhim bi Agrobacterium. Trong thc t, Agrobacterium khng c kh nng xm nhp vo cy mt l mm, c th protein VirA khng nhn bit cc tn hiu do cy mt l mm tit ra. Ni chung, Agrobacterium -

Agrobacterium . Hin nay, ngi ta cng thnh cng trong vic chuyn gen vo mt s cy mt l mm nh la, ma... qua trung gian A. tumefaciens. A. tumefaciens, do T-DNA hp

rhizogenes

A. rhizogenes A. .

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
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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 TDNA t vi khun vo t bo thc vt. Cc loi protein rt cn thit cho qu trnh ct T-DNA khi plasmid, cm ng thay i mng t bo thc vt m chng tip xc, tham gia di chuyn phn T-DNA qua mng vi khun ti t bo cht ca t bo thc vt, vn chuyn ti nhn ri cui cng xm nhp vo genome ca cy ch. Thc cht, ch ring T-DNA ca plasmid c chuyn vo genome t bo thc vt, m khng cn phn no khc. Qu trnh dn truyn ch do sn phm ca cc gen vir v gen chv quyt nh m khng lin quan n cc gen khc trn T-DNA. Tuy nhin, chui DNA 25 bp (RB v LB ca T-DNA) c vai tr l v tr cm ng cho cc sn phm ca t hp cc gen vng vir, c bit l protein t gen virE mang chng dn truyn vo t bo thc vt. Chng hot ng nh cc tn hiu nhn bit v khi ng qu trnh dn truyn. Trc ht gen virA trong t hp gen vng vir c phosphoryl ha nh tc ng ca cc hp cht phenol nh acetosyringone gii phng ra t cc t bo thc vt tn thng. Sn phm ca qu trnh ny li tip tc phosphoryl ha gen virG. Sn phm ca gen virG lin tip lm hot ha ton b cc gen vir cn li, m hai gen cui cng c hot ha l gen virB v virE. Trc , khi gen virD c hot ha, sn phm ca n cm ng nhn bit RB v LB ca T-DNA v lm t phn 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.

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V. Sp xp v khuch i cc gen trong genome

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

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

Z
Ct HO

HML MATa

Xm ln si

Ko di u 3

Chuyn trng thi

Y
Y

HML

MAT

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
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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 Nterminus, 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. Nterminus 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.
Peptide tn hiu N-terminus H2N 20 360 100 Vng tng ng Vng bin thin COOH 20 ui k nc C-terminus

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

Protein

C-terminal amino acid Ethanolamine Glycan Glucosamine Inositol

Phospholipase C

Mng

1,2-Dimyristoylglycerol

Hnh 2.10. Cu trc ca m neo glycolipid ca VSG

- Hot ng ca gen VSG Gen m ha cho mt VSG c gi l bn gen gc (basic copy gene). Cc gen ny c phn thnh hai nhm ty thuc vo v tr ca chng trn nhim sc th.
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+ Cc gen nm telomere (khong 5-15 kb)>200 gen. + Cc gen nm cch telomere hn 50 kb. Tng t nh nm men, cc gen m ha cho VSG nm ri rc trong genome v trng thi khng hot ng. Mt gen bt k c hot ha ch khi n c sao chp vo v tr hot ng (expression site) trong khi nguyn bn ca n vn c bo tn v tr tnh. Bn sao ca bn gen gc vo v tr hot ng c gi l ELC (bn sao hot ng-expression linked copy). V tr hot ng nm gn telomere. Nh vy, vic chn mt bn gen gc sao chp to bn sao hot ng s ph thuc vo v tr ca bn gen gc telomere hoc nm pha trong telomere. C hai gi thit mt gen tr nn hot ha nh sau: - V tr hot ng khng thay i m ch c cc ELC thay th cho nhau. Bn sao ca mt bn gen gc s thay th cho bn sao ca mt gen khc ti v tr , iu ny xy ra tng t nh cc gen cassette tnh c sao chp vo cassette hot ng trong trng hp vi nm men. - V tr hot ng thay i, khi cn tng hp mt VSG mi, gen v tr hot ng c b ngng v gen mt v tr khc (gn telomere) c khi ng. Mt s phn t mRNA tng ng vi cc VSG khc nhau c phn lp v c xc nh trnh t nucleotide (thng qua cDNA). iu ngc nhin l phn oligonucleotide u 3 ca mi gen VSG u khc vi phn 3 ca cc mRNA c phin m t cc gen . Mt khc, cc gen ny khng c phn 5 ging nh cc mRNA. Nh vy, cc mRNA khng c tng hp hon ton trn khun mu cc gen ny. Phn 3 ca cc mRNA tng ng vi phn 3 ca v tr hot ng ELC, trong khi phn 5 (gm 35 nucleotides) c tng hp t nhng on DNA khc v c gn vo mRNA (hin tng trans-splicing). 2. Khuch i cc gen S lng bn sao ca mt s gen cng c th c tng ln tm thi trong qu trnh pht trin cc t bo soma. Vic tng s lng ca mt gen c bit no ph thuc vo tng iu kin c th ca t bo v xy ra khng ph bin. Cc bn sao c th nm tp trung thnh mt nhm gm bn sao ny ni tip bn sao khc hoc c th tn ti nh nhng on DNA c kh nng ti bn c lp. Chng hn:
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- 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

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ca t bo. V d in hnh l vi khun A. tumefaciens cm ng to khi u t bo thc vt b chng xm nhim. Khi DNA l c a vo t bo eukaryote, n c th tn ti ngoi nhim sc th hoc c hp nht trong genome. Nu xy ra theo trng hp th hai, genome s mang nhng t bin di truyn v nhiu khi DNA l vn tip tc hot ng lm xut hin cc tnh trng mi. Vic a cc gen l vo t bo soma hoc t bo sinh dc m vn duy tr hot ng ca nhng gen c gi l chuyn nhim (transfection). C th biu hin tnh trng mi nh hot ng ca gen l a vo t bo sinh dc c gi l c th chuyn gen. Qu trnh ny c th lm thay i s n nh ca genome. DNA sau khi c tim vo trong cc t bo trng ng vt c th c hp nht trong genome v truyn cho th h sau nh mt thnh phn di truyn bnh thng. Kh nng a mt gen chc nng c trng c th tr thnh mt k thut y hc s dng cho vic cha bnh di truyn.

Ti liu tham kho/c thm

1. V Th Thng Lan. 2002. Sinh hc phn t. NXB i hc Quc gia H Ni, H Ni. 2. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 3. Cantor CR and Smith CL. 1999. Genomics. John Wiley & Sons, Inc. New York, USA. 4. Dale JW and Von Schanzt M. 2002. From Gene to Genome. John Wiley & Sons, Ltd. West Sussex, UK. 5. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 6. Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J. 2004. Molecular Cell Biology. 5th ed. WH Freeman and Company, New York, USA. 7. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Loscik R. 2004. Molecular Biology of the Gene. The Benjamin Cummings/Cold Spring Habor Laboratory Press, San Francisco, CA, USA. 8. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company Inc. New York, USA.

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Chng 3

Cu trc v chc nng ca gen

I. nh ngha gen
Chng ta c th im qua nhng mc chnh trong lch s nghin cu v gen nh sau: Mendel (1865) l ngi u tin a ra khi nim nhn t di truyn. Johansen (1909) xut thut ng gen (t genos, ngha l sn sinh, ngun gc) ch nhn t di truyn xc nh mt tnh trng no . Sau , Morgan trong nhng nm 1920 c th ha khi nim v gen, khng nh n nm trn nhim sc th v chim mt locus nht nh, gen l n v chc nng xc nh mt tnh trng. Vo nhng nm 1940, Beadle v Tatum chng minh gen kim tra cc phn ng ha sinh v nu gi thuyt mt gen-mt enzyme. Tuy nhin, trng hp hemoglobin l mt protein nhng li gm hai chui polypeptide do hai gen xc nh, do gi thuyt trn buc phi iu chnh li l mt gen-mt polypeptide. Vo nhng nm 1950, DNA (deoxyribonucleic acid) c chng minh l vt cht di truyn. M hnh cu trc DNA ca Watson v Crick c a ra v l thuyt trung tm (central dogma) ra i. Gen c xem l mt on DNA trn nhim sc th m ha cho mt polypeptide hay RNA. Cui nhng nm 1970, vic pht hin ra gen gin on sinh vt eukaryote cho thy c nhng on DNA khng m ha cho cc amino acid trn phn t protein. V th, khi nim v gen li c chnh l mt ln na: Gen l mt on DNA m bo cho vic to ra mt polypeptide, n bao gm c phn pha trc l vng 5 khng dch m (5 untranslation) hay cn gi l vng ngc hng (upstream) v pha sau l vng 3 khng dch m (3 untranslation) hay cn gi l vng cng hng (downstream) ca vng m ha cho protein, v bao gm c nhng on khng m ha (intron) xen gia cc on m ha (exon). Hin nay, c th nh ngha gen mt cch tng qut nh sau: Gen l n v chc nng c s ca b my di truyn chim mt locus nht nh trn
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nhim sc th v xc nh mt tnh trng nht nh. Cc gen l nhng on vt cht di truyn m ha cho nhng sn phm ring l nh cc mRNA c s dng trc tip cho tng hp cc enzyme, cc protein cu trc hay cc chui polypeptide gn li to ra protein c hot tnh sinh hc. Ngoi ra, gen cn m ha cho cc tRNA, rRNA v snRNA...
Bng 3.1. Tm tt lch s nghin cu v di truyn hc

Mc thi gian 1850

Nm

Cc s kin chnh

1865 1871

Gen l cc nhn t ht Khm ph ra nucleic acid Nhim sc th l cc n v di truyn Gen nm trn nhim sc th Nhim sc th l cc dy sp xp mch thng ca gen t bin l nhng thay i vt l ca gen S ti t hp xut hin bi hin tng vt cho DNA l vt liu di truyn Gen m ha cho protein Trnh t protein u tin DNA c dng xon kp DNA ti bn theo phng thc bn bo th M di truyn l b ba Cc gen ca sinh vt eukaryote b gin on DNA c th c phn tch trnh t Genome ca vi khun c phn tch trnh t Genome ngi c phn tch trnh t

1900

1903 1910 1913 1927 1931 1944 1945

1950

1951 1953 1958 1961 1977 1977 1995

2000

2001

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II. L thuyt trung tm

1. S xc nh di truyn cu trc bc mt ca protein Cu trc khng gian ca chui polypeptide c xc nh bi trnh t sp xp ca cc amino acid tc cu trc bc mt. Nh vy, mc d c nhiu mc cu trc khng gian khc nhau, nhng cu trc bc mt tc trnh t sp xp cc amino acid chi phi ton b cc mc cu trc khc. Vic xc nh di truyn phn t protein trng thi t nhin c y hot tnh sinh hc ch quy t li ch yu xc nh cu trc bc mt l . 2. Cc enzyme mt hot tnh do t bin Nhiu nghin cu cho thy, vic mt hot tnh enzyme nhiu khi khng phi do vng mt ca enzyme, m ch do cc bin i trn phn t (modification). C trng hp t bin dn n nhng thay i tinh vi, enzyme vn c hot tnh nhng s biu hin khc nu thay i iu kin. Chng hn: nm mc Neurospora crassa, enzyme tyrosinase do gen T xc nh, xc tc cho phn ng chuyn ha tyrosine thnh dihydroxyphenylalanine. Alelle T+ ca dng hoang di sn xut tyrosinase c hot tnh nhit bnh thng v c 60oC. Mt t bin TS sn xut tyrosine c hot tnh nhit bnh thng, nhng li mt hot tnh 60oC. Nh vy, trong a s trng hp, t bin ca mt gen khng lm bin mt enzyme m ch bin i cu trc dn n thay i hot tnh. Cc t bin ca cng mt gen c th gy ra nhng bin i khc nhau trn enzyme. Cc hin tng chng t rng cu trc ca enzyme chu s kim sot trc tip ca gen. 3. Bn cht cc bin i di truyn ca protein Bn cht chnh l quan h mt gen-mt polypeptide. Nh nu trn, ngi ta khm ph ngi c nhng gen to ra hemoglobin (Hb) khi bin d s to ra nhng hemoglobin bt thng do sai hng cc chui polypeptide hoc (Bng 3.2 v 3.3) v gy ra cc bnh di truyn.

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Bng 3.2. Cc loi hemoglobin chui polypeptide

Th t amino acid Tn amino acid Loi hemoglobin Hb I

1 Val

2 Leu

16 Lys Asp

57 Glu

58 His

68 Asp

116 Glu

141 Arg

Hb Nocfolk Hb M Boston Hb B Philadelphia Hb O Indonesia

Asp Tyr Lys Lys

trng hp ny, th t cc amino acid c th b sai lch. T nhng d liu trn ta thy cc dng t bin to mt Hb bt thng l thay mt amino acid ny bng mt amino acid khc.
Bng 3.3. Cc loi hemoglobin chui polypeptide

Th t amino acid Tn amino acid Loi hemoglobin Hb S Hb C Hb E Hb M Minvauki Hb O Rp

1 Val

2 His

3 Leu

6 Glu Val Lys

26 Glu

67 Val

121 Glu

146 His

Lys Glu Lys

Qua hai chui polypeptide v chng ta thy c mt s dng hemoglobin bt thng ngi. Trn mi chui, ch trnh by nhng amino acid b thay i dng t bin. S th t ch v tr ca amino acid trong chui polypeptide. Mi hemoglobin bt thng c th c t cho mt ch cng tn (nu c) ca a phng c tm thy.
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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).
Cc v tr ca t bin trn DNA

H3N 1 15 22 49 Glu 175 177 Tyr Leu 183 211 213 Thr Gly Gly 234 235 Gly Ser 243 Gln

COO268
Cc gc amino acid b thay i

Lys Phe

STOP Leu Val Gln Met Cys Arg

Ile Arg Glu Val Cys Asp Leu STOP

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
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ca t bin c xc nh. Sn phm protein ca mi dng t bin c phn tch, v nhng thay i cc amino acid khc cng c xc nh. Ngi ta tm thy mi tng quan hon ton gia nhng khong cch ca cc t bin c tm thy trn gen vi khong cch ca amino acid b thay i trong phn t protein. 4.2. t bin 4.2.1. Khi nim Mt gen (DNA) c 4 loi base v mt phn t protein c 20 loi amino acid1, nhng gia chng c mi tng quan nh th no. u tin, ngi ta cho rng mt base qui nh mt amino acid, nhng nhng tnh ton cho thy khng hp l. V ch c 4 base trong DNA v 20 amino acid trong protein, cho nn mi codon phi cha t nht 3 base. Hai base cng khng th lm thnh mt codon bi v ch c 42 = 16 cp hp l ca 4 base. Nhng 3 base th c th bi v s c 43 = 64 b ba hp l. V s lng b ba hp l ln hn 20, cho nn s c trng hp mt vi codon ch nh cng mt amino acid. V d: UCU, UCC, UCA, UCG, AGU v AGC u cng m ha cho serine. T , ngi ta a ra khi nim m di truyn (tn hiu di truyn). M di truyn cho php c th t trn DNA bit th t trn chui polypeptide. M di truyn khng m h, c ngha vi mt trnh t chng hn ATA ta bit n ghi m cho mt amino acid g, v cng thy rng c nhiu m di truyn xc nh cho mt amino acid (Bng 3.4). 4.2.2. t bin im L t bin ch tc ng mt v tr, ni r hn l mt base. Khi thay i mt base trn DNA s to ra s thay i mt amino acid (Hnh 3.2).

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

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t bin d nhin xy ra trn DNA v c sao li trn mRNA trong phin m, ri trn protein trong dch m.
Bng 3.4. M di truyn chung

V tr th nht U U U U C C C C A A A A G G G G

V tr th hai U Phe (F) Phe (F) Leu (L) Leu (L) Leu (L) Leu (L) Leu (L) Leu (L) Ile (I) Ile (I) Ile (I) Met (M) Val (V) Val (V) Val (V) Val (V) C Ser (S) Ser (S) Ser (S) Ser (S) Pro (P) Pro (P) Pro (P) Pro (P) Thr (T) Thr (T) Thr (T) Thr (T) Ala (A) Ala (A) Ala (A) Ala (A) A Tyr (Y) Tyr (Y) STOP STOP His (H) His (H) Gln (Q) Gln (Q) Asn (N) Asn (N) Lys (K) Lys (K) Asp (D) Asp (D) Glu (E) Glu (E) G Cys (C) Cys (C) STOP Trp (W) Arg (R) Arg (R) Arg (R) Arg (R) Ser (S) Ser (S) Arg (R) Arg (R) Gly (G) Gly (G) Gly (G) Gly (G)

V tr th ba U C A G U C A G U C A G U C A G

Ch thch Nhng n v m (codon) c c theo chiu 5 STOP: codon kt thc (cn gi l v ngha). 3.

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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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AUG Met

ACU Thr

CGG Arg

AAG Lys

UCA Ser

CUA Leu

ACG Thr

AUU Ileu

AGG Arg

CUU Leu

UAC Tyr

... ...

AUG Met

ACU Thr

AGG Pro

AAG Lys

UCA Ser

CUA Leu

ACG Thr

AUU Ileu

AGG Arg

CUU Leu

UAC Tyr

... ...

AUG Met

ACU Thr

CGG Arg

AAG Lys

UGA Kt thc

CUA

ACG

AUU

AGG

CUU

UAC

...

AUG Met

ACU Thr

CGG Arg

ACA Thr

GUG Val

ACU Thr

AAC Asp

GAU Asp

UAG

GCU

UUA

...

Kt thc

AUG Met

ACU Thr

CGG Arg

AGU Ser

GAC His

UAA Kt thc

CGA

UUA

GGC

UUU

AC

...

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
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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 polyribonucleotide 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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Phin m

Dch m

DNA
Sao chp

mRNA

Protein

Hnh 3.3. L thuyt trung tm ca Crick

Phin m

Dch m

DNA
Phin m ngc Sao chp

mRNA
?
(virus)

Protein
(prion)

Hnh 3.4. Nhng b sung mi vo l thuyt trung tm ca Crick

6. DNA v m di truyn Chng ta bit c s lin quan ng tuyn tnh gia DNA v phn t protein, t d dng d on rng trnh t c hiu ca cc amino acid trn phn t protein s c m ha bng nhm cc nucleotide trn phn t DNA. C tt c 4 loi base, nu cc base c nhm i tc 2 nucleotide m ha cho mt loi amino acid th tt c ch c 16 t hp, khng cho 20 loi amino acid. Nh vy, n v m ha (codon) phi gm 3 nucleotide (xem phn 4.2). Vn tip theo l xc nh chnh xc cc codon no m ha cho tng amino acid. Nirenberg v Matthaei s dng enzyme tng hp nhn to RNA. Khi dng ch mt loi nucleotide l U s nhn c RNA l poly(U), nu ch dng A s nhn c poly(A). Nm 1961, Nirenberg v Matthaei dng poly(U) thay cho khun mu mRNA tng hp protein trong h thng v bo (c amino acid, enzyme tng hp protein, nhng khng c DNA...), sn phm thu c l chui polypeptide polyphenylalanine ch cha mt loi amino acid l phenylalanine. iu chng t codon UUU m ha cho phenylalanine. y l codon u tin c xc nh. Sau , h cng chng minh c rng AAA m ha cho lysine, GGG cho glycine v CCC cho proline.

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Nm 1964, Khorana tm ra phng php tng hp mRNA nhn to vi trnh t lp li (nh AAG AAG AAG...) v nh n gii quyt xong cc vn cn cha r rng. Bng m di truyn (Bng 3.4) cho thy trong 64 codon, c 3 codon UAA, UAG, UGA khng m ha cho amino acid c gi l v ngha (nonsense), ng thi l codon kt thc (termination) tc du chm cu, chm dt chui polypeptide. M di truyn c tnh suy bin (degeneration) tc mt amino acid c nhiu codon m ha, ch tr methionine v tryptophane ch c mt codon (tng ng l ATG v TGG). Cc codon ng ngha tc m ha cho cng mt amino acid thng c hai base u tin ging nhau, nhng khc nhau ci th ba. V d: CCU, CCC, CCA v CCG tt c u m ha cho proline. Trn thc t, U v C lun lun tng ng nhau v tr th ba, cn A v G tng ng nhau trong 14 trn 16 trng hp. Tr mt s ngoi l, m di truyn c tnh ph bin (universal) tc ton b th gii sinh vt c chung b m di truyn.

III. Cu trc v chc nng ca gen

Khi nghin cu cc quy lut di truyn Mendel v hc thuyt di truyn nhim sc th, gen c quan nim nh mt im trn nhim sc th, va l n v chc nng xc nh mt tnh trng, va l n v t bin, va l n v ti t hp. Cng vi s pht trin ca di truyn hc, khi nim v gen c c th ha thm, cu trc v chc nng ca gen c hiu chi tit hn. 1. Cu trc gen Mt gen thng c xem gm c hai phn, phn mang m di truyn c phin m sang phn t mRNA v phn DNA lm nhim v iu khin hot ng ca gen (Hnh 3.5). C hai phn u c cu trc c bn kh ging nhau mi sinh vt prokaryote v eukaryote. Tuy nhin, gen eukaryote cn c cu trc c th lin quan n cc c ch kim sot khc nhau i vi hot ng ca gen. Cu trc mt gen in hnh ni chung u c promoter, v tr RNA polymerase hot ng khi u phin m. i khi nm rt xa gen, c th thy cc enhancer (vng tng cng) hoc silencer (vng c ch) c vai tr
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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.
Vng 5 ngc hng Vng promoter
Cc enhancer Hp CCAAT Hp TATA

n v phin m Cc exon

Vng 3 cng hng

Cc enhancer

Chui 5 Cc intron khng m ha

Chui 3 khng m ha

Hnh 3.5. Cu trc chung ca mt gen

Vng DNA mang m di truyn s c phin m sang phn t mRNA. Qu trnh ny thc hin theo chiu 5 3 trn si mRNA ang c tng hp. Khng phi mi phin m di truyn trn phn t mRNA u c dch m sang phn t protein. Hai u 5 v 3 ca phn t mRNA gm mt s nucleotide khng c dch m m li lin quan n tnh bn vng ca phn t mRNA hoc tham gia kim sot qu trnh dch m. Hai on ny gi l vng khng dch m 5 v 3 (untranslated region). Vng khng dch m 5 nm trc im khi u dch m (3 nucleotide AUG m ha cho methionine u tin ca chui polypeptide) v vng khng dch m 3 nm sau im kt thc dch m (stop codon c th l UAA, UGA v UAG). Do t bo prokaryote khng c cu trc nhn nn qu trnh phin m (tng hp mRNA) v dch m (tng hp protein) xy ra ng thi. Cn phn t mRNA ca eukaryote c phin m trong nhn, sau phi ct b intron v gn cc exon li, chu bin i ti cc u 5 v 3 trc khi vn chuyn ra ngoi t bo cht dng lm khun mu tng hp protein. Hot ng ca mt gen c nh gi thng qua qu trnh phin m (tng hp mRNA) v qu trnh dch m (tng hp protein). Hot ng ny c kim sot rt cht ch bng cc c ch khc nhau mi giai on, nh bt u v kt thc phin m, qu trnh bin i mRNA, quyt nh tnh bn vng v kim tra li thng tin di truyn trn cc phn t ny... Do cu trc

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sp xp cc gen prokaryote khc vi gen eukaryote nn s phi hp gia cc c ch iu khin mang tnh cht ring bit cho tng loi genome. Cc gen prokaryote thng sp xp nm gn nhau v chu s iu khin chung ca mt promoter, tc l chng c phin m sang cng mt phn t mRNA. Cu trc ny c gi l operon. Nh vy, mt operon gm hai hay nhiu gen nm cnh nhau trn mt nhim sc th. Thng thng, l cc gen cng tham gia vo mt con ng chuyn ha, v d nh cc gen m ha cho cc enzyme cn thit cho qu trnh chuyn ha glucose. Do c chung promoter iu khin cho mi gen nm trong mt operon cho nn ch c mt loi phn t mRNA c tng hp t mt operon (mang thng tin di truyn ca tt c cc gen nm trong ). Ni cch khc, qu trnh phin m ca cc gen trong mt operon xy ra ng thi v phn t mRNA c trng cho operon c gi l mRNA-polycistron. Tuy nhin, iu cn ghi nh l qu trnh dch m trn cc phn t mRNA-polycistron xy ra hon ton c lp vi nhau. Mi on tng ng vi mt gen trn phn t ny u c v tr bm ca ribosome, c m bt u v kt thc tng hp chui polypeptide ring bit. Do , tc tng hp cc protein trn cc phn t mRNA-polycistron hon ton khc nhau (Hnh 3.6).
Operon

Operator

Gen cu trc

Gen a Promoter

Gen b

Gen c

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

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cho thy gen c cu to phc tp, s bin i ca gen c th dn n nhiu trng thi allele khc nhau. 2.1. Hin tng allele gi Theo quan nim c in gen l n v ti t hp. Nu c th mang hai allele ln a1/a2 ca mt dy a allele s to thnh hai loi giao t l a1 v a2, lai phn tch vi b m ng hp t ln s ch cho kiu hnh t bin a1 v a2 m khng c dng ti t hp hoang di. V d:
B m Giao t Cc con lai a1/a2 a1 v a2 a1/a1 a1

a1/a1 v a2/a1 - c hai u l kiu hnh t bin

Tuy nhin, nhiu th nghim cho thy nu tng s c th th nghim ln 10.000 hoc 100.000, th c th pht hin c dng kiu hnh hoang di do ti t hp. V d: Trng hp locus mt qu trm rui gim, c 18 allele. Khi tng s c th nghin cu ln nhiu ln, ngi ta pht hin cc allele xp thnh 3 nhm A, B v C. Cc allele ca cng mt nhm, khi lai ln nhau, khng cho kiu hnh ti t hp hoang di mt bnh thng, m ch c kiu hnh mt qu trm. Nhng lai allele ca nhm ny vi allele ca nhm khc s c xut hin kiu hnh hoang di do ti t hp. Hin tng ny c gi l allele gi. Hin tng allele gi cho thy gen phn chia nh v mt ti t hp, c th xy ra ti t hp gia cc phn trong gen. Lc u, hin tng allele gi c coi l trng hp ngoi l, nhng khi tng s c th nghin cu ln nhiu ln th r rng l hin tng ph bin. N c tm thy nhiu i tng khc nhau nh nm men S. cerevisiae, ng, b cu, chut, bacteriophage... 2.2. Locus rII ca bacteriophage T4 Nghin cu chi tit v cc t bin rII ca bacteriophage T4 lm sng t hn v cu trc gen. Bacteriophage T4 dng hoang di r+ c kh nng xm nhim ng thi hai chng E. coli B v K, trong khi cc t bin rII ch xm nhim chng B m khng xm nhim chng K (Hnh 3.7).
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Chng vi khun E. coli B Dng hoang di K

Vt tan (plaque) nh

Vt tan nh

t bin rII

Vt tan ln

Khng c vt tan

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

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

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Kt qu th nghim cho thy, gen c th phn chia nh v mt t bin. Cc on rIIA v rIIB c gi l cistron, n v chc nng nh nht m bo kh nng xm nhim chng K. Thut ng cistron thc cht l gen, ngy nay n ch c tnh cht lch s, t c s dng. Theo quan nim hin nay, rIIA v rIIB l hai locus. Hai khi nim mi c a ra l muton-n v t bin v recon-n v ti t hp. Benzer tm thy 2.000 im t bin trn on gen c nghin cu, chng phn b khng u nhau, c nhng im tp trung nhiu t bin hn. Chiu di gen khong 900 nucleotide. n v t bin muton y tng ng vi 900/2.000. S t bin ghi nhn c th thp hn so vi thc t nn muton c th tng ng vi mt cp nucleotide. Ging nh vy recon c th tng ng vi mt cp nucleotide. Tm li, gen l n v chc nng, c th chia nh bi cc n v t bin ti t hp. 3. Th nghim chc nng allele Mun nghin cu cu trc bn trong mt gen, phi tm hiu nhiu allele ca gen . Nhiu t bin c kiu hnh ging nhau nhng khng allele vi nhau. Th nghim chc nng allele c s dng xc nh xem hai t bin c allele vi nhau khng. y chnh l th nghim m Benzer dng lp bn locus rII. Th nghim ny cn c gi l th nghim b sung (complementary test) v n cho bit sai hng chc nng hai t bin c b sung tc b tr cho nhau c khng. Phng php th ny cng c gi l th nghim u-lch (cis-trans test). S d nh vy l v php th nghim ny so snh hiu qu kiu hnh ca cc gen t bin hai v tr khc nhau trn nhim sc th tng ng. v tr lch (trans) cc t bin nm trn hai nhim sc th, cn v tr u (cis) cc t bin nm trn cng mt nhim sc th. Trng hp sai hng hai gen khc nhau nn c th b sung c, cn trng hp sai hng cng mt gen khng b p c s dn n kiu hnh t bin.

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cis

trans

1
a1 I: Hai t bin trn mt cistron + a2 + Gen A + + Gen B a1 + + a2 Gen A + + Gen B

3
a1 II: Hai t bin trn hai cistron + + Gen A + Gen B + + Gen A b Gen B + b a1 + +

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

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

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ca chu trnh. Nu bin i di truyn lm sai hng chc nng s dn n xut hin t bin mt kh nng tng hp adenine.
ade3 ade5 A ade2 ade6 B

I
ade4

III IV
ade7

V
ade8 ade1

VI

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

Ti liu tham kho/c thm

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

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Chng 4

Ti bn DNA
I. Chng minh ti bn DNA theo c ch bn bo th
1. C ch ti bn bn bo th 1.1. C ch ti bn prokaryote c im c bn ca s ti bn l ti bn theo phng thc bn bo th (semiconservative replication). Ti bn bn bo th ngha l trong hai chui ca tt c cc phn t DNA bao gi cng c: - Mt chui ca DNA c (t mt trong hai chui ca DNA m). - Mt chui ca DNA mi (mi c tng hp). Mi mt ln ti bn u c s tch ri ca hai chui ca DNA m, ng thi mi chui m tin hnh sao chp cho mt chui con, chui ny sau li kt hp vi chui m. V tr m xon kp v tng hp DNA mi cng mt lc trn DNA gi l chc ba ti bn (replication fork) do cu trc ca vng ti bn c hnh ch Y. S tng hp DNA mi gn lin vi vic m xon DNA c. 1.2. C ch ti bn eukaryote S ti bn t bo eukaryote phc tp hn so vi t bo prokaryote nhng c ch ca s ti bn eukaryote cng tng t nh prokaryote v tin hnh theo cc nguyn tc: - Hai hng. - B sung, i song song, theo chiu 5 3. - Khng lin tc mt trong hai chui. - Cn nhng RNA primer. Tuy nhin, c mt s im khc nh sau: - Trong khi prokaryote ch c mt im khi u, th s ti bn eukaryote bt u cng mt lc nhiu im khi u. iu ny l cn thit do DNA ca eukaryote c chiu di rt ln. - Vn tc pht trin ca chc ba ti bn eukaryote (khong 50 nucleotide/s) ch bng 1/10 so vi E. coli.
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2. Th nghim ca Meselson v Stahl Nhng th nghim ca Meselson v Stahl (1957) chng minh l thuyt ti bn DNA theo kiu bn bo th (Hnh 4.1). Cc tc gi trn nui cy E. coli trong nhiu th h trong mt mi trng cha 15NH4Cl lm ngun cung cp nitrogen duy nht. Bng cch ny, DNA c tng hp c 15 N (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.
DNA c tch chit v ly tm cn bng theo grandient mt CsCl

Cc phn t b m gc

DNA nng (15N) H Chui mi Chui m DNA lai (15N/14N) Cc phn t con th h th nht H

DNA nh (14N) DNA lai

Cc phn t con th h th hai

L H

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.

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- 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.
Si i ca con

Si i ca b m

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

2. Chc ba ti bn Hnh 4.4 m t cu trc ca chc ba ti bn. Nh vo phng php phng x nh t ghi, ngi ta nhn thy s ti bn thc hin theo hai hng (bidirectional synthesis). ng thi cng chng minh c vi khun E. coli (prokaryote) c duy nht mt im gc ti bn, l im m hai chui xon kp ca DNA m c tch ra, tng ng vi hai chc ba ti bn pht trin ngc chiu nhau. Chui DNA sau khi tch ra c dng lm khun mu cho s tng hp DNA mi (Hnh 4.5).
Sinh hc phn t
78

A
Chuyn ng ca chc ba ti bn
Gc

im t (nick) 1 Quay trong hng cun Mt im t c bt kn, mt im t khc c to ra

Hng cun

im t 2

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.

5 3 5 (a) 3 (b) 5 3 3 5

(c)

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.

Sinh hc phn t

79

Si i b m khng ti bn

Hng di chuyn ca chc ba ti bn

Si con c ti bn

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.

Gc ti bn Ti bn ch trong mt hng

Gc ti bn

Ti bn ng thi trong c hai hng

Ti bn khng nh hng Gc ti bn

Ti bn theo hai hng

Chc ba ti bn

Chc ba ti bn

Gc ti bn

Chc ba ti bn

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

Hnh 4.7 v 4.8 m t phng thc hp nht cc vng ti bn DNA rui gim (D. melanogaster). Qu trnh ti bn din ra ng thi trn hng chc ngn v tr khc nhau ca phn t DNA v to thnh cc vng ti bn, cc vng ti bn sau s m rng theo hai hng cui cng hp nht vi nhau to thnh hai phn t DNA.
Sinh hc phn t
80

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.

S ti bn bt u v theo hai hng

Gc ti bn

Gc ti bn

S tng hp khi u gc th hai v cng theo hai hng

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
Sinh hc phn t
81

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).
Cc nucleotide c b sung vo nhm 3-OH, chim ch ca si c u tn cng 5-P S ko di tip tc ca u 3

Hng quay

3-OH 5-P Nuclease ct DNA to ra nhm 3-OH v nhm 5-P Si c u tn cng 5-P cng c sao chp

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

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

Si ch

Kp Polymerase III dimer Helicase Chc ba ti bn DNA b m

Khun mu si ch

Primase Khun mu si th RNA primer

Cc protein lin kt DNA si n (SSB)

Ligase

Polymerase I

on Okazaki

Hnh 4.10. Qu trnh ti bn DNA

2. Ko di-Tng hp chui Okazaki Giai on ko di bao gm s tng hp cng mt lc hai chui DNA. Mt chui c ti bn cng hng pht trin ca chc ba s lin tc (si ch), cn chui kia s khng lin tc bao gm cc on Okazaki (si th). Cc nucleotide gn vo u 3 t do, v ko di chui ra nh enzyme DNA polymerase III theo chiu 5 3. Trong giai on ny, nhiu enzyme tham gia vo s tng hp hai chui DNA ti chc ti bn. DNA helicase tip tc tch hai chui DNA m, DNA gyrase m xon, protein SSB n nh nhng chui DNA n c tch ra, DNA ligase gn cc on Okazaki trn si th. -

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( .

5 5 3 nhng ng ). Trong khi di chuyn tng qung, enzyme primase s tng hp nhng on RNA primer ngn (11 1 nucleotide) t DNA c tip ni nh DNA polymerase III. Khi on Okazaki mi c hon chnh, RNA primer c tch ra nh DNA polymerase I (hot tnh exonulease 5 3) v c thay th bi DNA cng nh tc dng ca cng enzyme. Cc khe h cn li gia cc on Okazaki 4.11). 3. Kt thc Cui cng, hai chc ba ti bn gp nhau pha i din ca nhim sc th vng ca E. coli. Ngi ta bit rt t v nhng phn ng ca giai on ny. C th hot ng ca mt loi DNA topoisomerase l cn thit tch hai phn t DNA vng c tng hp. Qu trnh phn i hai phn t DNA trong t bo con khi phn chia t bo cng cha c bit r.

IV. Khi nim mi

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

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Si ch DNA b m Cc on Okazaki DNA polymerase

Ligase

Si ch Si th

Hng ti bn

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

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

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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).

Si th RNA primer

Primase

RNA primer

DNA polymerase III

on Okazaki k tip c tng hp DNA polymerase I

DNA ligase

Hnh 4.12. Tng hp cc on Okazaki. Qu trnh ny i hi s gn mi, ko di, loi b RNA, lm y cc khong trng v hn cc im t. Primase tng hp on mi RNA, DNA polymerase III ko di on mi RNA thnh on Okazaki si th, DNA polymerase I s dng dch m im t thay th on mi RNA bng DNA, v cui cng DNA ligase hn im t. Sinh hc phn t
86

Nh vy, vic ti bn DNA cn cc enzyme sau: - Helicase: m xon tch si DNA ang xon. - SSB: gn trn DNA mt si si lun lun tnh trng m. - RNA polymerase (primase): tc ng hnh thnh on mi RNA. - DNA polymerase I: loi b on mi RNA. - DNA polymerase III (khc vi loi trn) tc ng tng hp DNA bng cch ko di on mi RNA. - DNA ligase: gn cc on Okazaki tng hp vi nhau. T cc nghin cu v ti bn DNA ca bacteriophage M13, ngi ta a ra s gi thuyt v s bin i DNA mt si ca M13 thnh dng sao chp ti bn (RF). V quan st trn chc ba ti bn ang pht trin, ta c th theo di c s ti bn lin tc v khng lin tc ca cc on DNA mi b sung cho si DNA khun mu. V. Enzyme ti bn 1. DNA polymerase y l enzyme ch yu ca s ti bn, chu trch nhim tng hp hai chui DNA chc ti bn. C ba loi DNA polymerase khc nhau, c k hiu l I, II v II. C ch v vai tr ca cc enzyme DNA polymerase cng rt khc nhau.
Mg2+ (DNA)n + dNTP DNA polymerase (DNA)n+1 + PPi

1.1. DNA polymerase I E. coli

4.13).
87

Sinh hc phn t

5 3

DNA khun mu

3 5

Primer

Mg2+ DNA polymerase I 4dNTP

4.13.

E. coli cn c hot tnh exonuclease (hot tnh ct cc chui nucleotide cc u t do ca DNA) 3 5 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. 5 eo DNA.

Khi u, enzyme DNA polymerase I c xem nh gi vai tr ch yu trc tip trong ti bn DNA. Nhng sau , ngi ta quan st thy E. coli t bin khng c DNA polymerase I m vn c s ti bn, v pht hin rng chnh DNA polymerase III gi vai tr ti bn DNA. Nghin cu tnh cht v so snh ba loi enzyme trn ngi ta nhn thy DNA polymerase III mi l enzyme thc s iu khin tng hp DNA. Trong khi DNA polymerase I ch c vai tr loi b RNA mi nh ct u 5 3 v thay vo ch RNA mi bng DNA khc, cn DNA polymerase II cha r tc dng, c l n tham gia vo qu trnh sa cha (thay mt on DNA hng bng mt on DNA bnh thng) hoc thay th DNA polymerase I khi c kh khn trong tng hp DNA.

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150 dNTP. 2. Cc topoisomer v DNA topoisomerase 2.1. Topoisomer Gi s c hai phn t DNA mch vng c cng trnh t nucleotide. Nhng hai phn t ny c th c s vng (linking number-Lk) khc nhau trong phn t. S vng y c nh ngha l s ln ca mt chui DNA qun xung quanh mt chui khc. Nhng trng hp ny c gi l topoisomer. Topoisomer c cc dng sau: 2.1.1. Dng lng lo (relaxed DNA) dng ny sc cng ca xon kp l ti thiu. l dng cu trc n nh nht ca phn t. 2.1.2. Dng siu xon (supercoiled DNA) Trc ca xon kp c th cun xung quanh mnh to thnh mt siu xon. C hai dng siu xon sau: - Siu xon dng (+). S vng tng, xon kp xon cng chiu (xon phi) to thnh dng siu xon dng (positive supercoil). - Siu xon m ( ). S vng gim, xon kp xon theo chiu ngc li (chiu tri) to thnh dng siu xon m (negative supercoil), Phn ln nhng phn t DNA trong t nhin dng siu xon m. 2.2. DNA topoisomerase Enzyme DNA topoisomerase (l mt enzyme nuclease thun nghch) c tc dng thay i s Lk ca DNA. Cc DNA topoisomerase c tc dng thm vo hoc loi bt nhng siu xon trong phn t DNA xon kp. C hai loi DNA topoisomerase sau: 2.2.1. DNA topoisomerase I DNA topoisomerase I tn ti c prokaryote ln eukaryote v c tc dng sau:
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- Ct mt chui DNA v tho mt vng xon sau v tr ct. - Ni li chui DNA b ct bi lin kt phosphodiester mi. DNA sau khi c ti to li c cu trc lng lo hn (Hnh 4.14).

im t

Chuyn si nguyn qua ch t gy v gn li

Lk = n

Lk = n+1

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

2.2.2. DNA topoisomerase II prokaryote, DNA topoisomerase II c tn l DNA gyrase. DNA topoisomerase II c tc dng ct tm thi hai chui ca DNA, c tc dng sp xp li siu xon, to ra siu xon tri ( ) ca chui DNA xon kp (Hnh 4.15). Phn ng ny cn 1 ATP. eukaryote, DNA topoisomerase II cng c tm thy nhng t c nghin cu. 3. Helicase v protein SSB 3.1. Helicase

th ng ny cn s c mt ca ATP (Hnh 4.16).

Sinh hc phn t

. Phn

90

B phn ti bn

Ti bn Siu xon dng (+)

t gy DNA

Topoisomerase II

Chuyn DNA i qua ch t gy Siu xon m ( )

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

3.2. Protein SSB

(Hnh 4.17) .
Sinh hc phn t
91

DNA helicase

ATP ADP + P i

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 5-PO4 2ATP (i vi eukaryote) hoc NAD+ (i vi vi khun).

O
DNA-3-OH + O-P-O-5-DNA
Ligase 2ATP

O
DNA-3-O-P-5-DNA

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92

A
Lin kt ca cc SSB b sung

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

Enzyme + ATP hoc Enzyme + NAD Enzyme- AMP

O- O-P- O- Adenine-Ribose O

- O-P-OHO O

-O
P O

O O

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

93

Ti liu tham kho/c thm

1. Phm Thnh H. 2003. Di truyn hc. NXB Gio dc, H Ni. 2. L c Trnh. 2001. Sinh hc phn t ca t bo. NXB Khoa hc v K thut, H Ni. 3. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 4. Karp G. 2002. Cell and Molecular Biology: Concepts and Experiments. 3 ed. John Wiley & Sons, Inc. New York, USA.
rd

5. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 6. Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J. 2004. Molecular Cell Biology. 5th ed. Freeman and Company, New York, USA. 7. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California, USA. 8. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company, New York, USA.

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Chng 5

Phin m
Phin m l qu trnh tng hp RNA t khun mu DNA. Qu trnh ny v phng din ha hc v enzyme rt ging vi qu trnh ti bn DNA. C hai u lin quan n cc enzyme tng hp mt chui nucleic acid mi b sung vi khun mu DNA. Tt nhin, hai qu trnh ny c nhng khc bit quan trng, m ng ch nht l chui mi trong qu trnh phin m c to thnh t cc ribonucleotide thay v cc deoxyribonucleotide. Cc nguyn tc c bn ca qu trnh phin m c thit lp da vo nghin cu trn prokaryote (E. coli) nhng dng nh cc nguyn tc ny cng c tnh ph bin cho c eukaryote. Tuy nhin, do s khc bit v cu trc genome v h thng enzyme nn s phin m prokaryote v eukaryote cng c nhng c trng nht nh.

I. Cc c im c bn ca qu trnh phin m
1. S phin m to ra RNA b sung vi mt si DNA Cc ribonucleotide ni tip ca RNA c xc nh da trn nguyn tc b sung vi cc nucleotide trn DNA khun mu. Khi s bt cp chnh xc xy ra, ribonucleotide tip theo c lin kt ng ha tr vi chui RNA ang hnh thnh nh phn ng c enzyme xc tc. Nh vy, sn phm phin m c ko di tng ribonucleotide mt v b sung chnh xc vi si DNA c dng lm khun mu. Qu trnh phin m d c tnh chnh xc cao nhng vn km hn nhiu so vi qu trnh ti bn DNA (t l mc li l 1/10.000 nucleotide so vi 1/10.000.000 trong ti bn). l do s thiu mt c ch sa sai hu hiu, mc d trong qu trnh tng hp RNA cng c hai dng sa sai tn ti. Tuy nhin, v cc RNA c phin m khng bao gi c sao chp li nn cc sai st xy ra khng nh hng n vic truyn t thng tin cho th h sau. Sinh hc phn t
95

2. S phin m l mt phn ng enzyme Nhng enzyme chu trch nhim cho qu trnh phin m c t bo prokaryote v eukaryote u c gi l RNA polymerase ph thuc DNA (DNA-dependent RNA polymerase), gi tt l RNA polymerase. RNA polymerase xc tc hnh thnh cc cu ni phospho-diester ni cc ribonucleotide thnh mt chui thng. Enzyme dch chuyn tng bc dc theo DNA khun mu v ko di chui RNA theo hng t 5 3, ngha l cc ribonucleotide c thm vo u 3 ca chui RNA ang hnh thnh. Cc c cht c s dng tng hp RNA l ATP, GTP, CTP v UTP. Cng ging nh trong s ti bn DNA, nng lng cho phn ng c cung cp t s thy phn cc cu ni giu nng lng ca cc c cht ni trn. 3. S phin m ch sao chp chn lc mt s phn ca genome v to ra nhiu bn sao S la chn vng no phin m khng phi xy ra ngu nhin. Mi vng phin m in hnh bao gm mt hoc nhiu gen, c nhng trnh t DNA c hiu hng dn khi u v kt thc phin m. i vi mt vng c chn phin m, c th c mt n hng trm thm ch c nghn bn sao RNA c to ra. S tng hp phn t RNA sau c bt u trc khi phn t RNA trc hon thnh. T mt gen n c, trong vng mt gi c th tng hp c hn mt nghn phn t RNA (i vi eukaryote). S la chn vng no phin m v mc phin m u c iu ha. V vy, trong nhng t bo khc nhau hoc trong cng mt t bo nhng nhng thi im khc nhau s c nhng nhm gen khc nhau c phin m. 4. Ch mt trong hai si n ca phn t DNA c dng lm khun mu Vic gn ca RNA polymerase vo promoter ca gen s quyt nh vic la chn si no trong hai si n ca DNA lm khun mu. Promoter, ngoi vic mang v tr gn RNA polymerase cn cha ng thng tin xc nh si no trong hai si n ca DNA c phin m v xc nh v tr bt u phin m. Sinh hc phn t
96

5. S phin m c khi pht khng cn mi RNA polymerase c th khi u s tng hp RNA trn khun mu DNA m khng cn mi nh DNA polymerase. iu ny i hi ribonucleotide u tin c mang n v tr bt u phin m phi c gi n nh trn DNA khun mu trong khi ribonucleotide th hai ang c a n xy ra phn ng trng hp.

II. Cc giai on ca qu trnh phin m

RNA polymerase tin hnh qu trnh phin m mt gen thng qua nhiu bc v c chia thnh ba giai on: khi u, ko di, v kt thc. 1. Giai on khi u u tin, RNA polymerase gn vi promoter ca gen (cng vi cc yu t khi u) to thnh phc hp promoter-polymerase. Mt khi c hnh thnh, phc hp ny s c s thay i cu trc cn thit cho vic tin hnh giai on khi u. Giai on khi u ny bao gm ba bc nh sau: 1.1. Phc hp ng (closed complex) Khi RNA polymerase va mi gn vi promoter th phc hp to thnh trng thi ng. trng thi ny, DNA vn l chui kp v enzyme gn vo mt pha ca vng xon. 1.2. Phc hp m (open complex) Lc ny DNA xung quanh im bt u phin m c m xon v lin kt gia cc cp base b sung b ph v. Hai si DNA vi di khong 14 bp xung quanh v tr bt u phin m tch ri nhau ra v to nn vng phin m. Vic m DNA gii phng si khun mu. Hai nucleotide u tin c mang n v tr bt u c hot ha, chng xp hng trn khun mu v c ni vi nhau. RNA polymerase bt u chuyn dch dc theo si khun mu, m xon pha trc v tr trng hp v ti gn hai si DNA pha sau. Bng cch ny, cc ribonucleotide tip theo c gn Sinh hc phn t
97

vo chui RNA ang pht trin. on RNA ban u vi s lng ribonucleotide t hn 10 s b loi b v enzyme bt u tng hp li. 1.3. Phc hp tam nguyn bn (stable ternary complex) Mt khi enzyme i c khong hn 10 bp, n c xem l thot khi promoter. Lc ny mt phc hp gm ba thnh phn l enzyme, DNA v RNA c hnh thnh v qu trnh phin m chuyn sang giai on ko di. 2. Giai on ko di Mt khi RNA polymerase tng hp c mt on RNA khong 10 base, n chuyn sang giai on ko di. S chuyn giai on ny i hi nhng bin i hn na v cu hnh ca RNA polymerase lm cho n cng gi cht khun mu hn na. Trong qu trnh ko di, enzyme ny thc hin mt lot nhiu nhim v rt quan trng ngoi vic tng hp RNA. N m xon DNA trc mt v ti gn DNA pha sau, n tch dn chui RNA ang hnh thnh ra khi khun mu khi ang di chuyn dc theo gen, v n cn thc hin chc nng c sa sai ( qu trnh ti bn DNA, cc chc nng ny do nhiu enzyme khc nhau thc hin). 3. Giai on kt thc Khi RNA polymerase phin m ht chiu di ca gen (hoc cc gen), n dng li v gii phng sn phm RNA. Trong mt s t bo, c nhng trnh t c hiu thc y giai on kt thc. Trong mt s t bo khc, ngi ta vn cha r yu t no iu khin enzyme ngng phin m v gii phng RNA khi khun mu.

III. Phin m prokaryote

1. Enzyme RNA polymerase prokaryote T bo prokaryote ch cha mt loi RNA polymerase. Enzyme ny c cu to bi nm tiu n v, gm hai tiu n v v cc tiu n v , , . Cc tiu n v ny lin kt vi nhau to thnh enzyme li.

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Ngi ta lm th nghim tinh sch enzyme li t t bo vi khun ri cho vo dung dch cha DNA vi khun v ribonucleotide th enzyme s gn vi DNA v tng hp RNA. Tuy nhin, RNA thu c trong th nghim ny khng ging vi RNA trong t bo v enzyme gn vo cc v tr khng thch hp trn DNA. Nu cho thm mt loi polypeptide tinh sch c gi l yu t trc khi enzyme gn vi DNA th s phin m s bt u ti nhng v tr chnh xc nh trong t bo. Vic gn yu t vo enzyme li lm tng i lc ca enzyme vi v tr khi ng trn DNA, ng thi lm gim i lc i vi cc vng khc. Khi enzyme li c gn thm yu t s tr thnh dng holoenzyme. 2. Promoter ca gen prokaryote Promoter ca gen vi khun nm ngay trc v tr khi u phin m. Bng cch phn tch cc trnh t DNA ny ca mt s lng ln cc gen vi khun ngi ta nhn thy c hai on ngn tng i ging nhau gia gen ny v gen khc, mi on gm su nucleotide. on th nht nm cch v tr khi u khong 35 bp, l s bin i ca trnh t TTGACA. on th hai nm cch v tr khi u khong 10 bp, l s bin i ca trnh t TATAAT. Chng ln lt c gi l vng 35 v vng 10 (hp Pribnow). i vi cc promoter mnh (v d gen m ha rRNA), ngi ta cn tm thy yu t UP lm tng s gn ca RNA polymerase vo DNA. C mt s promoter thiu vng 35 v c thay bng yu t 10 m rng, bao gm vng 10 chun v thm mt on ngn u 5 ca n (v d gen gal ca E. coli). Yu t nhn dng cc vng 35 v 10 hoc yu t 10 m rng nh cc cu trc c bit ca chng. Ring yu t UP khng c nhn dng bi m c nhn dng bi mt vng u C tn cng ca tiu n v , gi l CTD (carboxyl terminal domain). 3. Vai tr ca enzyme RNA polymerase v promoter trong qu trnh phin m Khi khi u s phin m, RNA polymerase gn vi DNA vng 35 mt cch lng lo to thnh phc hp ng. Sau , enzyme gn vo Sinh hc phn t
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promoter cht hn v phc hp chuyn sang trng thi m. Mt vng DNA c m xon nm gia hai v tr 11 v +3, ngha l bao gm c v tr bt u phin m +1. Si n DNA lm khun mu c bc l sinh tng hp RNA. Mt khi RNA polymerase tng hp c 10 nucleotide (sau ln khi u) th yu t ri khi enzyme v c th n gn vo promoter khc khi u mt qu trnh phin m mi. S tch ri yu t cho php hnh thnh mt knh thot m thng qua phn RNA c tng hp chui ra khi enzyme v c ko di (Hnh 5.1).

Khi u

DNA

-35

-10

Ti xon

M xon

Ko di

RNA polymerase RNA 5

RNA c gii phng

Kt thc
5

Cu trc kp tc

Hnh 5.1. Cc giai on phin m prokaryote

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

Hnh 5.2. Cu trc kp tc

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 AU. 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. 4.2. Tn hiu kt thc ph thuc Rho Tn hiu ny l nhng thnh phn RNA m mun hot ng, i hi phi c yu t Rho. Yu t Rho l mt protein hnh nhn ( E. coli c khi Sinh hc phn t
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lng phn t khong 50 kDa) gm su tiu n v ging ht nhau. Yu t ny c hot tnh ATPase: khi gn vo sn phm phin m, n s s dng nng lng t s thy phn ATP ko RNA ra khi khun mu v polymerase. Yu t Rho iu khin phn t RNA c bit nh tnh c hiu. Th nht l tnh c hiu ti nhng v tr n gn, l nhng on khong 40 nucleotide m khng gp li thnh cu trc bc hai, chng thng giu C. Tnh c hiu th hai l yu t Rho gim gn vi bt k sn phm phin m no ang c dch m (ngha l nhng sn phm ang gn ribosome). vi khun, phin m v dch m c song hnh cht ch: dch m khi pht ngay trn RNA ang c tng hp k t khi RNA bt u chui ra khi RNA polymerase. V vy Rho ch kt thc nhng sn phm vn ang c phin m qu u tn cng ca gen hoc operon.

IV. Qu trnh phin m eukaryote

1. Cu trc gen eukaryote Cu trc mt gen m ha cho protein ca eukaryote bao gm cc vng sau: 1.1. Vng 5 kim sot biu hin gen Vng ny bao gm cc trnh t nucleotide iu ha biu hin gen v hot ha s phin m, bao gm: - Promoter. L nhng trnh t nh v u 5 khng dch m ca gen c chc nng xc nh v tr bt u phin m, kim sot s lng mRNA v i khi c tnh c hiu m (tissue-specific). Promoter c th di n vi nghn bp. Vng ny thng cha mt trnh t bo th gi l hp TATA nm cch v tr phin m khong 25-30 bp. Hp ny gip xc nh chnh xc v tr bt u phin m. Ngoi ra, cn c hp CCAAT nm cch v tr bt u phin m khong 75-80 bp. Hp ny t ph bin hn hp TATA, n c chc nng tng hiu qu phin m. Mt s gen qun gia m ha cho cc enzyme hin din tt c cc t bo thng thiu c hai hp ny v promoter rt giu GC. Ngoi ra, cn c cc thnh phn c hiu khc.

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- V tr gn vng c hiu m. L trnh t DNA tng tc vi protein c hiu m ch huy gen cu trc sn xut ra tng protein c hiu ca tng m. - V tr gn vi vng tng cng phin m (enhancer). Cn gi l gen tng cng, l nhng trnh t DNA c gn vi cc tc nhn hot ha kch thch phin m ca cc gen k bn, chng c th tc ng qua mt khong cch xa v c th tc ng theo hai pha (t 5 hoc 3 ti). 1.2. Vng c phin m Bao gm cc exon v intron nm xen k. y l mt c im phn bit vi gen ca prokaryote. Cc exon v intron u c phin m nhng ch c cc exon l c dch m. Cc intron c bt u bng GT v kt thc bng AG. Cc intron chim phn ln trong mi gen v chng s c loi b khi RNA mi c tng hp, cn cc exon c ni vi nhau to nn mRNA hon chnh (mature mRNA). hai u ca vng c phin m (coding region) cn c vng 5 khng dch m (5 untranslation region) v vng 3 khng dch m (3 untranslation region). Vng 5 khng dch m c tnh t v tr bt u phin m cho n codon khi u ATG. Vng 3 khng dch m bt u t codon kt thc n v tr gn ui poly(A). 1.3. Vng 3 khng dch m Chc nng cha r, mt s gen vng ny mang cc trnh t iu ha chuyn bit. 2. Enzyme RNA polymerase ca eukaryote T bo eukaryote c n ba loi RNA polymerase l RNA polymerase I (pol I), RNA polymerase II (pol II), v RNA polymerase III (pol III). Trong , RNA polymerase II m nhn vic phin m cho hu ht cc gen, ch yu l gen m ha cho protein. RNA polymerase I phin m cc gen tin thn ca rRNA ln v RNA polymerase III phin m cc gen tRNA, mt s gen RNA kch thc nh ca nhn (small nuclear, snRNA) v RNA 5S. Cc RNA polymerase ca eukaryote cng bao gm nhiu tiu n v,

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

Bng 5.1. Cc tiu n v ca RNA polymerase

Prokaryote Vi khun Archaea A/A B D L K (+6) RNA pol I RPA1 RPA2 RPC5 RPC9 RPB6 (+9)

Eukaryote RNA pol II RPB1 RPB2 RPB3 RPB11 RPB6 (+7) RNA pol III RPC1 RPC2 RPC5 RPC9 RPB6 (+11)

3. Cc yu t gip RNA polymerase khi u phin m Trong khi RNA polymerase ca prokaryote ch cn thm mt yu t khi u l khi ng phin m th RNA polymerase ca eukaryote phi cn nhiu yu t khi u, cc yu t ny c gi l cc yu t phin m tng qut. Mt khc, s khi u phin m eukaryote cn phi i mt vi hin tng cc DNA c ng gi trong nucleosome v cc dng cao hn ca cu trc chromatin. iu ny i hi phi c nhiu yu t khc thm vo gip khi ng qu trnh phin m. 3.1. Vai tr ca cc yu t phin m tng qut Cc yu t phin m tng qut gip RNA polymerase vo ng v tr trn promoter, gip tch hai si n ca DNA ra phin m c bt u, v gii phng RNA polymerase khi promoter mt khi phin m c khi ng xong i vo giai on ko di. Cc yu t ny c gi l tng qut v chng gn trn tt c cc promoter c s dng bi RNA polymerase II. Do , chng c vit tt l TFII (transcription factor for polymerase II), bao gm TFIIA, TFIIB...

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Nh m t trn, nhiu promoter ca eukaryote cha hp TATA. Hp ny c nhn dng bi mt tiu n v ca TFIID l TBP (TATA binding protein: protein gn TATA). Phc hp TBP-DNA to nn mt ci nn thu ht cc TFII khc v RNA polymerase n promoter. In vitro, cc yu t phin m tng qut khc n gn vo promoter theo th t sau: TFIIA, TFIIB, TFIIF cng RNA polymerase II, TFIIE v TFIIH. Sau , vng promoter c m xon. Khc vi vi khun, s m xon y cn c nng lng cung cp t s thy phn ATP nh TFIIH (yu t ny c hot tnh ging helicase). Sau cng xy ra hin tng khi u sy ging nh prokaryote cho n khi c s bin i v cu hnh nhm gii phng RNA polymerase II khi promoter v i vo giai on ko di. Tuy nhin, y c mt bc m khng tm thy prokaryote l s gn thm cc gc phosphate vo ui ca RNA polymerase (ui ny cn c gi l CTD: carboxyl terminal domain). S phosphoryl ha ny cng c xc tc bi TFIIH nh hot tnh protein kinase ca n (Hnh 5.3). 3.2. Vai tr ca cc tc nhn hot ha, phc hp trung gian v enzyme bin i chromatin. Ngoi cc yu t phin m tng qut, RNA polymerase II cn cn s h tr ca cc yu t khc (Hnh 5.4). u tin, cn c cc tc nhn hot ha phin m (transcriptional activator) n gn vo cc trnh t c hiu trn DNA (cc enhancer) hp dn RNA polymerase II n v tr bt u phin m. S hp dn ny cn thit RNA polymerase II v cc yu t phin m tng qut vt qua tr ngi khi gn vi DNA c ng gi trong chromatin. Tip n, s khi u phin m in vivo cn cn s hin din ca cc protein to thnh phc hp trung gian (mediator complex). Phc hp ny cho php tc nhn hot ha tc ng tt ln RNA polymerase II v cc yu t phin m tng qut. Cui cng, s phin m cn cn cc enzyme bin i chromatin, bao gm phc hp ti to m hnh chromatin (chromatin remodeling complex) v enzyme histone acetylase. C hai c tc dng gip cho b my khi u phin m c th gn vo DNA trong chromatin mt cch d dng.

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TBP Hp TATA TFIIA

TFIIF

RNA polymerase II

TFIIE TFIIH

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

Tc nhn hot ha Phc hp trung gian Phc hp ti m hnh chromatin HAT

RNA polymerase II

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

4. Cc yu t kch thch RNA polymerase II hot ng trong giai on ko di Mt khi RNA polymerase II bt u chuyn sang giai on ko di th cc yu t khi u c loi b nh yu t phin m tng qut v phc hp trung gian. Thay vo , cc yu t kch thch giai on ko di c thu ht n, bao gm TFIIS v hSPT5. Ngoi ra, cn c cc yu t khc c huy ng phc v cho qu trnh bin i RNA mi c tng hp. Giai on ko di trong phin m c song hnh cht ch vi cc bc bin i RNA mi c tng hp. Nh phn tch trn, c mt bc quan trng din ra khi chuyn t giai on khi u sang ko di l s phosphoryl ha ui CTD ca RNA polymerase II. S phosphoryl ha ny khng nhng gip RNA polymerase II thot khi cc protein v tr bt u phin m m cn cho php cc protein khc n kt hp vi ui gip qu trnh ko di phn t RNA v bin i RNA tin thn.

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5. Qu trnh bin i cc RNA mi c tng hp eukaryote, s phin m ch l bc u tin trong mt lot cc phn ng bao gm c s bin i hai u ca mRNA tin thn v loi b cc intron to thnh mRNA hon chnh. 5.1. S gn m vo u 5 Ngay khi RNA polymerase II va mi to ra khong 25 ribonucleotide ca RNA, u 5 ca phn t RNA ny c bin i bng cch gn thm mt ci m l guanine c bin i ha hc. Phn ng gn m c thc hin bi ba loi enzyme l: - Phosphatase c tc dng loi mt gc phosphate khi u 5 ca RNA mi sinh. - Guanylyl transferase gn GMP bng lin kt o ngc (5 vi 5 thay v 5 vi 3) vo u 5 ca RNA ang c tng hp. - Methyl transferase gn nhm methyl vo guanosine (Hnh 5.5). Trong nhn, m gn vi mt phc hp protein c gi l CBC (CAP-binding complex: phc hp gn m) gip RNA c x l tt v c chuyn ra ngoi. M 5 cn c vai tr quan trng trong qu trnh dch m mRNA trong bo tng. 5.2. S gn ui poly(A) vo u 3 v kt thc phin m S gn ui poly(A) vo u 3 c lin kt vi s kt thc phin m. Cc enzyme cn thit cho cc qu trnh ny c tp trung trn ui CTD ca RNA polymerase II, bao gm CPSF (cleavage and polyadenylation specificity factor: yu t c hiu tch RNA v gn ui polyA) v CstF (cleavage stimulation factor: yu t kch thch tch RNA). Khi RNA polymerase di chuyn n cui gen, n gp nhng trnh t c hiu (c gi l tn hiu polyA). Trnh t ny c phin m thnh RNA, thc y chuyn CPSF v CstF n gn vo on RNA ny. Sau cc protein khc s c tp trung n khi u s tch ri RNA v gn ui poly(A). S gn ui c thc hin bi enzyme poly(A) polymerase (PAP). Enzyme ny thm khong t vi chc n 200-250 adenine vo u 3 ca Sinh hc phn t
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RNA c tch ra. Enzyme PAP s dng ATP lm c cht v hot ng ging RNA polymerase, tuy nhin khng c khun mu. Ngi ta vn cha r yu t no quyt nh chiu di ca ui nhng qu trnh ny c lin quan n cc protein gn c hiu vi ui poly(A). Sau khi RNA c tch ra v gn ui poly(A), RNA polymerase vn cha kt thc phin m ngay. N cn tip tc di chuyn dc theo khun mu v to ra mt phn t RNA th hai c th di hng trm nucleotide trc khi kt thc. Sau , RNA polymerase c tch ra khi khun mu, gii phng RNA mi v RNA ny s b ging ha.
base base

3 HO 5 5

3 RNA triphosphatase

3 HO 5

3 Guanylyl transferase

5 5 3 HO Methyl transferase H3C 7 methyl 3

3 HO

5 5 3

Hnh 5.5. Phn ng gn m vo u 5 ca RNA

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5.3. Qu trnh ct ni gen (splicing) y l qu trnh loi b cc intron v ni cc exon li vi nhau. Qu trnh ny c thc hin phn ln bi cc RNA thay v protein. Cc phn t RNA ny tng i ngn (di 200 nucleotide) c gi l snRNA. C nm loi lin quan n dng ct ni chnh l U1, U2, U4, U5 v U6. Mi snRNA c kt hp vi nhiu protein hnh thnh snRNP. C ba trnh t nm trn intron ng vai tr quan trng trong qu trnh ct ni l: v tr ct ni u 5, v tr phn nhnh l mt trnh t giu cc pyrimidine bao quanh mt nucleotide adenine gn u 3, v v tr ct ni u 3. u tin, v tr ct ni u 5 c nhn dng bi U1 snRNP bng s bt cp cc base b sung. V tr phn nhnh cng c nhn dng bi BBP (branch-point binding protein: protein gn v tr phn nhnh) v U2AF. U2 snRNP n thay th BBP (nh U2AF gip ). S bt cp base gia U2 snRNA v v tr phn nhnh lm tha ra mt gc A khng bt cp v sn sng phn ng vi v tr 5. Sau U4 v U6 snRNP cng U5 snRNP n gn vi phc hp trn. Dng U1 ri khi phc hp v U6 th ch U1 ti v tr ct ni u 5. U4 c gii phng U6 tng tc vi U2. iu ny lm cho v tr ct ni u 5 v v tr phn nhnh c t k nhau v to iu kin cho phn ng ct ni xy ra. Nucleotide adenine c hiu v tr phn nhnh tn cng v ct intron v tr u 5. ng thi c mt lin kt ng ha tr xy ra gia A v tr phn nhnh v u 5 ca intron to nn mt cu trc hnh thng lng. u 3 ca exon trc ni vi u 5 ca exon k tip v thng lng intron c gii phng (Hnh 5.6).

V. Phin m ngc
Phin m ngc l qu trnh tng hp DNA da trn khun mu RNA. Qu trnh ny c xc tc bi mt loi enzyme c bit gi l enzyme phin m ngc (RT: reverse transcriptase). Enzyme ny c c hot tnh DNA polymerase v hot tnh RNase H. Cng ging nh DNA polymerase trong qu trnh ti bn DNA, enzyme phin m ngc i hi phi c primer (mi) c bit tng hp nn DNA mi.

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snRNP 5 3

snRNPs

Hnh 5.6. Qu trnh ct ni gen

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

(1)

5 R

U5

PBS DNA tRNA

U5 U5 U3 R 3

B gen RNA ca retrovirus

(2)

3 R 5 R

PBS 3 R tRNA
U5

(3)

5 PBS

U3

(4)
5 PBS PBS

3 R
U3

U5

tRNA

R 3

(5)

3 5 PBS

U3 U3

U5

tRNA

R 3

PBS

(6)

3 5 PBS 3 5 PBS 3 5

U3

U5

tRNA

(7)

U3 R U3 R

U5 U5

tRNA 3 PBS tRNA

(8)

U3 U3

R R

U5 U5

(9)
5
U3 R

PBS 3
U5

3 PBS 5

U3

U5

3 PBS

(10)
3 5
U3 U3 R R U5 U5

PBS

DNA ca retrovirus
U3 U3 R R U5 U5

5 3

Hnh 5.7. Qu trnh phin m ngc ca retrovirus

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Ti liu tham kho/c thm

1. H Hunh Thy Dng. 1998. Sinh hc phn t. NXB Gio dc, H Ni. 2. Alberts B, Johnson A, Lewis J, Raff M, Roberts K and Walter P. 2002. Molecular Biology of the Cell. 4th ed. Garland Science Publishing, Inc. New York, USA. 3. Flint SJ. 2000. Principles of Virology: Molecular Biology, Pathogenesis, and Control. ASM Press, Washington, USA. 4. Gelehrter TD, Collins FS and Ginsburg D. 1998. Principles of Medical Genetics. 2nd ed. Williams & Wilkins Company, Baltimore, USA. 5. Griffiths AJF, Wessler SR, Lewontin RC, Gelbart WM, Suzuki DT and Miller JH. 2005. An Introduction to Genetic Analysis. WH Freeman & Co. New York, USA. 6. Karp G. 2002. Cell and Molecular Biology. John Wiley & Sons, Inc. New York, USA. 7. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Losick R. 2004. Molecular Biology of the Gene. Pearson Education, Inc./Benjamin Cummings Publishing, San Francisco, USA.

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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: Sinh hc phn t
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- UGA m ha cho tryptophan thay v bo hiu chm dt vic tng hp protein. - AGA v AGG khng m ha cho arginine m bo hiu chm dt tng hp protein. - AUA m ha cho methionine thay v m ha cho isoleucine. 2. Cc quy tc chi phi m di truyn C ba quy tc iu khin s sp xp v s dng cc codon trn mRNA l: - Cc codon c c theo hng 5'3'. V vy chui m ha cho dipeptide NH2-Thr-Arg-COOH c vit l 5'-ACGCGA-3'. - Cc codon khng chng ln nhau v vng dch m ca mRNA khng cha cc khong trng. - Thng tin c dch m theo mt khung c (reading frame) c nh. V mt nguyn tc, cng mt trnh t RNA c th c ba khung c khc nhau. Tuy nhin, trn thc t ch c mt trong ba khung c ny cha thng tin thc s, chnh codon khi u xc nh khung c ng cho mi trnh t mRNA.

II. Cc ribosome
Ribosome l b my i phn t iu khin s tng hp protein. N c cu to bi t nht l 3 phn t RNA v trn 50 protein khc nhau1, vi khi lng phn t l 2,5 MDa (megadalton) i vi ribosome ca prokaryote v 4,2 MDa i vi ribosome ca eukaryote. 1. Thnh phn cu to ca ribosome Mi ribosome bao gm mt tiu n v ln v mt tiu n v nh. Tiu n v ln cha trung tm peptidyl transferase chu trch nhim cho vic hnh thnh cc cu ni peptide. Tiu n v nh cha trung tm gii m, l ni cc tRNA c gn amino acid c v gii m cc codon. Ngoi ra cn c trung tm gn cc yu t tiu n v ln.

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

116

Sinh hc phn t

Theo quy c, cc tiu n v c t tn theo tc lng ca chng di lc ly tm. n v o tc lng l Svedberg (tn ca nh pht minh my siu ly tm) v c vit tt l S. Ribosome ca prokaryote l ribosome 70S, trong tiu n v ln l 50S v tiu n v nh l 30S. Ribosome ca eukaryote l 80S, vi tiu n v ln l 60S v tiu n v nh l 40S. Mi tiu n v u c cu to bi cc RNA ribosome (rRNA) v cc protein ribosome. n v Svedberg li c s dng phn bit cc rRNA (Bng 6.1). Trong qu trnh dch m, tiu n v ln v tiu n v nh ca mi ribosome lin kt vi nhau v vi mRNA. Sau mi vng tng hp protein, chng li ri nhau ra.
Bng 6.1. Cc thnh phn cu to ca ribosome

Cc thnh phn cu to rRNA

Prokaryote Tiu n v ln (50S) 5S rRNA (120 Nu) 23S rRNA (2900 Nu) Tiu n v nh (30S) 16S rRNA (1540 Nu)

Eukaryote Tiu n v ln (60S) 5,8S rRNA (160 Nu) 5S rRNA (120 Nu) 28S rRNA (4700 Nu) Tiu n v nh (40S) 18S rRNA (1900 Nu)

Protein

34 protein

21 protein

49 protein

33 protein

2. Khi nim polyribosome Mc d mt ribosome ch c th tng hp mt polypeptide ti mt thi im, nhng mi mRNA c th c dch m ng thi bi nhiu ribosome. Mt mRNA mang nhiu ribosome c xem l polyribosome hay polysome. Mi ribosome n c tip xc vi khong 30 nucleotide, nhng do kch thc ln ca ribosome nn mt cho php trn mRNA l 80 nucleotide cho mi ribosome. Sinh hc phn t
117

3. Cc v tr gn tRNA trn ribosome Trn ribosome cha ba v tr gn tRNA l v tr A, P v E. Trong : - A l v tr gn aminoacyl-tRNA (tRNA c mang amino acid). - P l v tr gn peptidyl-tRNA (tRNA c mang chui polypeptide). - E (exit) l v tr gn tRNA m c phng thch sau khi chui polypeptide c chuyn sang aminoacyl-tRNA. Mi v tr gn tRNA c hnh thnh ti giao din gia tiu n v ln v tiu n v nh. Bng cch ny, cc tRNA c gn vo c th bt ngang qua khong cch gia trung tm peptidyl transferase ca tiu n v ln v trung tm gii m ca tiu n v nh. u 3' ca tRNA c nm gn tiu n v ln v vng i m gn tiu n v nh.
NH3

Chui polypeptide ang tng hp

tRNA c gii phng t v tr E

Trung tm peptidyltransferase

C CC

A A A U CG GG G U U U A G C U E P A

3 Trung tm gii m

Chuyn ng ca ribosome

Hnh 6.1. Cc thnh phn chc nng ca ribosome

4. Cc knh ca ribosome l cc knh cho php mRNA i vo v i ra khi ribosome, v knh cho php chui polypeptide mi sinh i ra khi ribosome. mRNA i vo v i ra khi trung tm gii m ca ribosome thng qua hai knh hp ti tiu n v nh. Trong , knh vo c chiu rng ch cho RNA khng bt cp i qua. c im ny m bo cho mRNA c dui thng khi n i vo trung tm gii m, bng cch loi b mi tng tc bt cp base b sung ni phn t. Sinh hc phn t
118

Mt knh xuyn qua tiu n v ln to li thot cho chui polypeptide mi c tng hp. Kch thc ca knh hn ch c s gp ca cc chui polypeptide ang tng hp. V vy, protein ch c th hnh thnh cu trc bc ba sau khi n c gii phng khi ribosome.

III. S hnh thnh aminoacyl-tRNA

1. Bn cht ca s gn amino acid vo tRNA Qu trnh gn amino acid vo tRNA l qu trnh hnh thnh mt lin kt acyl gia nhm carboxyl ca amino acid v nhm 2'- hoc 3'-OH ca adenine u 3' ca tRNA. Lin kt ny c xem l mt lin kt giu nng lng. Nng lng gii phng ra khi lin kt b ph v gip hnh thnh cu ni peptide lin kt amino acid vi chui polypeptide ang c tng hp. 2. S nhn din v gn amino acid vo tRNA S nhn din v gn amino acid vo tRNA tng ng c thc hin bi mt enzyme gi l aminoacyl-tRNA synthetase. Qu trnh ny din ra nh sau: u tin, amino acid c adenylyl ha bng cch phn ng vi ATP, kt qu to thnh amino acid c gn adenylic acid qua cu ni ester giu nng lng gia nhm COOH ca amino acid v nhm phosphoryl ca AMP, ng thi gii phng ra pyrophosphate. Sau , amino acid c adenylyl ha ny (vn ang gn vi synthetase) phn ng tip vi tRNA. Phn ng ny chuyn amino acid n u 3' ca tRNA gn vi nhm OH, ng thi gii phng AMP. Phn ng tng hp ca qu trnh ny nh sau: Amino acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi

3. Tnh c hiu ca aminoacyl-tRNA synthetase Hu ht cc t bo u c mt enzyme synthetase ring bit chu trch nhim cho vic gn mt amino acid vo mt tRNA tng ng (nh vy c tt c 20 synthetase). Tuy nhin, nhiu vi khun c di 20 synthetase. Trong trng hp ny, cng mt synthetase chu trch nhim cho hn mt loi amino acid. Sinh hc phn t
119

S nhn din amino acid chnh xc l da vo kch thc, s tch in v gc R khc nhau ca cc amino acid. S nhn din tRNA da vo cc trnh t nucleotide khc nhau ca tRNA. T l sai st trong qu trnh gn amino acid vi tRNA tng ng l kh thp. 4. Phn loi aminoacyl-tRNA synthetase C hai loi tRNA synthetase. - Loi I bao gm cc synthetase gn cc amino acid nh Glu, Gln, Arg, Cys, Met, Val, Ile, Leu, Tyr, Trp vo nhm 2'-OH. - Loi II gm cc synthetase gn cc amino acid nh Gly, Ala, Pro, Ser, Thr, His, Asp, Asn, Lys, Phe vo nhm 3'-OH.

IV. Cc giai on ca qu trnh dch m

Qu trnh dch m c bt u bng s gn ca mRNA v mt tRNA khi u vi tiu n v nh t do ca ribosome. Phc hp tiu n v nhmRNA thu ht tiu n v ln n to nn ribosome nguyn vn vi mRNA c kp gia hai tiu n v. S tng hp protein c bt u ti codon khi u u 5' ca mRNA v tin dn v pha 3'. Khi ribosome dch m t codon ny sang codon khc, mt tRNA gn amino acid k tip c a vo trung tm gii m v trung tm peptidyl transferase ca ribosome. Khi ribosome gp codon kt thc th qu trnh tng hp chui polypeptide kt thc. Chui ny c gii phng, hai tiu n v ca ribosome ri nhau ra v sn sng n gp mRNA mi thc hin mt chu trnh tng hp protein mi. Qu trnh dch m c chia thnh ba giai on l khi u, ko di v kt thc. 1. Giai on khi u 1.1. prokaryote 1.1.1. Cc yu t khi u (IF: initiation factor) C cc yu t khi u xc tc cho tiu n v nh trong vic hnh thnh phc hp khi u. l IF1, IF2, IF3. Mi yu t khi u c tc dng nh sau:

Sinh hc phn t

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- IF1 gip tiu n v nh gn vo mRNA v ngn cn cc tRNA gn vo vng thuc v tr A trn tiu n v nh. - IF2 l mt protein gn v thy phn GTP. IF2 thc y s lin kt gia fMet-tRNAifMet v tiu n v nh, ngn cn nhng aminoacyl-tRNA khc n gn vo tiu n v nh. - IF3 ngn cn tiu n v nh ti lin kt vi tiu n v ln v gn vi cc tRNA mang amino acid. IF3 gn vo tiu n v nh vo cui vng dch m trc, n gip tch ribosome 70S thnh tiu n v ln v tiu n v nh. Khi tiu n v nh c gn ba yu t khi u, n s gn tRNA khi u v mRNA. S gn hai RNA ny l hon ton c lp vi nhau. 1.1.2. Bc 1: Tiu n v nh gn vo codon khi u S lin kt gia tiu n v nh vi mRNA c thc hin thng qua s bt cp base b sung gia v tr gn ribosome v rRNA 16S. Cc mRNA ca vi khun c mt trnh t nucleotide c hiu gi l trnh t ShineDalgarno (SD) gm 5-10 nucleotide trc codon khi u. Trnh t ny b sung vi mt trnh t nucleotide gn u 3' ca rRNA 16S. Tiu n v nh c t trn mRNA sao cho codon khi u c t ng vo v tr P mt khi tiu n v ln gn vo phc hp. 1.1.3. Bc 2: tRNA u tin c mang methionine bin i n gn trc tip vi tiu n v nh Mt tRNA c bit c gi l tRNA khi u n gn trc tip vi v tr P (khng qua v tr A). tRNA ny c anticodon (b ba i m) c th bt cp vi AUG hoc GUG. Tuy nhin tRNA ny khng mang methionine cng nh valine m mang mt dng bin i ca methionine gi l Nformyl methionine. tRNA khi u ny c gi l fMet-tRNAifMet. Trong hoc sau qu trnh tng hp polypeptide, gc formyl c loi b bi enzyme deformylase. Ngoi ra, aminopeptidase s loi b methionine cng nh mt hoc hai amino acid k tip u chui polypeptide.

Sinh hc phn t

121

Tiu n v 30S Cc yu t khi u

IF2-fMet-tRNAi + mRNA

IF3 fMet

mRNA

Phc hp khi u 30S Tiu n v 50S

IF1 + IF2 fMet

Phc hp khi u 70S

Hnh 6.2. Khi u dch m prokaryote

1.1.4. Bc 3: Hnh thnh phc hp khi u 70S Bc gn thm tiu n v ln to thnh phc hp khi u 70S din ra nh sau: khi codon khi u v fMet-tRNAifMet bt cp vi nhau, tiu n v nh thay i hnh dng lm gii phng IF3. S vng mt IF3 cho Sinh hc phn t
122

php tiu n v ln gn vo tiu n v nh ang mang cc thnh phn trn. Nh c tiu n v ln gn vo, hot tnh GTPase ca IF2-GTP c kch thch thy phn GTP. IF2-GDP to thnh c i lc thp i vi ribosome v tRNA khi u dn n s gii phng IF2-GDP cng nh IF1. Nh vy phc hp khi u cui cng c to thnh bao gm ribosome 70S c gn ti codon khi u ca mRNA, vi fMet-tRNAifMet ti v tr P, cn v tr A ang trng. Phc hp ny sn sng tip nhn mt tRNA mang amino acid vo v tr A bt u tng hp polypeptide (Hnh 6.2). 1.2. Eukaryote 1.2.1. Bc 1: S hnh thnh phc hp tin khi u 43S Giai on khi u i hi s h tr ca hn 30 protein khc nhau, mc d eukaryote cng c nhng yu t khi u tng ng vi prokaryote. Cc yu t khi u ny c k hiu l eIF. Khi ribosome ca eukaryote hon thnh mt chu trnh dch m, n tch ri ra thnh tiu n v ln v tiu n v nh t do thng qua tc ng ca cc yu t eIF3 v eIF1A (tng t vi IF3 prokaryote). Hai protein gn GTP l eIF2 v eIF5B lm trung gian thu ht tRNA khi u gn methionine (ch khng phi N-formyl methionine nh prokaryote) n tiu n v nh. Chnh yu t eIF5B-GTP l tng ng vi IF2-GTP ca prokaryote. Yu t ny lin kt vi tiu n v nh theo phng thc ph thuc eIF1A. Ri eIF5B-GTP gip thu ht phc hp eIF2-GTP v MettRNAiMet n tiu n v nh. Hai protein gn GTP ny cng nhau a MettRNAiMet vo vng thuc v tr P ca tiu n v nh. Kt qu, hnh thnh phc hp tin khi u 43S. 1.2.2. Bc 2: S nhn dng m 5 ca mRNA Qu trnh ny c thc hin thng qua eIF4F. Yu t ny c ba tiu n v, mt tiu n v gn vo m 5', hai tiu n v khc gn vi RNA. Phc hp ny li c gn vi eIF4B lm hot ha mt enzyme RNA helicase ca mt trong nhng tiu n v ca eIF4F. Helicase ny tho xon tt c cc cu trc bc hai c hnh thnh u tn cng ca mRNA. Phc hp eIF4F/B v mRNA li thu ht phc hp tin khi u 43S n thng qua tng tc gia eIF4F v eIF3. Sinh hc phn t
123

5 Met M

mRNA Cc yu t khi u + GTP Met-tRNAi Tiu n v 40S

ATP

ADP + Pi Met

Tiu n v 60 S

Cc yu t khi u Met

Phc hp khi u 80S

Hnh 6.3. Khi u dch m eukaryote

1.2.3. Bc 3: Tiu n v nh tm thy codon khi u bng cch qut xui dng t u 5' ca mRNA v s hnh thnh phc hp khi u 80S Mt khi c gn vo u 5' ca mRNA, tiu n v nh v cc yu t lin kt vi n di chuyn dc theo mRNA theo hng 5' 3' cho n khi gp trnh t 5'-AUG-3' u tin m n nhn dng l codon khi u. Codon Sinh hc phn t
124

c nhn dng bng s bt cp base b sung gia anticodon (b ba i m) ca tRNA khi u v codon khi u. S bt cp ny thc y phng thch eIF2 v eIF3 cho php tiu n v ln gn c vo tiu n v nh. S gn ny dn n phng thch cc yu t khi u cn li thng qua s thy phn GTP di tc dng ca eIF5B. Cui cng, Met-tRNAiMet c a vo v tr P ca phc hp khi u 80S. Lc ny, ribosome trong t th sn sng tip nhn aminoacyl-tRNA vo v tr A (Hnh 6.3). 1.2.4. Nhng yu t khi u dch m gi mRNA eukaryote dng vng Ngoi vic gn vo u 5' ca mRNA, cc yu t khi u cn lin kt cht ch vi u 3' thng qua ui poly(A). iu ny c thc hin bi s tng tc gia eIF4F v protein gn poly(A) bc bn ngoi ui poly(A). Vic tm thy cc yu t khi u dch m c vai tr "vng ha" mRNA theo phng thc ph thuc ui poly(A) gii thch c quan st trc y l mt khi ribosome kt thc s dch m mt mRNA m c vng ha thng qua ui poly(A) th ribosome mi c phng thch ny l ribosome l tng ti khi u dch m trn cng mRNA. 2. Giai on ko di 2.1. Bc 1: Aminoacyl-tRNA c a n v tr A nh yu t ko di EFTu Mt khi tRNA gn amino acid th EF-Tu n gn vo u 3' ca aminoacyl-tRNA. EF-Tu ch c th gn vi aminoacyl-tRNA khi n lin kt vi GTP. EF-Tu-GTP a aminoacyl-tRNA vo v tr A ca ribosome. Ch phc hp aminoacyl-tRNA-EF-Tu-GTP no c anticodon b sung vi codon ca mRNA ti v tr A th mi c gi li trn ribosome. Sau , EF-Tu tng tc vi trung tm gn yu t ca ribosome nm trn tiu n v ln v thy phn GTP, ri EF-Tu c phng thch khi tRNA v ribosome, aminoacyl-tRNA nm li ti v tr A. 2.2. Bc 2: Hnh thnh cu ni peptide Aminoacyl-tRNA ti v tr A c quay vo trung tm peptidyl transferase v cu ni peptide c hnh thnh. Phn ng ny c xc tc bi peptidyl transferase, ngy nay n c xc nh l rRNA, c bit l Sinh hc phn t
125

rRNA 23S ca tiu n v ln. V vy, peptidyl transferase cn c gi l ribozyme. Trong qu trnh hnh thnh cu ni peptide, cu ni gia amino acid v tRNA v tr A khng b ph v. u 3' ca c hai tRNA c a n gn nhau v nhm amine ca amino acid v tr A tn cng nhm carboxyl ca amino acid v tr P. Kt qu l tRNA v tr A mang mt dipeptide, trong khi tRNA v tr P b kh acyl. Sau xy ra s chuyn dch (xem bc 3): peptidyl-tRNA (ang mang dipeptide) chuyn sang v tr P, v v tr A sn sng tip nhn mt aminoacyl-tRNA mi. Cu ni peptide tip theo c hnh thnh theo cch ging ht trn, trong nhm amine ca amino acid mi lin kt vi nhm carboxyl u C tn cng ca chui polypeptide ang tng hp. Thc cht, y l qu trnh chuyn chui polypeptide ang tng hp t peptidyl-tRNA v tr P sang aminoacyl-tRNA v tr A. V vy, phn ng to cu ni peptide c gi l phn ng peptidyl transferase. Nh vy, chui polypeptide c tng hp theo chiu t u N n u C. Trong qu trnh ny, khng c s thy phn nucleoside triphosphate. Nng lng c cung cp t s ph v cu ni acyl giu nng lng gia chui polypeptide ang tng hp v tRNA. 2.3. Bc 3: S chuyn dch (translocation) Mt khi phn ng peptidyl transferase xy ra th tRNA trong v tr P khng gn vi amino acid na, v chui polypeptide ang hnh thnh c lin kt vi tRNA trong v tr A. mt vng ko di polypeptide mi xy ra, tRNA v tr P phi chuyn n v tr E v tRNA v tr A chuyn n v tr P. ng thi, mRNA phi di chuyn qua 3 nucleotide ribosome tip xc vi codon tip theo. Nhng s di chuyn ny c gi l s chuyn dch. Bc u tin trong chuyn dch c song hnh vi phn ng peptidyl transferase. Khi chui peptide c chuyn sang tRNA v tr A, u 3' ca tRNA ny hng n vng v tr P ca tiu n v ln, trong khi u anticodon vn cn nm v tr A. Tng t, tRNA v tr P (m khng

Sinh hc phn t

126

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-GGDP, 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).
Phc hp bc ba

Aminoacyl-tRNA gn vo v tr A

Hnh thnh cu ni peptide EF-G Chuyn v Pi

AminoacyltRNA gn vo v tr A tRNA gii phng v tr E

Hnh 6.4. Ko di dch m

Sinh hc phn t

127

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 EFTu-EF-Ts. Phc hp sau cng c tch thnh EF-Ts t do v EF-Tu-GTP. 3. Giai on kt thc 3.1. Cc yu t gii phng kt thc dch m Chu k gn aminoacyl-tRNA ca ribosome, s hnh thnh cu ni peptide, v s chuyn dch xy ra lin tc cho n khi mt trong ba codon kt thc vo v tr A. Cc codon ny c nhn din bi cc yu t gii phng (RF: release factor) (Hnh 6.5). C hai loi yu t gii phng: - Cc yu t gii phng loi I nhn din codon kt thc v thc y s thy phn tch chui polypeptide ra khi peptidyl-tRNA ti v tr P. Prokaryote c hai yu t gii phng loi I l RF1 v RF2, trong RF1 nhn din codon kt thc UAG v RF2 nhn din UGA, cn UAA c nhn din bi c RF1 v RF2. Eukaryote ch c mt yu t gii phng gi l eRF1 nhn din c c ba loi codon kt thc. - Cc yu t gii phng loi II kch thch s tch yu t gii phng loi I ra khi ribosome sau khi chui polypeptide c gii phng. Ch c mt yu t gii phng loi II, c gi l RF3 prokaryote v eRF3 eukaryote. Yu t gii phng loi II c iu ha bi GTP.

Sinh hc phn t

128

RF1

Phn tch peptidyl-tRNA

RF1

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 Sinh hc phn t
129

vi trung tm gn yu t ca tiu n v ln. S tng tc ny kch thch thy phn GTP. V khng cn yu t loi I na nn RF3-GDP c i lc thp vi ribosome v b phng thch ra ngoi. 3.3. S quay vng ca ribosome Sau khi phng thch chui polypeptide v cc yu t gii phng, ribosome vn cn gn vi mRNA cng vi hai tRNA ti v tr P v v tr E. ribosome tham gia vo qu trnh tng hp polypeptide mi, tRNA v mRNA phi i khi ribosome v hai tiu n v ca ribosome phi ri nhau ra. Tp hp nhng s kin nh vy gi l s quay vng ribosome (ribosome recycling). prokaryote, c mt yu t gi l yu t quay vng ribosome (RRF: ribosome recycling factor). RRF gn vo v tr A, n bt chc tRNA. RRF li ko EF-G n ribosome v EF-G kch thch gii phng nhng tRNA ti v tr P v E. Sau , EF-G v RRF c phng thch khi ribosome cng vi mRNA. IF3 c th tham gia vo s gii phng mRNA ng thi n cng cn cho s tch ri hai tiu n v ca ribosome. Kt qu to ra tiu n v nh gn IF3 v tiu n v ln t do. Ribosome by gi c th tham gia vo vng dch m mi.

V. Cc nhn t c ch dch m
Qu trnh dch m bao gm rt nhiu bc v tun theo mt quy tc l khng c mt bc no c th xy ra khi bc trc n cha hon thnh. Tuy nhin, iu ny to ra mt im yu trong dch m l nu chng may c mt bc khng hon thnh th ton b qu trnh phi b ngng li. V vy, dch m thng l im ch tc ng ca nhiu loi khng sinh v cc c t (Bng 6.2). Khong 40% cc loi khng sinh l nhng yu t c ch dch m. Cc khng sinh ny tiu dit vi khun gy bnh ngi hoc ng vt do n tc ng lm ngng dch m vi khun nhng khng nh hng n dch m eukaryote, do qu trnh dch m vi khun v eukaryote c nhng im khc nhau c ngha, v d ribosome khc nhau v kch thc v cc thnh phn. Nhiu khng sinh gn chn lc vi ribosome ca vi khun v c ch nhiu bc trong dch m nhng khng nh hng n ribosome ca Sinh hc phn t
130

eukaryote. V d tetracycline gn vo v tr A ca ribosome vi khun v c ch s i vo ca cc aminoacyl-tRNA nhng khng hiu qu trn ribosome ca eukaryote. Nhng khng sinh khc nhau th c ch cc bc khc nhau trong qu trnh dch m nn chng thng c s dng nghin cu s hot ng ca b my dch m, c bit l puromycin. Do cu trc ba chiu ca puromycin ging vi u 3' ca aminoacyl-tRNA nn n c th gn vo v tr A v c ch s i vo y ca cc aminoacyl-tRNA.
Bng 6.2. S c ch dch m ca mt s khng sinh v c t

Khng sinh c t Tetracycline

T bo ch Prokaryote

ch mc phn t

Hu qu

V tr A/30S Gn v tr A/30S

c ch gn tRNA vo A Ngn cn chuyn dch ca tRNA t A sang P Gim tnh chn lc ca s bt cp base gia codon v anticodon Ngn cn a tRNA ti A vo phn ng peptidyl transferase chnh xc Kt thc sm s tng hp polypeptide c ch chui polypeptide i ra khi ribosome Ngn cn phng thch EF-G-GDP khi ribosome
131

Hygromycin B

Prokaryote Eukaryote

Paromycin

Prokaryote

V tr tng tc codon-anticodon/30S

Chloramphenicol

Prokaryote

Trung tm peptidyl transferase/50S

Puromycin

Prokaryote Eukaryote

V tr A/tiu n v ln Knh thot peptide/ 50S

Erythromycin

Prokaryote

Fusidic acid

Prokaryote

EF-G

Sinh hc phn t

Thiostrepton

Prokaryote

Trung tm gn yu t /50S

nh hng s gn ca IF2 v EF-G vi trung tm ny Ngn cn thay i hnh dng lin quan thy phn GTP, phng thch EF-Tu Ngn cn s hot ha cc yu t dch m GTPase c ch chc nng EFTu c ch hot tnh peptidyl transferase

Kirromycin

EF-Tu

Ricin, -Sarcin Diptheria

Prokaryote Eukaryote Eukaryote

Bin i ha hc RNA trung tm gn yu t Bin i ha hc EFTu Trung tm peptidyl transferase/60S

Cycloheximide

Eukaryote

Cu ni peptide c th c hnh thnh gia puromycin ti v tr A v chui polypeptide ti v tr P. Tuy nhin, puromycin khng gn vo v tr P nn s chuyn dch khng xy ra v chui polypeptide gn puromycin ri khi ribosome d qu trnh tng hp cha kt thc. Do cu trc ca tRNA l tng t nhau mi sinh vt nn puromycin c ch dch m c vi khun v t bo eukaryote, hu qu l n tiu dit c t bo vt ch cng vi vi khun. Thnh thong, puromycin cng c dng iu tr ung th nhm tiu dit t bo khi u.

Ti liu tham kho/c thm

1. Alberts B, Johnson A, Lewis J, Raff M, Roberts K and Walter P. 2002. Molecular Biology of the Cell. 4th ed. Garland Science Publishing, Inc. New York, USA. 2. Griffiths AJF, Wessler SR, Lewontin RC, Gelbart WM, Suzuki DT and Miller JH. 2005. An Introduction to Genetic Analysis. WH Freeman & Co. New York, USA.

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3. John LB, Carey JC, Bamshad MJ and White RL. 2003. Medical Genetics. Mosby Publishing, Missouri, USA. 4. Karp G. 2002. Cell and Molecular Biology. John Wiley & Sons, Inc. New York, USA. 5. Pierce BA. 2003. Genetics: A Conceptual Approach. WH Freeman & Co. New York, USA. 6. Watson JD, Baker TA, Bell SP, Gann A, Levine M and Losick R. 2004. Molecular Biology of the Gene. Pearson Education, Inc./Benjamin Cummings Publishing, San Francisco, USA.

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Chng 7

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

I. Khi qut v cc c ch sa sai

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

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sung vi si th nht) c dng lm khun mu sa cha nhng sai hng . Mt s c ch sa cha nh sau: - H thng sa cha nhn bit cc trnh t DNA khng thch hp vi cc cp base chun v thay th chng. - H thng sa cha-ct b (excision-repair system) loi i mt on DNA v tr sai hng v sau thay th n. - H thng sa cha-ti t hp (recombinant-repair system) s dng phng thc ti t hp thay th vng si i b sai hng. Cc h thng sa cha cng phc tp nh b my ti bn ca n, iu cho thy tm quan trng ca chng i vi s sng ca t bo. Khi h thng sa cha phc hi mt sai hng ca DNA, th khng c mt hu qu xu no xy ra. Nhng mt t bin c th to ra hu qu xu khi DNA b hng. Hnh 7.1 tm tt mt s c ch sa cha DNA nh sau: - Mt vi enzyme phc hi trc tip cc loi sai hng c bit ca DNA. - Mt s phng thc sa cha bng cch ct b base, sa cha bng cch ct b nucleotide, v sa cha ghp i lch, tt c chc nng ca chng thc hin bng cch loi b v thay th nguyn liu. - Cc h thng chc nng c th ti t hp to ra mt bn sao khng b sai hng thay th cho mt si i b sai hng. - Phng thc ni u khng tng ng ni li cc u si i b hng. - Mt s DNA polymerase khc nhau cn thit trong vic tng hp li cc on DNA thay th. 1. Cc bin i xy ra trn phn t DNA Trn DNA c th xy ra cc bin i ngu nhin nh sau: - Gy hay t mch. Phn t DNA c chiu ngang rt mnh, bn thn n li thng xuyn cun xon v gin xon nn d xy ra t gy mt si. Tuy nhin, kh nng t cng lc hai si him khi gp hn.

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Phc hi trc tip sai hng

Sa cha bng cch ct b base

Sa cha bng cch ct b nucleotide

Sa cha bng cch ct b ghp i lch

Sa cha bng cch ti t hp

Ni u khng tng ng

DNA polymerase xc tc cc tiu n v

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.
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- Gn cc nhm mi vo base bng lin kt cng ha tr lm thay i tnh cht nh trng hp methyl ha (gn nhm CH3 vo mt base). - Bin mt base ny thnh mt base khc lm bt cp sai. V d: qu trnh kh amine (desamination) ca cytosine bin n thnh uracil. - Cc base c th tn ti hai dng keto v enol nn c th dn n bt cp sai. V d: dng enol ca cytosine c th bt cp vi adenine. - To cc thymine dimer. - Lin kt cho gia cc mch (interstand crosslink). Trn y l cc bin i ngu nhin, nu c tc ng ca cc tc nhn gy t bin cc bin i s xy ra nhiu hn. 2. Khi qut cc c ch sa cha mc phn t S nguyn vn ca phn t DNA mang thng tin di truyn c ngha sng cn i vi t bo, nht l t bo prokaryote. t bo vi khun, c n 50% DNA khng b bin i thm ch sau khi ti bn n 100 triu ln. S n nh cao ca DNA t bo c c nh hng lot cc c ch bo v s nguyn vn v sa cha ngay lp tc bt k sai hng no va xut hin. Cc h thng sa sai rt a dng v c hiu qu rt cao E. coli. C khong 100 locus tham gia trc tip hoc gin tip vo vic bo v DNA v sa sai. Ch vi DNA, t bo phi u t rt ln cho s n nh ca thng tin di truyn. Nu nh s ti bn ch xy ra khi phn bo, th cc c ch sa sai phi hot ng lin tc. Ti bn c thc hin vi c ch v c bn ging nhau cc loi t bo v trong mi ln nhn i DNA. Trong khi , sai hng xy ra rt a dng, nn cc c ch sa sai nhiu hn v vi s lng gen tham gia ln hn. Hn na, cc c ch di truyn c bn nh ti bn, ti t hp DNA u c s tham gia ca cc c ch sa sai v ngc li sa sai cn n ti bn v ti t hp. 3. Bin i lm tng tn s t bin Nh cc c ch sa sai nn bnh thng DNA t bo c n nh rt ln, thng s thay th base c tn s 10-9-10-10. Tuy nhin, ngi ta pht hin c cc dng t bin ngu nhin tng cao hn hn, chng c gi

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l mutator (nhn t gy t bin). Trong nhiu trng hp, cc kiu hnh mutator lin quan n sai hng trong h thng sa sai. E. coli, cc locus mutator mutH, mutL, mutU v mutS tc ng n cc cu phn ca h thng sa sai do ghp i lch (mismatch-repair) sau ti bn nn lm cho tn s t bin ngu nhin tng cao. Ngoi mutator, s sai hng lm tng tnh nhy cm vi tia t ngoi (ultraviolet) v tng sai hng khi ti t hp.

II. Cc kiu sa cha

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

Pyrimidine dimer trong DNA b chiu tia UV

Phc hp DNA v enzyme quang ti hot ha

Hp th nh sng

Gii phng enzyme hon li DNA t nhin

Hnh 7.2. Quang ti hot ha

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

A T

Li ti bn to ra s ghp i lch A-C

GATC CTAG
Si DNA cn sa cha c adenine khng c methyl ha

A C

A GATC CTAG
Si khng c methyl ha b ct gy

GATC CTAG
Methyl ha

A T
Tng hp DNA sa cha li

GATC CTAG

A T

Hnh 7.3. Sa cha ghp i lch trong ti bn

S nhn bit v sa cha cc sai lch do bt cp base sai trn DNA c pht hin E. coli, nm men v t bo ng vt c v. vi khun E. coli, c ba h thng enzyme khc nhau c s dng sa cha cc sai lch bng cch:
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- 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 Nglycosylase. 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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DNA

DNA glycosylase

V tr AP

AP endonuclease

DNA polymerase

NTPs Deoxyribose phosphate + dNMPs

DNA ligase

DNA mi

Hnh 7.4. Phng thc ct b base

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Cu trc si kp b sung cho nhau ca DNA cho php, nu thng tin b mt do ct b sai hng trn mt si c th chp li c nh da vo si n b sung kia. Tuy nhin, mt s sai hng lin quan cng lc c hai si cng c th c sa cha nh vo mt on nucleotide tng ng tn ti u trong genome. S mt on hay xen on nucleotide, hay lin kt cho gia cc si DNA c th c phc hi nh s thay th on tng ng bng ti t hp. Thm ch s t i c hai si cng ch, c coi l nng nht trong cc sai hng, c th c gn lin li nh ligase hay ti t hp (Hnh 7.5). 4. c sa i vi cc base bt cp sai C ch c sa i vi cc base bt cp sai (proofreading for basepair 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 3 5 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.

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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.

Sai hng Base t bin b ghp i lch v/hoc vn vo cu trc

To khc Endonuclease ct trn c hai mt ca base b sai hng

Ct b Exonuclease loi b DNA gia cc im t

Tng hp Polymerase tng hp DNA thay th

Ligase hn im t

Hnh 7.5. Ct b nucleotide. Sa cha v ct b mt on DNA c (cc) base sai hng. Sinh hc phn t
144

- 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.
Cc base trn mt si DNA b sai hng

S ti bn to ra mt bn sao c l thng i din vi si sai hng v mt bn sao bnh thng

L thng c sa cha bng cch thu hi mt trnh t t bn sao bnh thng

L thng bn sao bnh thng c sa cha

Hnh 7.6. Sa sai nh ti t hp v ti bn Sinh hc phn t

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6. H thng SOS H thng SOS (cha khong 30 gen khng lin kt v thng b c ch bi protein LexA) s hot ng khi t bo b cc tc nhn gy t bin to nhiu sai hng trn DNA. Trong trng hp DNA b hng ngng ti bn, phn ng SOS s phc hi ti bn bng phng thc ti bn errorprone. vi khun E. coli ngi ta quan st thy s ph hy DNA lm m ra khong 20 gen ca h thng SOS, c kim sot m bi cht c ch LexA (LexA repressor). Trong trng hp c nhiu sai hng cn cp cu, LexA b kch thch, thay i cu hnh, t ct v mt hot tnh c ch. Lc cc gen ca h thng SOS c m ra. Nu qu trnh sa sai thc hin khng kp th t bo phi chp nhn hoc b t bin hoc cht. Protein RecA khi ng h thng SOS nh sau: - Hp SOS l trnh t DNA (operator) di khong 20 bp c nhn bit bi protein ca cht c ch LexA. - Sai hng ca DNA lm cho RecA khi ng phn ng SOS, cha cc gen m ha cho nhiu enzyme sa cha. - RecA hot ha hot tnh t phn gii t ca LexA. - LexA ngn chn h thng SOS, nhng phn ng t phn gii ca n hot ha cc gen ca h thng ny. 6.1. Sa cha theo c ch error-prone Phn t DNA c th b sai hng nng n khi b chiu tia t ngoi hoc khi chu tc ng ca cc tc nhn gy ung th (carcinogens). Lc , khng phi ch mt si n DNA b hng m c hai si u b t gy v mt i mt s nucleotide. Thng tin di truyn ca on hng b mt hon ton nn khng c si khun mu sa cha theo cc c ch thng thng. Trong trng hp ny, t bo cn li mt gii php duy nht tng kh nng sng st l sa cha DNA mt cch ngu nhin vi t l sai st (t bin) cao nhng d sao vn duy tr c s sng. y chnh l c ch sa cha DNA error-prone (cn c xem nh l s pht sinh t bin SOS). Nh vy, sa cha DNA theo c ch ny cng l mt trong nhng nguyn nhn to nn tnh a dng ca DNA.
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Khi phn t DNA b t bin, mt s protein c bit s lm nhim v dng qu trnh tng hp DNA sa cha theo cc c ch khc nhau. Bi v nu t bo tip tc phn chia (tc l DNA tip tc c nhn ln) th t l t bin cao s xut hin khin s t bo sng st gim xung. Mt s trng hp sai hng c sa cha bng c ch error-prone: - DNA kt hp vi cc base khng b sung trn si con (bt cp sai). - DNA b sai hng khng c sa cha do DNA polymerase III b gi li trong sut qu trnh ti bn. DNA polymerase V (c m ha bi gen umuD v umuC ca E. coli), hoc DNA polymerase IV (c m ha bi gen dinB ca E. coli) c cm ng trong h thng SOS, tng hp mt on b sung cho si DNA b hng. DNA polymerase V l mt phc hp cha hai protein tiu n v UmuD v mt protein tiu n v UmuC (Trong qu trnh sa cha error-prone, protein RecA, hot ng nh mt co-protease, gin tip t xc tc ct protein UmuD thnh mt on c hot tnh, UmuD). DNA polymerase IV v V l mt phn ca mt h ln, bao gm cc DNA polymerase l nhng enzyme cn thit trong sa cha DNA b sai hng do chng c hot tnh polymerase. Cc protein tiu n v UmuD (thc t l UmuD) v UmuC c kh nng gn cc nucleotide vo si DNA bt chp khng tn ti si khun mu. Nh vy, chng c th c ch hot tnh t sa exonuclease 3 5 ca DNA polymerase hoc chnh chng l mt loi DNA polymerase c bit (DNA polymerase V). Khi t bin xy ra trn gen m ha cho UmuD v UmuC, cc t bo vi khun s sng st t hn t bo bnh thng di tc dng ca tia t ngoi. Mt vi plasmid mang gen mucA v mucB, tng ng vi gen umuD v gen umuC, khi c a vo trong vi khun s lm tng tnh khng vi tia t ngoi. Ngoi ra, khi c hai si n DNA u chu t bin nhng nu trong genome cn c t nht mt bn sao ging ht on b hng, lc on b hng c th c phc hi nh c ch trao i cho dng bn sao lm khun mu. 6.2. Protein LexA Hot tnh ca RecA gy ra s phn gii sn phm protein ca gen lexA. LexA l mt protein nh (22 kDa) kh n nh trong cc t bo khng
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b x l, ni n c chc nng nh mt cht c ch nhiu operon. Phn ng phn gii l khng thng xuyn; LexA c hot tnh protease mun c hot ha bi RecA. Khi RecA c hot ha, n s gy ra phn ng t phn gii ca LexA lm bt hot chc nng c ch ca LexA, v cm ng phi hp tt c operon m n c lin kt. Phng thc ny c minh ha hnh 7.7.

VNG IU HA

CC GEN CH

Gen recA b c ch

gen lexA CM NG CA RecA

Gen ch b c ch

RecA khi ng s phn gii LexA

RecA c hot ha

Gen recA c cm ng

Gen lexA

Gen ch c biu hin

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

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sai hng. S tng tc ca n vi RecA l nhanh: phn ng SOS xy ra trong mt vi pht x l sai hng. Tm li. RecA v LexA l cc mc tiu chung trong hp SOS: RecA khi ng s phn gii ca LexA, nhn t c ch recA v chnh n. V th, phn ng SOS to ra s khuch i ca c hai protein RecA v cht c ch LexA. Kt qu l khng cn mu thun nh lc u na. Vic tng mc biu hin ca protein RecA cn thit cho vai tr trc tip ca n trong cc phng thc sa cha-ti t hp. V s cm ng, nng ca RecA c tng ln 50 ln so vi nng ban u ca n khong 1.200 phn t/t bo. Nng cao cc t bo c cm ng c ngha l c RecA m bo rng tt c protein LexA b phn gii.

III. Bo v DNA: H thng ct hn ch (R)-Bin i (M)

Ngoi cc h thng sa sai, t bo cn c h thng bo v DNA. Cc vi khun c h thng min nhim hiu qu DNA l, l: h thng ct hn ch-bin i (restriction-modification system) hin din trong nhiu loi vi khun, trong DNA c bin i bi cc enzyme c hiu. S bin i xy ra nhm mc ch bo v DNA chng li s phn hy enzyme (ct hn ch) bi cc endonuclease ca chnh chng. S bin i bao gm mt kiu c trng ca phn ng methyl ha cc gc nucleotide trong DNA. Bng cch dng S-adenosylmethionine nh l mt cht cho, cc enzyme methyl ha (methylase) s hot ng trn DNA si i (dsDNA). Cc v tr m c s bin i cng c nhn bit bi cc enzyme hn ch (restriction enzyme, RE) tng ng, tuy nhin cc enzyme hn ch khng th ct DNA trong cc v tr c bin i ny mt hoc c hai si DNA. Cc vi sinh vt prokaryote ln eukaryote u c cc enzyme methyl ha gn nhm CH3 nhng im nht nh trn phn t DNA. Cc enzyme ny c tnh c hiu cao chuyn ha cho tng dng vi khun, v th DNA ca mi dng vi khun ch c methyl ha nhng v tr c bit nht nh. Nh , mi vi khun d dng phn bit DNA ca bn thn chng vi DNA ngoi lai (foreign DNA) (v d: DNA ca bacteriophage) xm nhp vo trong t bo. Cc enzyme ct hn ch l mt loi endonuclease ca mi
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dng vi khun (xem chng 9), khng ct DNA ca chng v c methyl ha nhng im cn thit m ch ct DNA ngoi lai (ca bacteriophage) do chng khng c methyl ha nhng v tr nht nh. Cc c ch ch yu bin i ca DNA ca n mt cch c hiu v ct hn ch (restriction) cc phn t DNA khng c nh du. H thng R-M ngn chn DNA ngoi lai khng c hot ng chc nng nhng c th thc hin ti t hp. H thng R-M c hai tnh cht cn bn: - Hot tnh restriction (ct hn ch) c hiu chng s xm nhp ca DNA ngoi lai. - Hot tnh bo v DNA ca bn thn n. H thng R-M ln u tin c pht hin khi nghin cu hin tng nhim bacteriophage vo E. coli. S bin i thng l methyl ha nhm 6NH2 ca adenine nhng trnh t nucleotide c hiu trong h thng kiu II, C5 ca cytosine c methyl ha. Ct hn ch c thc hin do cc h thng endonuclease. Chng nhn bit cc im c hiu t nht mt si DNA khng b bin i. Endonuclease ct t si nhiu im nhn bit v sau cc on ngn c ct bng nucleotide khc. Phn ln cc DNA b bin i c cc c tnh di truyn khng n nh. S methyl ha thng mt qua ti bn trong t bo ch. Cc nghin cu cho thy chng E. coli B c 3 gen lin kt cht vi nhau m ha cho 3 sn phm khuch tn: cc polypeptide phc v cho s ct hn ch, cho s bin i v to s c hiu ca im nhn bit. DNA si kp nhy cm hn vi s bin i v ct hn ch. Cc bacteriophage si n nh M13 v X 174 t b tc ng hn. S bin i ca mt si DNA min nhim DNA l. Do vy, DNA ti bn bn bo tn c bo v bi methyl ha si khun. 1. Cc methylase ca R-M Methylase l enzyme c hot tnh xc tc gn mt nhm methyl vo mt phn t, v d: DNA methyltransferase c chc nng methyl ha cc gc C trong DNA.

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Enzyme methylase to ra mt cp adenine methyl ha (hoc cytosine) c v tr i xng cho nhau. Cc methylase c lin quan cht ch vi cc kiu ct hn ch. - Kiu I. Cc RE c th tin hnh c hai s bin i v ct hn ch. Mt RE kiu I bao gm ba tiu n v khc nhau (R, M v S) chu trch nhim tng ng cho vic ct hn ch, bin i v nhn bit trnh t. V d: EcoB v EcoK (t E. coli chng B v K), cc trnh t nhn bit ca hai enzyme ny l TGA(N)8TGCT i vi EcoB v AAC(N)6GTGC i vi EcoK (trong N l mt trong bn loi nucleotide). Nu v tr nhn bit khng c methyl ha trong mt si, th enzyme RE s methyl ha si , nhng nu c hai si u khng c methyl ha th enzyme RE s ct DNA (cn c ATP). Phn ng ct xut hin mt v tr khng c hiu >1000 bp tnh t trnh t nhn bit; r rng l enzyme duy tr lin kt v tr nhn bit ca n, v DNA c di chuyn qua mt v tr th hai trn enzyme hng ti mt v tr ct hn ch. - Kiu II. Cc RE l dng chung nht trong vi khun; chng ch thc hin phn ng ct hn ch-cn s bin i c tin hnh bi mt enzyme ring bit. Trnh t nhn bit cho hu ht cc enzyme ny l cc trnh t palindrome ngn (v d: EcoRI nhn bit GAATTC) v thc hin phn ng ct (khng cn ATP) trong hoc gn k vi v tr nhn bit. Mt vi RE kiu II ct c hai si cng v tr to ra u bng , trong khi cc RE khc li to ra u dnh. Cc RE kiu II l cng c hu ch trong cng ngh DNA ti t hp. - Kiu III. Cc RE ny tng t cc enzyme kiu I. Mi RE kiu III c hai tiu n v; n nhn bit mt trnh t i xng ngn, v to ra mt u so le (cn c ATP) v tr khong 24-26 bp so vi v tr nhn bit. V d enzyme EcoPI, c m ha bi prophage ca bacteriophage P1. 2. Adenine v cytosine methyltransferase E. coli S methyl ha A v C thc hin rng ri tt c cc loi DNA. prokaryote (k cc bacteriophage v plasmid), vai tr th hin r l phn hy DNA ngoi lai. Vai tr ph l phn bit DNA b m vi si con mi c tng hp trong phc hi cc ch bt cp sai. prokaryote, cc sinh vt khng c h thng ct hn ch, vai tr ch yu ca methyl ha c l s iu ha biu hin ca gen.
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Bn cnh cc methylase ca h thng R-M, cn c cc methyltransferase khc E. coli. Cc nucleotide c methyl ha khng gn trc tip vo DNA. Cc methylase chuyn nhm methyl t S-adenosine methionine sang adenine v cytosine nhng im c hiu trn DNA. 3. S methyl ha DNA ca eukaryote S methyl DNA ca eukaryote c nhiu im khc vi prokaryote: - Base b bin i ch yu eukaryote l 5-methylcytosine. ng vt c v, ch c 5-methylcytosine l base b bin i, trong 90% l trnh t i xng CpG. - Do cha hiu bit r h thng R-M eukaryote nn ngi ta cho rng c l methyl ha khng gp phn bo v chng s xm nhp ca DNA ngoi lai. S methyl ha ca DNA ng vt c v gp phn vo s iu ha biu hin ca gen v bit ha t bo. Cc nghin cu cho thy c s tng quan thun gia methyl ha thp (hyphomethylation) vi hot tnh ca gen v ngc li gia s methyl ha mnh vi bt hot ca gen. S vng mt ca cc nhm methyl trn DNA ca rui gim chng t rng sinh vt bit ha cao c th hot ng khng cn c ch methyl ha. S methyl ha ng vt c v c th ng vai tr: - Thay i trng thi ca DNA v c ch i lc ca repressor vi operator. - S methyl ha c truyn t theo dng t bo soma. - Methyl ha c th to s khc nhau gia cc m. - 5-azacytidine, cht ng ng vi cytidine, tc ng lm t bo nguyn bo si (fibroblast) bit ha thnh t bo c, c l do c ch c ch methyl ha DNA v nh vy hot ha gen tng ng.

Ti liu tham kho/c thm

1. Phm Thnh H. 2003. Di truyn hc. NXB Gio dc, H Ni. 2. V Th Thng Lan. 2002. Sinh hc phn t. NXB i hc Quc gia H Ni, H Ni.

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3. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 4. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 5. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California, USA. 6. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company, New York, USA.

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Chng 8

iu ha biu hin gen

Nh chng ta bit ba qu trnh thit yu cho s tn ti ca t bo, l: ti bn, phin m v dch m. Tuy nhin, t bo khng th tn ti c lp vi mi trng chung quanh. Nh vy, s ny sinh mt vn quan trng: t bo s iu chnh hot ng ca mnh nh th no cho ph hp vi cc bin i ca mi trng bn ngoi c th tn ti thch ng? Chng ny s cp n cc phng thc iu chnh , tc l cc c ch iu ha s biu hin ca gen cc sinh vt prokaryote v eukaryote. S biu hin ca cc gen chu s kim sot ca cc c ch iu ha. Cc c ch ny gi vai tr rt quan trng cho cc hot ng sng, p li nhng bin i ca mi trng bn trong v bn ngoi c th. Biu hin gen ca cc t bo prokaryote v eukaryote cng c s khc nhau ng k. Vic iu ha c thc hin nhiu mc khc nhau v lin quan n tng giai on pht trin. Theo quan nim v operon, cc gen iu ha (regulatory gene) gi vai tr quan trng trong vic ng v m cc gen cu trc (structural gene) c th biu hin tng hp protein ng lc, ng ni theo nhu cu c th ca t bo. Trong mi t bo, tt c cc gen u khng hot ng ng thi. V d: t bo E. coli c khong 107 phn t protein gm 3.000 loi khc nhau. Nhiu loi protein c n 500.000 phn t, tuy nhin mt s loi khc ch khong 10 phn t. Nh vy, khng phi loi protein no cng c tng hp vi s lng ln nh nhau v t bo phi c nhng c cht tng hp protein mt cch tit kim v hp l nht. Mt s gen hot ng thng xuyn cung cp sn phm lin tc, mt s khc ch biu hin nhng giai on nht nh trong chu trnh sng v c th ch hot ng trong iu kin mi trng khng bnh thng. Mt s protein cn c tng hp vi s lng ln, mt s khc ch cn c mt phn t. Do vy, hot tnh ca gen c iu ha bi nhiu c ch khc nhau c hiu qu tt nht trong vic s dng ngun nng lng ca t bo.
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I. Cc hin tng iu ha
duy tr ni cn bng (homeostasis) v s pht trin ca c th, cc sinh vt c cc c ch iu ha khc nhau. Cc kiu iu ha u bt ngun t s biu hin ca cc gen. 1. iu ha thch nghi Mt s amip (ameba) biu hin s thay i hnh thi v sinh l c bit p li cc iu kin mi trng khc nhau. Khi cc amip c cho vo nc, chng chuyn t dng amip sang dng c lng bi. Khi mi trng thiu dinh dng chng c th chuyn thnh cc dng tng t nh biu b. Vi khun trong mi trng dinh dng ti thiu c kh nng tng hp amino acid. Nhng khi b sung amino acid vo mi trng nui, vi khun s ngng tng hp amino acid. Lc ngun amino acid t ngoi b sung vo ht, t bo vi khun li t tng hp li amino acid cho bn thn. Cc bin i nu trn l thun nghch, chng t s thay i chc nng y khng phi do bin d di truyn. Cc hin tng trn cn cho thy vic xut hin hay bin mt cc cu trc mi khng lm nh hng n tim nng di truyn sn c. C th cho rng, c trng hp mt s gen hot ng, nhng cng c trng hp mt s gen ngng biu hin. Cc hin tng c cp trn u do c ch iu ha thch nghi (adaptive regulation) chi phi. 2. Hot ng ni tip ca cc gen Khi bacteriophage xm nhim vi khun, DNA ca n lc u s ti bn, sau cc protein khc nhau mi c tng hp nn to thnh v. Nh vy, c cc gen sm to ra enzyme ti bn DNA v cc gen mun xc nh cc thnh phn v protein. iu chng t c c ch iu ha chc nng ca gen din ra theo mt trnh t nghim ngt. y l kiu iu ha ni tip (sequential regulation). Hot ng ni tip ca cc gen cn th hin r trong qu trnh pht trin c th ca cc sinh vt eukaryote a bo. 3. Bit ha t bo Nhiu sinh vt bc cao nh con ngi cha nhiu t t bo bt ngun t mt hp t do phn chia nguyn nhim. T mt hp t ban u n khi
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trng thnh, c th ngi c khong 200 loi t bo khc nhau. Mi loi t bo ch biu hin mt phn thng tin ca mnh. Qu trnh chuyn mn ha chc nng ca t bo c gi l s bit ha hay phn ha (differentiation). Tuy c s bit ha, nhng t bo vn gi nguyn vn kh nng di truyn ca mnh. Mt v d rt r l nui cy m t bo thc vt (plant tisue and cell culture): ngi ta c th nui cy mt phn m phn sinh trong mi trng dinh dng tng hp cho n khi chng pht trin thnh cy in vitro hon chnh (plantlet), cc cy ny sau c a ra trng trong iu kin t nhin v ra hoa kt qu. 4. Khi qut v iu ha prokaryote v eukaryote C s khc nhau ng k gia prokaryote v eukaryote trong iu ha biu hin ca gen. Cc t bo eukaryote c cu to phc tp hn nhiu nn c ch iu ha cng phc tp hn prokaryote. prokaryote, mc ch ca s iu ha biu hin gen l nhm iu chnh h enzyme cho ph hp vi cc tc nhn dinh dng v l ha ca mi trng, m bo c hai yu cu chnh ca t bo l sinh trng v sinh sn. S iu ha y rt linh ng v c tnh thun nghch. eukaryote, do t bo khng tip xc trc tip vi mi trng, nn s iu ha y khng cn nhm mc ch i ph vi cc bin ng ngoi bo. S iu ha eukaryote hng n vic chuyn bit ha tng loi t bo vo tng cu trc v chc nng ring v v th khng mang tnh thun nghch. Ba thnh phn chnh ca s iu ha biu hin gen l: 1) Tn hiu gy ra p ng lm thay i biu hin gen; 2) Giai on c thc hin s iu ha trong qu trnh t ti bn n dch m; v 3) C ch phn t ca s iu ha biu hin gen. 4.1. S biu hin ca gen prokaryote B my di truyn ca sinh vt prokaryote l mt DNA mch vng cha mt s lng gen gii hn c phin m trng thi tip xc trc tip vi t bo cht (Hnh 8.1). Chu trnh t bo ngn v khng c s bit ha t bo. V th, hot ng ca cc gen c iu ha do cc nhu cu ca t bo khi cn thit. Tc
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ng ca cc nhn t mi trng lm nhng gen tng ng c m phin m, dch m tng hp protein hay c hiu qu ngc lm dng li.
RNA polymerase DNA mRNA Polyribosome

0,25 m Chiu dch m DNA RNA polymerase

Polyribosome

Ribosome mRNA Polypeptide

Hnh 8.1. S biu hin gen prokaryote

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

mRNA trong t bo cht S t gy ca mRNA Dch m

T BO CHT

Polypeptide Nhng bin i ha hc hu dch m v vn chuyn ti t bo

Protein hot tnh S t gy ca protein

Protein b t gy

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II. Cc mc iu ha
Cc c ch iu ha s biu hin ca gen c th tc ng mt hay nhiu mc khc nhau. S iu ha c th xy ra mc gen bng s kim sot thi gian v tc phin m. Cc c ch khc c th hot ng lc dch m hoc sau dch m. 1. Mc cht nhim sc Ngay trn cht nhim sc c th thc hin cc kiu sau: - DNase ct mt s vng trn genome lm tho xon cc gen biu hin. Hai vng c lu l cc vng nhy cm (sensible) v siu nhy cm (hypersensible). - Cc vng nhy cm c lin quan n cc gen c hot tnh cao v nhng gen qua biu hin ri (nh cc gen hot ng phi). Cc vng siu nhy cm lin quan n cc gen c hot tnh rt cao (nh cc gen histone). - DNA Z (DNA tri) l dng cu trc siu xon c th lin quan n ng m gen. - Methyl ha cc base. cc prokaryote s methyl ha c th thc hin i vi A v C, cn eukaryote s methyl ha ch thc hin vi C v tr th 5. Methyl ha lm gen ngng hot ng. V d: nhim sc th X bt hot ngi thuc loi siu methyl ha. Ni chung, s thay i cu hnh (reconfiguration) c th nh hng n s biu hin ca gen. 2. Mc phin m y l s iu ha nh hng trc tip n vic m hoc ng ca gen. Kiu iu ha ny thng gp trong iu ha trao i cht, cng nh cc qu trnh bit ha t bo. - S tc ng ca cc trnh t cis (gn k, lin k) nm trn cng mch DNA nh enhancer (vng tng cng) lm tng s phin m. - iu ha bi cc nhn t trans (cch qung, t xa) do cc nhn t khng nm cng trn mt mch DNA. - Chn la promoter thch hp. - S suy yu/suy thoi.
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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 (proenzyme 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

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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.

III. iu ha biu hin gen prokaryote

Cc gen c phin m to RNA, c gi l cc gen cu trc. Cc protein c dch m t mRNA c th l enzyme hoc khng phi enzyme. Trong s cc protein khng phi enzyme c cc protein iu ha (regulatory protein), chng tng tc vi cc trnh t DNA c hiu kim sot hot tnh phin m ca cc gen cu trc. Cc gen tng hp cc protein iu ha c gi l cc gen iu ha (regulatory gen). Pha trc mi gen cu trc (hoc mt nhm gen) c mt trnh t promoter, ni RNA polymerase nhn bit (Hnh 8.3). C ch iu ha prokaryote ch yu c thc hin thng qua operon. y l khi nim ch tn ti prokaryote.

Operon Operator Gen cu trc Gen iu ha Promoter Phin m RNA polymerase Gen a Promoter Gen b Gen c

Phin m

mRNA Dch m mRNA Dch m Protein iu ha Cc protein (enzyme)

Cc con ng ha sinh Tin cht X Cc sn phm trung gian Sn phm Y

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

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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.

TTGACA 35 bp

TATAAT 10 bp +1

mRNA

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.

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Cc protein cn thit cho biu hin gen c gi l cht hot ha. Chng c th gn vi cc im khi s nm bn trong ca promoter ca operon hay im tng cng hoc c th gn nhng trnh t xa operon. Vic gn ca protein iu ha vo im khi u (initiator) hay enhancer, kch thch s phin m ca cc gen cu trc, c gi l c ch kim sot dng. S kch thch cc gen iu ha phn ng c th l t cc phn t tng i nh nh ng, amino acid n cc phn t ln hn nh cc phc hp hormone steroid v cc protein th th (receptor). Cht lm cho gen phin m c gi l cht cm ng, c tc ng ngc vi cht km hm. Cc gen cm ng thng tham gia vo cc phn ng thoi dng (catabolic reaction), nh phn hy cc polysaccharide thnh ng n. Cc gen c ch thng tham gia vo cc phn ng bin dng thc hin vic tng hp cc cht nh amino acid t cc tin cht n gin hn. 3. iu ha thoi dng: Kim sot m-cm ng Trong thoi dng, cc cht thc n c phn hy d dng to nng lng hoc cc cht cn thit cho qu trnh tng hp. C ch iu ha y l s c mt ca c cht (v d lactose) dn ti tng hp cc enzyme phn hy. V d in hnh cho trng hp ny l operon lactose ca E. coli. galactosidase l enzyme c chc nng i. Chc nng u tin ca n l thoi dng lactose thnh glucose v galactose. Chc nng th hai ca n l chuyn lin kt 1-4 ca glucose v galactose thnh lin kt 1-5 ca allolactose. Bnh thng enzyme ny khng hin din nng cao trong t bo, khi vng mt lactose trong mi trng. Ngay sau khi cho lactose vo mi trng nui khi khng c glucose, enzyme ny bt u c to ra. S vn chuyn lactose xuyn qua mng t bo c hiu qu nh protein vn chuyn galactoside permease. Protein cng xut hin vi nng cao khi c lactose trong mi trng. S iu ha ca operon lactose cn ph thuc vo nng glucose trong mi trng. Nng glucose ny li kim sot nng bn trong t bo ca phn t nh cAMP (cyclic adenosine monophosphate), l cht bt ngun t ATP v lm tn hiu bo ng cho t bo. T bo c xu hng s dng glucose hn l lactose lm ngun carbon v glucose c bin dng trc tip cung cp carbon v to nng lng. Cc enzyme bin dng
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glucose thuc loi cu trc v t bo tng trng ti a vi ngun glucose. Khi ngun glucose cn, t bo phn ng li bng cch to ra c-AMP. Vic tng nng c-AMP trong t bo gy nn hng lot s kin, trong s hin din ca lactose, dn n s phin m cc gen cu trc ca operon lactose. 3.1. Cu trc ca operon lactose H thng lactose (lactose system) bnh thng (Hnh 8.4) gm c gen iu ha (i hoc R) v operon mang trnh t promoter (P) locus operator (O) v ba gen cu trc cho -galactosidase (Z), permease (Y) v transacetylase (A). Nhiu t bin cc locus ny c pht hin. 3.2. Hot ng ca h thng - iu kin cm ng (c lactose). Lactose c chuyn vo t bo rt yu v ch c vi phn t permease lm vic. Khi vo trong t bo, mt s lactose (lin kt -1,4) c chuyn thnh allolactose (lin kt -1,6) nh galactosidase. Allolactose l cht cm ng, n gn vo protein km hm v gy bin i cu hnh to phc hp allolactose-repressor. Phc hp ny mt kh nng gn operator. Lc ny operon c m, RNA polymerase bt u phin m cc gen cu trc. Ton b s kin din ra nh trn hnh 8.4b. - iu kin khng cm ng (khng c lactose). Gen iu ha ca operon thng xuyn tng hp protein km hm (repressor protein) mc thp, v n c promoter t hiu qu. S tng hp cc protein ny b tc ng do nng lactose trong t bo. Ngc li, promoter bnh thng ca operon lac gn vi RNA polymerase rt c hiu qu. Khi khng c ng lactose, protein iu ha hot ng (active regulator protein) cn gi l protein km hm gn vo promoter hay c trnh t operator v protein km hm chim on ny. Nh vy, s phin m ca tt c cc gen cu trc ca operon lac b dng (Hnh 8.4a). Do s lng permease tng, nn lactose vo t bo vi s lng ln v c phn hy bi -galactosidase. Khi lactose c s dng ht, cc protein km hm gn tr li vo operator lm operon b ng; s phin m cc gen cu trc b dng.

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Bn thn gen iu ha lacI ch c mt promoter (Pi) v gen cu trc ca protein km hm. Promoter ny yu, khi cc protein km hm c s lng ln, n b cc protein ny gn vo lm dng phin m.
(a) Khng c lactose
Gen iu ha lacI RNA polymerase Khng th lin kt Operon Operator lacO Gen cu trc

X
lacP

lacZ

lacY

lacA

pI

Phin m v dch m

Khng phin m

Protein iu ha hot ng (protein km hm)

(b) C lactose

RNA polymerase

Operator lacO

X
Phin m v dch m

Phin m v dch m

Protein iu ha hot ng

Enzymes
Protein iu ha bt hot

-Galactosidase

Permease

Transacetylase Glucose

Allolactose -Galactosidase Lactose

Galactose

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.

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Qu trnh tng hp tryptophan bt u t tin cht tryptophan l chorismic acid, tri qua 5 giai on k tip do enzyme xc tc. H thng tng hp amino acid tryptophan E. coli l v d in hnh v operon b km hm do s kim sot m. 4.1. Cu trc v hot ng H thng tryptophan cng c cu trc tng t h thng lactose gm gen iu ha trpR v operon tryptophan (promoter, operator v 5 gen cu trc). Cc gen cu trc xc nh 5 enzyme c xp theo th t tng ng vi chc nng xc tc theo trnh t cc phn ng ca chui bin dng tryptophan (Hnh 8.5).
(a) Nng tryptophan thp Gen iu ha (trpR) RNA polymerase Operator Cu gen cu trc 5 UTR trpE trpD trpC trpB trpA Promoter

PR
Phin m v dch m

X
Phin m v dch m

mRNA

Protein iu ha bt hot

Enzymes

Chorismate (trp repressor)

Tryptophan

Operator (b) Nng tryptophan cao

PR
Phin m v dch m Khng phin m

Protein iu ha bt hot

Hnh 8.5. Operon tryptophan Sinh hc phn t

167

S khc nhau cn bn vi h thng lactose l gen iu ha. Gen iu ha ca h thng tryptophan tng hp thng xuyn aporepressor protein, l cht km hm m ring n khng c hot tnh. Khi tryptophan d tha n tr thnh cht corepressor (ng km hm) v kt hp vi aporepressor thnh phc hp km hm (holorepressor) c hot tnh. Phc hp ny gn vo operator ca operon tryptophan (trp) lm dng phin m cc gen cu trc. Khi nng tryptophan thp, n tch khi phc hp km hm v aporepressor mt hot tnh. Lc ny cc operator li c m v RNA polymerase dch m 5 gen cu trc tng hp 5 enzyme to tryptophan (Hnh 8.5). S iu ha kiu ny cn gi l iu ha c ch ngc (retroinhibition) do sn phm cui cng c mi lin h ngc (feed-back). Nh vy, hot ng ca h thng ny ngc li vi h thng lactose: khi c tryptophan th operon b ng, thiu tryptophan th gen c m. 4.2. S suy yu (attenuation) Kiu iu ha th hai c pht hin operon tryptophan c gi l s suy yu. u 5 ca mRNA a gen (polycistronic) ca operon ny c 5 enzyme. on ny c gi l trnh t leader (trnh t ch huy). Mt phn ca trnh t ny c phin m to leader peptide gm 14 amino acid, m chc nng n nay cha r. C ch kim tra m s tng hp mRNA c bt u nhng kt thc sm vi chiu di mRNA ngn hn c gi l s suy yu. V d: cc t bo E. coli ang tng trng trong mi trng thiu mt amino acid no , nhng cha cc enzyme cn c cho s tng hp ca tt c 20 amino acid cn thit to ra protein. Khi thm mt amino acid (tryptophan) vo mi trng s lm gim ng k s tng hp ca cc enzyme cn cho s to amino acid . Phn ng ny c tnh thch ng v duy tr cc ngun enzyme khng cn cn na khi sn phm cui cng ca chui sinh tng hp (tryptophan) ang c trong t bo (c ch ngc). Operon tryptophan c phng thc iu ha hot ng gen th hai, hon ton c lp vi h thng repressor-operator (cht km hm on iu hnh). operon tryptophan, s tng hp mRNA bt u t 161 base trc codon khi s ca trpE, enzyme cu trc u tin c phin m. t bin do mt on DNA ngay pha trc trpE, gii phng trpE khi s km hm ca phc hp represser-operator. Cc t bin ny lm tng mc biu
Sinh hc phn t
168

hin ca c operon ln gp su ln. Tinh sch cc DNA c phin m in vitro cho thy phn ln mRNA kt thc ngay base 139 v khng bao gi t ti trpE. y, c s km hm phin m ngay trc gen cu trc v khi mt n s biu hin ca gen c hiu qu hn. on di ca mRNA nm trc trpE c gi l leader (trpL) v on km hm phin m c gi l attenuator (trpa, ch khng phi trpA). Cui cng, quan st cho thy s suy yu dao ng theo nng ca tryptophan thp, nhiu phn t mRNA c to ra hn qua attenuator v ton b operon c phin m. S suy yu l mt phng thc khc kim sot s biu hin ca gen.

Ribosome phin dch nhanh mRNA khi tryptophan phong ph dn ti to thnh cu trc stem-and-loop c ch RNA polymerase v kt thc phin m

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

C ch suy yu thc hin s kt hp gia phin m v dch m. RNA polymerase bt u phin m cc gen m ha cho cc enzyme sinh tng hp cn thit to ra tryptophan. V d: khi hin din tryptophan n nhanh chng t ti im (trn RNA mi c tng hp) gi l attenuator v dng phin m ti y. Attenuator c chc nng nh im kt thc ch khi tryptophan hin din. S phin m dng khi trnh t leader t attenuator. Nu c
Sinh hc phn t
169

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

Sinh hc phn t

170

Hot t

L-Arabinose

(bn ngoi)

Gen c ch

araC mRNA

Cc v tr iu chnh Cc gen cu trc

araC

araO2
Gen iu ha

araO1

CAP

araI

araB

araA

araD

araBAD mRNA

Pemease system
L-Arabinose L-Arabinose

L-arabinose isomerase
L-Ribulose

L-arabinose isomerase
L-Ribulose-5-P

L-arabinose isomerase
D-Xylulose-5-P

(bn ngoi)

(bn trong) ATP ADP

Hnh 8.7. Operon arabinose ca E. coli

IV. iu ha hot tnh ca eukaryote

Cc c ch iu ha eukaryote c th xy ra 5-6 mc khc nhau (xem mc II). Trong phn ny ch nhn mnh thm mt s c im ca iu ha hot ng gen eukaryote: - cc operon ca prokaryote, cc gen iu ha v cc promoter thng nm gn nhau, nhng eukaryote cc gen iu ha t khi nm gn cc promoter do chng kim sot. - Cc enhancer l nhng trnh t cng nm trn mt phn t vi cc promoter c th c hng trm cp base pha trc hoc pha sau promoter m chng kch thch. - Trnh t iu ha 5 pha trc c promoter eukaryote thng rt di, c khi hng chc kilobase (kb). - C nhiu kiu iu ha dng cc nhn t c tc ng t xa (trans) l cc protein. S phin m c th c kch thch bi cc tn hiu khc nhau. S iu ha hot ng gen prokaryote phn ln p ng li cc tn hiu bn trong.
Sinh hc phn t
171

1. Cc promoter Tng t nh vi khun, cc promoter ca eukaryote cng nm pha trc im xut pht ca mRNA v c nhng trnh t c bo tn trong tin ha. Hp TATA, nh hng cho RNA polymerase bt u phin m, nm khong di 30 basepair (bp) ng vt c v, v 60-120 nm men. Hp TATA hot ng c hiu qu cng vi hai trnh t tng ng pha trc khong 40 bp l CCAAT v 110 bp l trnh t giu GC. S thay i hp TATA lm gim tc phin m. Hiu qu ca tc phin m c o bng s thay i ca tng base trong promoter, cc thay i base ngoi hp TATA v cc trnh t pha trc khng gy hiu qu i vi tc phin m, trong khi cc thay i nhng yu t c nu trn lm gim ng k tc phin m. Khc vi promoter ca prokaryote, cc promoter ca eukaryote kh m bo nhn bit cc tn hiu mt cch y RNA polymerase khi s phin m in vitro. Hp TATA v cc trnh t pha trc phi c nhn bit bi cc protein iu ha, v chnh cc protein ny gn vi cc im nht nh trn chng v hot ha s phin m. 2. Cc enhancer Enhancer l cc trnh t c tc ng cis (lin k), chng tng tc phin m ng k t cc promoter nm ngay trn cng mt phn t DNA. Tnh c o ca cc enhancer l ch chng c kh nng thc hin mt cch hiu qu khi mt khong cch rt xa, c khi n vi nghn cp base. Ngoi ra, chng c hot ng chc nng bt k hng no, d pha trc hay pha sau promoter. Xt trn mt gc no , cc enhancer tng t promoter. C th, chng c t chc gm mt dy cc trnh t c tc ng cis nhn bit cc nhn t tc ng t xa (trans) (Hnh 8.8). 3. Cc protein l nhn t c tc ng trans Mt vi protein, nhn bit hp CCAAT, c xc nh t bo ng vt c v. Cc nhn t ny c th c phn bit gia cc n v ca cc phn t CCAAT. Cc hp GC c nhn bit bi cc nhn t ph. Cc v d v cc tc ng trans (trans-acting factor) tm thy nm men v
Sinh hc phn t
172

ng vt c v. Cc nhn t trans c c im chung l gm hai vng cu trc v chc nng chnh: - Vng gn nhn t trans vo DNA. - Vng tc ng ln s phin m. ng thi cc nhn t trans c bn kiu tc ng nh: - ngn tay km (zinc-finger) - xon-vng-xon (helix-turn-helix) - xon-nt-xon (helix-loop-helix) - dy ko leucine (leucine-zipper) (Hnh 8.9).
Activators DNA Enhancers Hp TATA Promoter Gen

Cc nhn t phin m

RNA polymerase

3
Phc hp khi u phin m

RNA polymerase

Tng hp RNA

Hnh 8.8. Enhancer ca DNA eukaryote

c tnh chung ca cc cu trc va k l chng lun lun gn ln cc trnh t cis di dng dimer (gm hai dimer).

Sinh hc phn t

173

Trnh t cis v nhn t trans thng c cc tng tc c hiu do nhng lin kt yu hnh thnh gia cc phn t ca nhn t trans vi cc base ca trnh t cis. V d, trng hp tng tc gia cc hormone steroid vi cc enhancer. Nhiu protein c tc ng trans tham gia vo tin trnh pht trin ca c th rui gim, chng c vai tr c bit trong xc nh s sp xp cc on thn ca rui. Nh vy, cc gen eukaryote c hot ha bi hai trnh t DNA c tc ng cis l promoter v enhancer, chng nhn bit c cc nhn t protein c tc ng trans, cc nhn t trans cho php RNA polymerase khi s phin m v t tc phin m ti a.
A. Xon-nt-xon Khung Cp base ng-phosphate B. Xon-vng-xon

DNA

COOH

NH2 C. Ngn tay km D. Dy ko leucine

Hnh 8.9. Cc kiu tc ng ca nhn t trans Sinh hc phn t

174

4. Hormone V d r nht v cc cht iu ha ni ti ca hot tnh gen l hormone. l nhng cht to ra do mt lot t bo c hiu qu n cc t bo khc. Cc hormone thng c vn chuyn n cc phn ca c th nhng ch tc ng n cc t bo c cc receptor tng ng. S tng tc gia hormone vi receptor gy ra tn hiu tc ng n cc vng c trng ca DNA, gy hot ha gen hoc mt nhm gen tng ng. Cc hormone c th kch thch phin m bi mt trong cc c ch sau: - Hormone c th lm cho DNA tch khi histone v to iu kin cho RNA polymerase bt u phin m. - C th lm cht cm ng (inducer) gy bt hot phn t receptor. - C th gn trc tip vi on DNA c hiu to thun li cho vic gn RNA polymerase hoc protein l nhn t phin m (protein transcription factor). - C th hot ha effector protein lm thnh phc hp gn DNA v kch thch gn RNA polymerase. - C th gn vi protein to phc hp hot ha kch thch gn RNA polymerase. 5. Kim sot cc cht thng gp trong nhn Mt s nhm phn t hin din rt nhiu trong mi t bo eukaryote, nh cc histone, cc cht ca b my dch m, cc cu phn ca mng Vi chng trnh khc nhau c thc hin duy tr s lng ln ca chng trong t bo. Cc chng trnh gm: - S phin m lin tc v lp li trong t bo. - S lp li cc gen. - S khuch i ngoi nhim sc th cc trnh t gen c hiu. 5.1. S phong ph Cc gen m ha cho rRNA v tRNA c nghin cu k. Phn ng lai n gin DNA-RNA cho thy nhim sc th ca Xenopus c vng t chc hch nhn (nucleolus organizer) cha 450 bn sao ca DNA m ha
Sinh hc phn t
175

cho 18S v 28S rRNA. Ngc li, trong mi nhn c 20.000 bn sao ca cc gen m ha cho 5S rRNA v cc gen ny khng nm vng t chc hch nhn. Cc gen m ha cho histone cng c vi nhiu bn sao c lp li hng trm ln. S phong ph ca cc gen nu trn m bo s lng cn thit cho dch m khi phi tng hp hng triu phn t protein vo lc cn thit. 5.2. S khuch i gen Mt v d v s khuch i gen trong nhn l cc ch phnh (puff) ca nhim sc th khng l hay a si (polytene chromosome) tuyn nc bt ca rui gim. cc ch phnh ny, DNA nguyn nhim sc (euchromatic) c khuch i khong vi nghn ln. Trong nhn t bo trng ca Xenopus, c hng trm nhn con ngoi nhim sc th (extrachromosome nucleoli) vi kch thc khc nhau, mi nhn con cha cc vng trn rDNA kch thc khc nhau, m vai tr n nay cha r. Cc DNA vng trn ny sn sinh nhiu rRNA mc hp l.

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

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Tm li, s lng DNA ca cc t bo bit ha v cn bn ging vi cc hp t ban u v cha nguyn vn thng tin di truyn pht trin thnh c th nguyn vn. 2. Cc t bo bit ha tng hp cc nhm protein khc nhau hiu c s khc nhau gia cc t bo bit ha, cn xt nhiu vn sau: - Th nht, nhiu qu trnh l chung cho tt c cc t bo nn c nhiu protein ging nhau. Nhng protein ny gm s lng nhiu, d phn tch nh phn ln cc protein cu trc ca thnh t bo v nhim sc th; mt s protein cn bn ca cc bo quan (li ni sinh cht, b my Golgi, cc ribosome). Nhiu protein c s lng khng nhiu nh cc enzyme khc nhau, tham gia vo cc phn ng trung tm ca qu trnh trao i cht, u hin din nh nhau tt c cc kiu t bo. - Th hai, mt s protein c s lng phong ph mt s t bo chuyn ha m vic pht hin chng cn c th nghim ring. V d, hemoglobin ch c th pht hin t bo mu. - Th ba, nu nh hn 2.000 loi protein c s lng di do c so snh gia cc kiu t bo bit ha ca cng mt vi sinh vt vi nhau bng in di hai chiu polyacrylamide, th mt s khc bit ng k s c tm thy. D s so snh gia hai dng t bo nui cy (nh cc dng t bo c v thn kinh) hay gia cc t bo ca cng mt m ca chut (nh gan v phi), i a s cc protein c pht hin u c tng hp c hai loi t bo v vi tc tng hp c th khc nhau n 105; ch c vi trm protein hin din vi s lng khc nhau ng k hai kiu t bo. Cc nghin cu cho thy cc t bo eukaryote bc cao tng hp t 10.000-20.000 protein khc nhau. Phn ln chng c s lng rt t v kh pht hin. Nu cc protein him (minor protein) ny khc nhau gia cc t bo vi mc tng t cc protein di do, th ch mt s lng nh ca chng cng to nn nhiu khc bit chuyn ha ln v hnh thi v sinh l ca t bo. T bo c th thay i s biu hin gen ca chng p li tn hiu bn ngoi.

Sinh hc phn t

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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.
Glucocorticoid receptor trong s hin din ca glucocorticoid hormone

Glucocorticoid hormone

gen 1

gen 1

gen 2

gen 2

gen 3 Gen biu hin mc thp Gen biu hin mc cao

gen 3

Hnh 8.10. Tc dng ca glucocortcoid lm tng mc biu hin ca gen Sinh hc phn t
178

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

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Ti liu tham kho/c thm

1. Phm Thnh H. 2003. Di truyn hc. NXB Gio dc, H Ni. 2. L c Trnh. 2001. Sinh hc phn t ca t bo. NXB Khoa hc v K thut, H Ni. 3. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 2002. Molecular Biology of the Cell. 3rd ed. Garland Publishing, Inc. New York, USA. 4. Karp G. 2002. Cell and Molecular Biology: Concepts and Experiments. 3 ed. John Wiley & Sons, Inc. New York, USA.
rd

5. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 6. Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J. 2004. Molecular Cell Biology. 5th ed. Freeman and Company, New York, USA. 7. Walker JM and Rapley R. 2000. Molecular Biology and Biotechnology. Chapman & Hall Limited, London, UK. 8. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California, USA. 9. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company, New York, USA

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Chng 9

Cng ngh DNA ti t hp

I. M u
Vo nm 1973, mt nhm cc nh khoa hc to ra c th sinh vt u tin vi cc phn t DNA ti t hp. Theo , Cohen (H Stanford, M) v Boyer (H California, M) cng cc cng s a c mt on DNA t mt plasmid ny vo mt plasmid khc, to ra mt plasmid hon ton mi, plasmid ti t hp. Sau , h a plasmid ti t hp vo trong cc t bo E. coli. Trong mt thi gian ngn, cc tc gi ny dng cc phng php ging nhau gn cc gen t hai loi vi khun khc nhau, cng nh chuyn cc gen t ch vo vi khun. Cc th nghim ny nh du mt cuc cch mng v cng quan trng trong lch s nghin cu khoa hc ca nhn loi. Cng ngh DNA ti t hp l mt tp hp cc k thut phn t nh v, phn lp, bin i v nghin cu cc on DNA. Thut ng ti t hp c dng thng xuyn do mc tiu ca n l phi hp DNA t hai ngun xa nhau. V d: cc gen t hai ngun vi khun khc nhau c th c lin kt li, hoc mt gen ngi c th c a vo nhim sc th vi khun. Cng ngh DNA ti t hp (cn gi l cng ngh di truyn, cng ngh gen hay k thut gen) hin nay bao gm mt mng li cc k thut phn t c dng phn tch, bin i v ti t hp hu nh mi trnh t DNA. 1. Tc ng ca cng ngh DNA ti t hp Cng ngh DNA ti t hp bin i su sc phng thc nghin cu gen. Trc y, thng tin v cu trc v t chc ca gen thu c bng cch kim tra biu hin kiu hnh ca chng, nhng nhng k thut mi to ra kh nng t c cc trnh t nucleotide. Trc y, cc nh di truyn phi ch i s xut hin cc t bin ngu nhin hoc cm ng phn tch hiu qu ca s sai khc di truyn, ngy nay h c th to ra t bin

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cc im nht nh mt cch chnh xc v xem chng thay i kiu hnh nh th no. Cng ngh DNA ti t hp cung cp cc thng tin mi v cu trc v chc nng ca gen v thay i nhiu khi nim c bn ca di truyn hc. V d: trong khi m di truyn c xem l rt ph bin, th by gi chng ta cn bit rng cc m khng ph bin cng tn ti trong DNA ty th. Trc y, chng ta ngh rng t chc ca cc gen eukaryote ging vi prokaryote, nhng by gi chng ta bit rng nhiu gen eukaryote b gin on bi cc intron. Ngy nay, chng ta bit y hn v cc qu trnh ti bn, phin m, dch m, bin i RNA (RNA processing) v iu ha gen thng qua vic s dng cc k thut ti t hp DNA. Cc k thut ny cng c dng trong nhiu trong nhiu lnh vc khc, bao gm ha sinh hc, vi sinh vt hc, sinh hc pht trin, sinh hc thn kinh, tin ha v sinh thi hc. Cng ngh DNA ti t hp cng c ng dng to ra nhiu sn phm thng mi, chng hn: thuc, hormone, enzyme v cc ging cy trng-vt nui. Mt nn cng nghip hon ton mi, cng nghip cng ngh sinh hc, pht trin chung quanh vic s dng cc k thut ny to ra cc sn phm mi. Trong y hc, cc k thut ti t hp DNA c dng thm d bn cht ca ung th, chn on cc bnh di truyn v nhim trng, sn xut thuc v iu tr cc ri lon di truyn. 2. Lm vic mc phn t K thut gen cho thy mt lot c hi, m ra cc phng thc cn thit (m trc y c th khng c) gn nh l hin nhin. Vn c bn l cc gen c kch thc qu nh v c hng ngn gen trong mi t bo. Thm ch, khi quan st trn knh hin vi mnh nht, th DNA xut hin nh l mt si dy b xu, cc nucleotide ring r khng th thy, v khng c mt du hiu no v cc ng nt vt l ch bt u v kt thc ca mt gen. minh ha vn ny, chng ta hy xem xt mt v d c trng v di truyn phn t nh sau: Gi thit rng chng ta mun phn lp mt gen c bit ca ngi v t n vo trong vi khun sn xut mt lng ln cc protein ngi c m ha. Vn u tin l tm c gen mong mun. Genome n bi ca ngi cha khong 3,3 t cp base ca DNA.
Sinh hc phn t
182

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

Sinh hc phn t

183

(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 h 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):
a
HindIII

u dnh

PvuII

u bng

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).

Sinh hc phn t
184

- 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. Eco 5 li 3 li (blunt) (v d: Pst : Bal 9.1).

Enzyme BamHI BglII CofI DraI EcoRI HaeIII

Ngun vi sinh vt Bacillus amyloliquefaciens Bacillus globigii Clostridium formicoaceticum Deinococcus radiophilus Escherichia coli Haemophilus aegypticus

Trnh t nhn bit

5-G GATCC-3 3-CCTAG G-5 5-A GATCT-3 3-TCTAG A-5 5-G CGC-3 3-CGC G-5 5-TTT AAA-3 3-AAA TTT-5 5-G AATTC-3 3-CTTAA C-5 5-GG CC-3 3-CC GG-5

Loi u Dnh Dnh Dnh Bng Dnh Bng

Sinh hc phn t

185

HindIII HpaII PstI PvuII SmaI XmaI

Haemophilus influenzae Haemophilus parainfluenzae Providencia stuartii Protrus vulgaris Serratia marcescens Xanthomonas malvacearum

5-A AGCTT-3 3-TTCGA A-5 5-C CGG-3 3-GGC C-5 5-CTGCA G-3 3-G ACGTC-5 5-CAG CTG-3 3-GTC GAC-5 5-CCC GGG-3 3-GGG CCC-5 5-C CCGGG-3 3-GGGCC C-5

Dnh Dnh Dnh Bng Bng Dnh

C hai kiu gn khc nhau: gn u bng v gn 4 hoc n u dnh. V d: Hnh 9.2 minh ha vic gn cc u dnh c ct bng enzyme HindIII. 2. Isochizomer

. : - Mbo Sau
5 GATC 3 3 CTAG 5

Sinh hc phn t

186

HindIII

HindIII

Khong trng trong khung ng-phosphate

Gn cc on

Ligase

Khong trng trong khung ng-phosphate

Ligase

Bam
5 GGATCC 3 3 CCTAGG 5

: Sal

(G

XhoI ct trnh t (C Sal XhoI:

5G 3CAGCT

TCGAG3 C5

5GTCGAG3 3CAGCTC5

Sinh hc phn t

187

III. Phng thc to dng

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

Sinh hc phn t

188

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.

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. Sinh hc phn t
189

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.

400

420

440

460

AGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCATAATCATGGTCAT

EcoRI

SacI

KpnI

SmaI XmaI

BamHI

XbaI

HincII AccI SalI

PstI

SphI

HindIII lacZ 1 ThrIleMetThr(Met)

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

Sinh hc phn t
190

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

Bin np T bo vi khun

Sao chp vi khun

Sao chp vi khun Nui cy vi khun cha mt lng ln t bo, sinh trng qua m 37oC s sn xut khong 109 t bo/mL

Hnh 9.5. Phng thc c bn to dng gen trong vi khun E. coli

Sinh hc phn t

191

Hnh 9.5 trnh by ton b phng thc sn xut DNA ti t hp (to dng gen). Plasmid c ct cc v tr xc nh bng enzyme hn ch. DNA ca mt genome ngoi lai c ct bi cng mt loi enzyme, mt s on trong c th c gen quan tm. Plasmid v cc on ca genome c phi trn v kt hp nh enzyme DNA ligase. Cc plasmid ti t hp c bin np vo vi khun bng ng nui cy plasmid v vi khun. 2. Bacteriophage vector

Cc bacteriophage c nhiu u im khi c s dng lm vector to dng. Vector dc s dng rng ri nht l bacteriophage gy nhim vo t bo E. coli. Mt trong nhng u im chnh ca phage l c hiu qu chuyn DNA vo trong t bo vi khun cao. u im th hai l mt phn ba ca genome phage l khng cn thit cho s xm nhim v sinh sn ca n trong t bo vt ch, khng c nhng gen ny, mt tiu th vn chuyn thnh cng DNA ca n vo vi khun v sinh sn c. Cc gen khng cn thit ny, khong >15 kb, c th c thay th bng mt on DNA ngoi lai khong 15-23 kb. u im th ba l DNA s khng b ng gi trong v tr khi n di t 40-50 kb; v th cc on DNA ngoi lai khng bao gi c chuyn vo t bo tr khi chng c chn vo trong genome phage , iu kin cn thit m bo on DNA ngoi lai s c ti bn sau khi n vo trong t bo vt ch. Cc gen cn thit ca genome phage c nh v trong mt cm. Cc chng ca phage , c gi l vector thay th, c bin i di truyn vi mt v tr RE duy nht cho EcoRI (chng phage th h mi EMBL 3 c ba v tr cho cc RE: EcoRI, BamHI v SalI) trn mt mt khc ca cc gen khng cn thit (Hnh 9.6). V th, c th loi b cc gen khng cn thit bng EcoRI. DNA ngoi lai c ct bng EcoRI s c u dnh b sung cho u ca cc on DNA ca phage cn thit (nhnh tri v phi), c th c kt ni bng DNA ligase. Genome ca phage c cc on si n ngn c gi l cc v tr cos cn cho s ng gi DNA trong u ca phage. Genome ti t hp ca phage sau c th c ng gi trong v protein v c b sung vo trong E. coli. Cc phage gy ra s xm nhim DNA ti t hp ca chng vo trong t bo vt ch, ni n s c ti bn. Ch cc on DNA c kch thc thch hp v mang cc gen

Sinh hc phn t

192

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.

EcoRI

45 kb

EcoRI

Nhim sc th ca Bacteriophage

Cc gen khng cn thit (vng trung tm)

>15 kb

Ct loi b cc gen khng cn thit

Nhnh tri

Nhnh phi Cc nhnh phi v tri c trn vi DNA ngoi lai cng c ct bng EcoRI

EcoRI
20 kb

EcoRI

Cc u dnh b sung ca DNA c gn vi nhau

Nhim sc th ca bacteriophage ti t hp sau c ng gi trong v protein ca phage

Hnh 9.6. Bacteriophage l mt vector to dng hiu qu

3. Cosmid vector Cc vector phage ch c th mang cc on DNA c kch thc khong 15-23 kb. Tuy nhin, cc cosmid vector li mang c cc on DNA c kch thc ln hn nhiu, khong 45 kb.
Sinh hc phn t
193

cos BamHI cos

Scal
DNA

Tet r ori Scal

cos BamHI ori Tetr cos (~45kb) bng BamHI

BamHI
cos ori Tetr cos

T4 DNA ligase

cos
50 kb

ori

Tet r cos

ng gi phage in vitro u cos DNA vector c ng gi 50 kb u

ui Si ui

Xm nhim

cos

ori Tet r
Plasmid

Hnh 9.7. To dng trong cosmid. Hai v tr cos gn v tr ct hn ch ScaI v BamHI. DNA ngun b ct bi BamHI phn on c kch thc khong 45 kb. Phn t plasmid DNA c ct bi BamHI v ScaI. Hai mu DNA ny c trn vo nhau v c gn bng T4 DNA ligase. Sau khi gn xong, nhng phn t ny c ng gi trong phn u ca phage , v nhng phn t c th ly nhim s c hnh thnh sau khi to ui. Sinh hc phn t
194

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

Vector to dng Plasmid Phage Cosmid

Kch thc DNA c th c to dng Khong 10 kb Khong 20 kb Khong 45 kb

Phng php sinh sn Ti bn plasmid Sinh sn phage Sinh sn plasmid

Phng php chuyn vo vi khun Bin np Xm nhim phage Xm nhim phage

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 : -

Sinh hc phn t

195

. -

ca .

. c 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). e H ca E. coli. Tng hp si cDNA th hai t khun mu si cDNA th nht nh enzyme DNA polymerase vi primer l vng cp tc ca n thu c phn t cDNA si i. - Ct vng cp tc bng enzyme nuclease S1 v dng enzyme Klenow sa cha hai u ca si i cDNA to ra u bng. - Gn cc on ni (linker) vo hai u ca cDNA si i trc khi to dng trong vector thch hp xy dng th vin cDNA. 5. Th vin genomic DNA -

) trnh t nucleotide quan tm (physical mapping).

Sinh hc phn t

196

. Th vin genomic DNA c nhiu ng dng, chng hn lp bn vt l (physical mapping) ca DNA v xc nh cc gen gy bnh hoc cc chui DNA quan tm cho nhng phn tch khc. Sn xut ra cc dng mang cc on chn DNA khc nhau nhng gi ln nhau c nhiu thun li v th vin c th c s dng trong qu trnh chromosome walking. Chromosome walking thng c thc hin vi th vin ca cosmid, phage hoc YAC2. Cc th vin genomic DNA cng cn thit cho vic xc nh cc gen gy bnh bng cch to dng chc nng (functional cloning). Theo hng ny, thng tin v chc nng ca gen c khai thc phn lp gen mong mun t th vin. Mt oligonucleotide c trnh t da trn chui amino acid tng phn c dng nh l mt probe (mu d) phn lp dng cDNA bng cch sng lc th vin cDNA. Dng cDNA ny sau c th c dng sng lc th vin genome nhm phn lp cc dng genomic DNA v cho php kho st c im ca chui genomic hon chnh.

IV. Biu hin gen ngoi lai trong vi khun

V mt l thuyt, k thut DNA ti t hp cho php a bt k mt gen no t mt sinh vt ny vo mt sinh vt khc. Vn quan trng l lm sao gen ngoi lai c th biu hin trong c th vt ch. biu hin tt c cc gen ngoi lai trong E. coli phi bt u bng vic gn on gen ngoi lai vo trong vector biu hin (thng l plas

E. coli. Vector ny phi c cc cu trc cn thit sau: - Cc trnh t m ha gen ch th (marker) m bo duy tr vector trong t bo. - Mt promoter kim sot phin m (v d: lac, trp hoc tac) cho php sn xut mt lng ln mRNA t cc gen c to dng.
2

YAC (yeast artificial chromosome): Nhim sc th nhn to ca nm men.

Sinh hc phn t

197

- Cc trnh t kim sot dch m nh vng lin kt ribosome c b tr thch hp v codon khi u AUG. - Mt polylinker a gen ngoi lai vo trong mt hng chnh xc vi promoter. Ch khi c cu trc y nh th, cc vector biu hin mang gen ngoi lai mi c bin np vo chng E. coli thch hp. Nu on gen ngoi lai khng nm gia promoter v v tr kt thc phin m, th n s khng c phin m. 1. Cc protein nguyn th ti t hp Cc protein nguyn th (native protein) c th c sn xut trong E. coli bng cch s dng promoter mnh v mt vng lin kt ribosome (ribosome binding 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. 1.1. Biu hin ca gen prokaryote-Promoter

E. coli . in hnh l promoter (lai) trp-lac. Promoter trp-lac cn gi l promoter tac (mt dng promoter lai gia promoter trp v promoter lac) c s dng thnh cng sn xut mt lng ln protein trong E. coli (Hnh 9.8). Promoter trp trp . Trong khi , promoter lac lac - -D-thiogalactoside (IPTG) vo mi tr , promoter (lai) trp-lac trp-35 n laclac lac (lac repressor). 1.2. Biu hin ca gen eukaryote-Promoter v vng E. coli

Sinh hc phn t

198

. Trong E. coli E. coli 16S rRNA.

Vng to dng 5S T1 T2 tac promoter Ptac

pKK177-3
(2,9 kb)
Amp 2000
r

ori

1000

Vng to dng

Ptac

EcoRI SmaI

SalI

HindIII PstI

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.

sau : AUG.
Sinh hc phn t
199

16S rRNA.

1 (Hnh 9.9). Vector pAS1 mang promoter PL v RBS ca gen cII ca bacteriophage

E. coli thch hp. Sng lc th bin np thu cc khun lc c mc phin m cao.

EcoRI 0

HindIII

1000 5000 Amp

r

PL

PL nuL nuR tR cII

RBS

pAS1
(5,8 kb)
ori

BamHI 2000 4000

3000 cII RBS (ribosome-binding sites: vng lin kt ribosome) PL cII Met AAGGAAATACTTACAT ATG GAT CC cII SD BamHI

Hnh 9.9. Vector pAS1. Vector pAS1 l mt plasmid di khong 5,8 kb mang promoter PL ca bacteriophage v v tr ct hn ch duy nht BamHI nh v codon khi u ATG ca gen cII ca bacteriophage .

Sinh hc phn t

200

2. Cc protein dung hp ti t hp 2.1. Protein dung hp Protein dung hp cn gi l protein lai c m ha bi mt gen lai (fusion gene) do s dung hp in vitro cc on gen khc nhau. V vy, protein dung hp s mang trnh t amino acid ca hai protein khc bit. Cc protein dung hp c xy dng cho cc mc ch khc nhau. S dung hp gen c thc hin bng cch gn phn m ha ca gen c to dng gn u tn cng 3 ca gen lacZ. Protein dung hp c nhng u im chnh sau: - Protein dung hp thng c sn xut vi hm lng ln do s khi u phin m v dch m c iu khin bi cc trnh t tiu chun ca E. coli. - Dung hp cc trnh t ngoi lai vi cc gen ca E. coli thng cho kt qu cc sn phm n nh hn cc protein ngoi lai nguyn th. 2.2. Vector biu hin cc gen dung hp vi gen lacZ Mt s h thng vector c pht trin biu hin cc gen dung hp vi gen lacZ, in hnh l cc vector h pUR (Hnh 9.10). Chn vector v v tr ct hn ch thch hp ngi ta c th tin hnh dung hp cho hu ht gen c to dng. 2.3. Pht hin cc protein dung hp Gn vector plasmid (v d: pUR) thch hp vi on DNA ngoi lai to ra mt s dung hp trong khung c. Bin np cc vector ny vo E. coli. Kim tra cc khun lc ring bit mang on DNA ngoi lai mong mun bng cch tch chit DNA ca vector plasmid, sau ct bng enzyme hn ch thch hp v in di kim tra trn agarose gel. Sng lc cc khun lc sn xut protein dung hp. 2.4. Tch chit cc protein dung hp sn xut khng th Protein dung hp c th c tch chit theo mt s cch: dng dch chit urea, sc k i lc aminophenylthiogalactoside, in di sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) hoc phi hp gia cc

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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.
EcoRI Vng to dng

5000

Pst I
r

Amp

1000 4000

pUR278
(5,2 kb)
ori lacZ

Plac
3000
lac UV5

2000

Vng to dng
lacZ pUR278 TGT CAA AAA GGG GAT CCG TCG ACT CTA GAA AGC TTA TCG ATG BamHI Sal I XbaI HindIII ClaI pUR288 TGT CGG GGA TCC GTC GAC TCT AGA AAG CTT ATC GAT GAT BamHI SalI XbaI HindIII ClaI pUR289 TGT CAG GGG ATC CGT CGA CTC TAG AAA GCT TAT CGA TGA BamHI SalI XbaI HindIII ClaI pUR290 TGT CAA AAA GGG GAT CCG TCG ACC TGC AGC CAA GCT TAT CGA TGA BamHI SalI PstI HindIII ClaI pUR291 TGT CGG GGA TCC GTC GAC CTG CAG CCA AGC TTA TCG ATG BamHI SalI PstI HindIII Cla I pUR292 TGT CAG GGG ATC CGT CGA CCT GCA GCC AAG CTT ATC GAT BamHI SalI PstI HindIII ClaI

Hnh 9.10. Cc vector h pUR. y l cc vector dung hp vi gen lacZ, c cc v tr to dng BamHI, SalI, PstI, XbaI, HindIII v ClaI u 3 ca gen lacZ. S chn vo ca on DNA ngoi lai (cDNA) trong cc v tr to dng thch hp cho php sn xut protein dung hp ca -galactosidase hot ng vi chui peptide c m ha bi DNA ngoi lai.

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3. Xc nh mc biu hin ca gen c to dng Ni chung, c ba cch thng c dng xc nh mc biu hin protein ngoi lai ca gen c to dng: - in di polyacrylamide gel c SDS xc nh protein c kch thc thch hp c sn xut mc cao trong cc t bo mang vector biu hin. Thng thng, protein quan tm c th quan st bng cch nhum gel vi Coomassie Brilliant Blue hoc bng AgNO3. Nu khng thy c bng protein mi khi dng cc thuc nhum ny, th nh du s trao i cht vi 100 Ci ca [35S]Met hoc [35S]Cys trn 1 mL dch nui cy trong 5 pht. Sau , s dng k thut SDS-PAGE hoc phng x t ghi c th pht hin c protein quan tm. - Phn tch Western blot bng cch dng cc khng th lin kt c hiu vi protein quan tm c thm tch ln mng nitrocellulose sau khi thc hin in di SDS-polyacrylamide gel. - Nu mc biu hin thp th nn t gen lacZ cng hng vi gen c biu hin. Nh vy, nu s phin m hoc dch m hn ch biu hin th nhng thay i trong h thng biu hin c th c kim sot bng nhng thay i trong hot tnh ca -galactosidase. Phn tch Western blot - K thut SDS-PAGE L k thut in di trn polyacrylamide gel vi s c mt ca SDS cho php phn tch cc phn t protein c khi lng khc nhau. SDS c in tch m rt ln v c kh nng lin kt vi mch peptide. Nh vy, s lng SDS tng tc vi protein t l vi kch thc phn t protein v in tch ca SDS bm vo c th lm bt c phn t protein no cng chuyn ng trong in trng t cc m sang cc dng. Do , bng phng php in di, c th phn tch ring bit cc phn t protein c khi lng phn t khc nhau. - Phn ng lai khng nguyn-khng th Phn ng khng nguyn-khng th c tnh c hiu rt cao. V vy, c th p dng phn ng ny pht hin s c mt v tinh sch protein. Khng th (antibody) c sn xut khi a khng nguyn vo ng vt th nghim (th, chut) v c tinh sch t mu ng vt sau khi gy
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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

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l gen khng khng sinh v cc t bo bin np sinh trng trn mi trng cha khng sinh tng ng.

1. Gn khng nguyn ln b mt 2. Ngn cc v tr trng bng protein khng c hiu 3. khng th th nht c hiu vi khng nguyn 4. khng th th hai c gn enzyme lin kt vi khng th th nht 5. B sung c cht 6. Phn ng to mu ch ra s hin din ca khng nguyn c hiu Phn ng lin kt khng nguyn-khng th

ELISA

SDS gel

Western blot

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

Ngoi ra, cc vector cn cha cc gen ch th b sung phn bit cc t bo bin np cha on chn ca DNA ngoi lai vi cc t bo cha cc vector t ti to li vng. V d: gen lacZ m ha enzyme -galactosidase. Mt dng (clone) cha cc chui DNA quan tm c bit c th c xc nh bi lai khun lc hoc vt tan. Mt lng nh khun lc bin np hoc vt tan c chuyn ln mng nitrocellulose hoc nylon bng cch ph
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(overlay) mng ny ln trn a agar. DNA c bin tnh v c nh trn mng bng un nng (baking) hoc chiu tia cc tm (UV-crosslinking), v sau c lai trong m cha probe nh du ng v phng x c trnh t b sung mt phn ca chui c xc nh. V d: C th mt oligonucleotide tng hp nhn to, c ngun gc t genomic DNA tng phn, cDNA hoc trnh t protein hoc sn phm PCR. Trong mt vi trng hp, probe c th c thit k da trn trnh t bt ngun t gen tng ng ca cc loi khc v thng c lai vi cng lc thp. Cc probe tha c ra khi mng vi phim X-quang. Theo hng ca phim (sau khi ra) so snh vi a agar gc, sau s c kh nng gn kt cc khun lc thc t vi cc khun lc lai dng tnh tng ng trn phim X-quang. 2. Sng lc th vin gen bng PCR PCR (polymerase chain reaction) cng c th c ng dng sng lc cc th vin genomic DNA hoc cDNA c xy dng trong cc plasmid vector hoc bacteriophage vector. u im chnh ca sng lc bng PCR so vi sng lc da trn lai truyn thng l t tn thi gian hn. Sng lc bng PCR c th c m bo trong 3-4 gi trong khi n c th mt mt vi ngy trc khi pht hin bng k thut lai phn t (molecular hybridization). K thut sng lc bng PCR th hin ch th v kch thc ca cc on chn DNA c to dng hn l trnh t ca on chn, tuy nhin cc primer PCR c trng cho on chn DNA ngoi lai cng c th c s dng. iu ny cho php kho st chnh xc hn c im ca cc dng t th vin cDNA v genomic DNA. Nguyn tc ca PCR PCR l mt k thut c s dng ph bin trong cng ngh sinh hc hin i v ng gp rt ln cho nhng tin b v sinh hc phn t, nh du mt b cc enzyme hn ch v k thut Southern blot. in vitro Sinh hc phn t

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20 nucleotide. Taq polymerase l mt loi enzyme DNA polymerase chu nhit (c vi khun chu nhit cao Thermus aquaticus) c dng tng hp cc on DNA mi trong mi 4 loi deoxyribo

, s lng

DNA 35).

u to ra cc on DNA c chiu di xc nh. Cc primer thn (synthetic oligonucleotide) c khong 10-20 nucleotide hoc di hn. Nu bit trnh t ca on gen cn khuch i th c th tng hp nhn to cc primer tng ng thc hin PCR v tch chng ra bng k thut in di. , qua t 10-6 g DNA ban u c th khuch i (amplification) ln ti trn 1 bao gm ba giai on c nhit khc nhau: - Gy bin tnh (denaturation) 90-95oC. Trong giai on bin tnh phn t DNA khun mu dng xon kp c tch thnh hai si n (single strands). - Gn primer (annealing) 40-65oC. Trong giai on ny cc primer gn vo cc v tr c trnh t tng ng DNA khun mu. t on nh (cc primer ) v trn cc i (khun mu v primer) enzyme Taq polymerase c th bt u qu trnh sao chp khun mu. - Ko di phn t (extension) 70-72oC. y l khong nhit ti thch cho Taq polymerase tin hnh tng hp DNA bt u t cc v tr c primer theo chiu 5 3.

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DNA

DNA polymerase Primers

Chui ch

Nucleotides: dATP dCTP dGTP dTTP

Nhit gy bin tnh tch hai si DNA

Primer

Lm lnh cc primer to lin kt hydrogen

Chu k 1 to ra 2 phn t

DNA polymerase b sung cc nucleotide vo u 3 ca mi primer

Chu k 2 to ra 4 phn t

Chu k 3 to ra 8 phn t

Hnh 9.12

(PCR)

3. Sng lc cc th vin cDNA bng cc probe khc nhau

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. .

ia in vitro . . . . in vitro

bin

S thoi bin (suy bin) ca m di truyn (degeneracy): Bnh thng c th c 64 t hp b ba m ha khc nhau ca 4 nucleotide trong khi ch c 20 amino acid ph bin v mt amino acid li c th c nhiu b ba m ha.
3

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acid quan tm (probe). Tro

gel 9.13).

DNA + enzyme hn ch

Cc on ct hn ch I 1 II III

Mng ny lon (hoc nitrocellulose) Gel Vt xp thm nc Dung dch kim

Giy thm

Chun b cc on ct hn ch

in di Giy thm c tch ra Ra tri cc probe khng c gn I 1II III

Thm tch

Phim

DNA c nh du phng x lm probe Lai vi probe nh du phng x

Phng x t ghi

. 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. Sinh hc phn t
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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.

VI. Cc ng dng ca cng ngh DNA ti t hp

Cng ngh DNA ti t hp hin nay c rt nhiu ng dng trong i sng. Cc ng dng ny bao gm sn xut dc phm v cc loi ha cht khc, cc vi khun c bit, cc cy trng nng nghip quan trng, v cc vt nui c bin i di truyn... K thut ny cng c s dng rng ri trong cc xt nghim y khoa v (trong mt vi trng hp) ang c dng kim tra cc khim khuyt di truyn ngi. Hng trm cng ty hin nay ang c bit pht trin cc sn phm thng qua bin i di truyn cc c th sng. Di y l cc ng dng chnh ca cng ngh DNA ti t hp. 1. ng dng trong dc phm Sn phm thng mi u tin c pht trin bng cng ngh DNA ti t hp l cc dc phm dng trong iu tr cc bnh v cc ri lon ngi. Nm 1979, T hp Eli Lilly bt u sn xut thng mi insulin ngi bng cng ngh DNA ti t hp. Trc thi im ny, tt c insulin dng trong iu tr bnh tiu ng c tch chit t ty ca cc vt nui cho tht. Mc d ngun insulin ny c tc dng tt cho nhiu ngi mc bnh tiu ng, nhng n vn khng phi l insulin ngi, v mt s ngi tri qua cc phn ng ph vi protein ngoi lai. Gen insulin ngi sau c chn vo plasmid v chuyn vo vi khun sn xut insulin ngi. Cc dc phm c sn xut thng qua cng ngh DNA ti t hp bao gm: hormone sinh trng ngi (cho tr em mc bnh ci), nhn t to t huyt (cha bnh

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kh ng mu), hot t plasminogen m (dng ha tan cc huyt khi trong bnh nhn b nhi mu c tim), interferon, interleukin, DNA vaccine 2. Cc vi khun c bit Cc vi khun c vai tr quan trng trong cc qu trnh cng nghip, bao gm sn xut ethanol t cc nguyn liu thc vt, lc khong t qung, x l nc thi v cc loi cht thi khc. Cc vi khun ang s dng trong cc qu trnh ny c bin i di truyn bng cng ngh DNA ti t hp sao cho chng c th hot ng hiu qu hn. Cc chng vi khun mi hu ch thu c t k thut hin i ny ang c pht trin phn hy cc ha cht c v cc cht gy nhim, tng cng thu hi du, tng nitrogen cho cy trng, v c ch sinh trng ca cc vi khun v vi nm gy bnh. 3. Cc sn phm nng nghip Cng ngh DNA ti t hp cng c mt nh hng quan trng n sn xut nng nghip, ni m hin nay n c dng to ra cc cy trng v vt nui mang cc c im c gi tr. Trong nhiu nm, cc nh bnh hc thc vt tha nhn rng thc vt b nhim bnh virus th nh sau s khng li s nhim trng cc chng mang c tnh mnh. V vy, cc nh di truyn hc to ra tnh khng virus trong cy trng bng cch chuyn cc gen v protein ca virus vo t bo thc vt. Cy b (squash) bin i di truyn c tn l Freedom II, mang cc gen ca virus 2 gy bnh khm da hu (watermelon mosaic virus 2) v virus gy bnh khm mu vng cy zucchini (zucchini yellow mosaic virus) chng s nhim trng virus. Mt hng khc c bin i di truyn l tnh khng su (pest) thc vt lm gim s ph thuc vo cc thuc tr su ha hc. c t protein t vi khun Bacillus thuringiensis git mt cch chn lc cc u trng ca cc su b cn trng nht nh nhng li khng gy c cho s sng ca cc ng vt hoang d, ngi v cc loi cn trng khc. Gen c t c phn lp t vi khun, lin kt vi promoter hot ng sau c chuyn vo trong ng, c chua, khoai ty v bng. Gen ny sn xut c t i vi cn trng trong cy, v su bm khi n cy b cht. Cng ngh DNA ti t hp cng cho php pht trin tnh khng cht dit c thc vt. Mt vn quan trng trong nng nghip l iu
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khin cc c di cnh tranh nc, nh sng v cht dinh dng vi cy trng. Mc d cht dit c c tc dng git c di, nhng chng cng c th gy nguy him cho cy trng. Cc gen cung cp tnh khng cho nhiu loi cht c di c chuyn vo c chua, u tng, bng, cy ci du v cc cy trng thng mi quan trng khc. Khi ng rung c canh tc cc cy trng ny c phun thuc dit c, th c di b git nhng cc cy trng bin i di truyn li khng b nh hng. Nm 1999, c hn 21 triu ha cy u tng bin i di truyn v 11 triu ha cy ng chuyn gen c trng trn th gii. Cng ngh DNA ti t hp cng c ng dng cho cc vt nui. V d: gen sn xut hormone sinh trng c phn lp t gia sc v c to dng trong E. coli, cc vi khun ny sn xut mt lng ln hormone sinh trng ca b, yu t lin quan n tng sn xut sa. Cc ng vt chuyn gen c pht trin mang gen m ha cho cc sn phm dc liu. V d: mt gen ngi m ha cho nhn t VIII (hnh thnh t huyt) c lin kt vi vng iu ha ca gen sn xut -lactoglobulin cu, mt protein to sa. Gen dung hp c tim vo phi cu, to ra mt con cu chuyn gen c kh nng sn xut trong sa ca n nhn t ng mu ca ngi. Mt phng thc tng t c dng chuyn gen 1-antitrysin, mt protein dng iu tr bnh nhn kh thng di truyn (hereditary emphysema), vo trong cu. Cu ci mang gen ny sn xut rt nhiu 1-antitrysin khong 15g trong 1 lt sa ca chng. 4. Thuc oligonucleotide Mt ng dng cng ngh DNA ti t hp trong thi gian gn y l pht trin cc thuc oligonucleotide c trnh t phn t DNA hoc RNA nhn to ngn c th c dng iu tr bnh. Cc antisense oligonucleotide b sung cho cc RNA khng mong mun, nh l RNA ca virus. Khi c b sung vo t bo, th cc antisense DNA ny lin kt vi mRNA ca virus v c ch s dch m ca chng. Cc DNA oligonucleotide si n lin kt cht ch vi cc trnh t DNA khc, to thnh mt phn t triplex DNA. S to thnh triplex DNA cn tr lin kt ca RNA polymerase v cc protein khc cn cho s phin m. Cc oligonucleotide khc l cc ribozyme, cc phn t RNA m chc nng nh l cc enzyme. Nhng phc hp ny lin kt vi cc phn t
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mRNA c hiu v ct chng thnh tng on, ph hy kh nng m ha protein ca chng. Mt s thuc oligonucleotide sn sng th nghim trong iu tr bnh AIDS v ung th. 5. Liu php gen i vi loi bnh di truyn, bnh do t bin gen ngi ta phi cn n s can thip ca liu php gen, mt hng cha bnh gn lin vi cc k thut tin tin trong lnh vc cng ngh sinh hc hin i nh cc vi thao tc gen, sa i thay th gen... Liu php gen (gene therapy) thc cht l phng php cha bnh bng gen. C nhiu khi nim khc nhau v liu php gen, nhng cch hiu chung nht l tp hp cc bin php s dng cc gen cn thit (cn gi l gen tr liu) nhm mc ch cha bnh cho con ngi. Trong , gen tr liu c th l: - Cc gen hot ng bnh thng (gen lnh) c th a vo t bo thay th gen hng, gen mt chc nng, khi phc hot ng bnh thng ca t bo v s sng ca c th. - L nhng gen c kh nng m ha mt protein c hiu, khi a vo t bo sng c th to nn cc loi protein c hiu. Cc loi protein c hiu c th c ch hot ng ca mt gen khc trong t bo, km hm kh nng phn chia ca t bo hoc gy cht cc t bo b bnh. - Nhng gen khi a vo t bo hot ng ng thi vi cc gen bnh (gen b t bin trong t bo) lm hn ch tc ng ca gen bnh hoc h tr, b p cho cc gen b hng. - Gen tr liu cn l cc gen bt hot c a vo t bo thay th cho mt gen lnh no , nhm hn ch sn phm khng cn thit ca gen lnh hoc to ra cho t bo mt trng thi mi, c tc dng chng li bnh tt. - Gen tr liu c th l nhng on oligonucleotide c tc dng km hm hot ng ca gen b hng, b t bin trong t bo. 6. Chn on bnh can thip sm Bng k thut chc i, kt hp ng thi phn tch mu b m bng enzyme hn ch, ngi ta c th chn on sm trc khi sinh (v enzyme hn ch c kh nng phn bit c gen t bin vi gen bnh thng). Cc on DNA ct ra, c phn tch qua phng php in di, em lai vi cc
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mu d DNA hoc RNA nh du bng ng v phng x 32P hoc hunh quang. nh phng x t ghi hoc tn hiu hunh quang cho ta thy cc on DNA c lai vi cc mu d. Cc on ny c tch ra nghin cu v xc nh t bin hay bnh thng bng enzyme hn ch, v mt s t bin di truyn c nh hng n cc v tr dnh cho enzyme hn ch. ng dng cc k thut sinh hc phn t trong lnh vc ny c nhiu thun li hn cc phng php kinh in. Th nht, c th rt ngn c thi gian nh ng dng k thut PCR, chng hn chn on bnh HIV-1, dng phng php PCR ch mt mt ngy so vi 3-4 tun nui cy ca phng php truyn thng. Th hai, i vi cc tc nhn gy bnh kh nui cy hay khng th nui cy th k thut sinh hc phn t l gii php duy nht, chng hn Chlamydia, Brucella, vim gan B,... Th ba, vic to dng mt gen dng lm probe n gin hn vic sn xut khng th c hiu, hn na vi mt probe ngi ta c th pht hin tt c cc kiu huyt thanh (serotype) ca tc nhn gy bnh, iu m ch mt khng th khng th lm c. Cui cng, nu trc kia phi cn n nhiu k thut (quan st di knh hin vi, nui cy trn mt lot mi trng, min dch hc...) th hng chn on bng sinh hc phn t ch cn mt k thut (lai phn t hoc PCR). i vi tc nhn l virus, cc k thut lai phn t v PCR cho php chn on nhiu nhm nh papillomavirus, HBV, HIV. c bit phng php lai ti ch cn cho php xc nh trc tip virus trn lt ct sinh thit. i vi cc tc nhn vi khun, c nhiu b kit chn on sinh hc phn t c thng mi ha nh: Mycobacterium tuberculosis, Mycobacterium pneumoniae, Salmonella, Streptococcus pneumoniae, Haemophilus influenzae... i vi cc tc nhn k sinh trng, ngi ta cng thnh cng trong chn on Plasmodium, Schistosoma, Trypanosoma, Toxoplasma, Leishmania bng lai phn t. Phng php PCR cn cho php pht hin tc nhn ngay trong vt trung gian truyn bnh. 7. DNA fingerprinting S dng cc trnh t DNA nhn dng mt ngi, c gi l DNA fingerprinting, l mt cng c rt mnh kho st ti phm v cc ng dng php l khc.
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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
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a hnh trong cc kiu ca cc on DNA c to ra khi phn t DNA c ct cng mt enzyme hn ch. Nu DNA t hai ngi c ct bi cng mt enzyme v kiu ca cc on DNA khc nhau c to ra, th hai ngi ny phi c s khc nhau v cc trnh t DNA ca h. Nhng s khc nhau ny c di truyn v c th c dng trong lp bn , tng t vi phng thc m trong s khc nhau v allele c dng lp bn cc gen thng thng. Theo truyn thng, bn gen c da vo vic s dng cc sai khc di truyn to ra nhng sai khc kiu hnh c th quan st d dng. ng tic l do hu ht cc tnh trng b nh hng bi a gen v mi trng, nn s lng cc tnh trng c c s di truyn n gin thch hp cho vic s dng bn b gii hn. RFLP cung cp mt s lng ln cc marker di truyn c th c dng lp bn gen ca sinh vt.

Ti liu tham kho/c thm

1. Dale JW and Von Schanzt M. 2002. From Gene to Genome. John Wiley & Sons, Ltd. West Sussex, UK. 2. Erlich HA. 1989. PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, USA. 3. Glick BR and Pasternak JJ. 2003. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 3rd ed. ASM Press, USA. 4. Lewin B. 2000. Gene VII. Oxford University Press, Oxford, UK. 5. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory, USA. 6. Primrose SB, Twyman R and Old RW. 2001. Principles of Gene Manipulation. 6th ed. Blackwell Science, Oxford, UK. 7. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook. Humana Press Inc. New Jersey, USA. 8. Surzycki S. 2000. Basic Techniques in Molecular Biology. SpringerVerlag, Berlin, Heidelberg, Germany. 9. Walker JM and Rapley R. 2000. Molecular Biology and Biotechnology. Chapman & Hall Limited, London, UK.

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Ph lc

Mt s thut ng c bn
Adenine (A). . Adenosine diphosphate (ADP). Mt ribonucleoside 5-diphosphate c cu to t adenine, ng ribose (5C) v hai gc phosphate. ADP c tc dng nhn phosphate trong chu trnh nng lng ca t bo. Adenosine triphosphate (ATP). Mt ribonucleoside 5-triphosphate c cu to t adenine, ng ribose (5C) v ba gc phosphate. ATP l phn t cha nng lng ha hc chnh ca t bo, (chloroplast). Cc gc phosphate ca ATP c mang cc lin kt khi b thy phn s phng thch mt nng lng t do ln.

. Allele. Mt trong nhiu dng khc nhau ca mt gen chim mt locus xc nh trn nhim sc th.

. Amber codon. . Amino acid. L mt phn t nh mang mt gc amine (-NH3) v mt gc carboxyl (-COOH) lin kt vi cng mt nguyn t carbon. Amino acid
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l n v cu trc c s ca chui polypeptide. C 20 amino acid khc nhau trn cc chui polypeptide . Aminoacyl-tRNA (aminoacyl-tRNA). Mt ester gia aminoacyl vi tRNA tng ng ca amino acid . AMP vng (cyclic AMP, cAMP). L mt phn t AMP m trong gc phosphate c lin kt vi v tr 3 v c 5 ca ng ribose. cAMP l thng tin th hai trong t bo. S to thnh cAMP (xc tc bi enzyme adenylate cyclase) c kch thch bi mt s hormone. Ampicillin (Amp). . Antisense. Trnh t khng m ha ca mt phn t DNA si i. Chui cy (chui ngc chiu) b sung vi mRNA v lm khun mu cho s tng hp mRNA. Antisense molecule. . t ch (epistasis). Tc dng tng h gia cc gen khi mt gen cn tr s biu hin kiu hnh ca mt gen khng alelle vi n. ATP-ase. Enzyme thy phn ATP thnh ADP v phosphate, thng phi hp vi s bin i nng lng. ATP-synthetase. Enzyme tng hp ATP t ADP v phosphate trong qu trnh phosphoryl oxy ha (oxidative phosphorylation) mng trong ca ty th. Baculovirus. . Bn ct hn ch (restriction map). Trnh t cc v tr nhn bit (recognition sites) ca tt c cc enzyme hn ch (restriction enzyme hay restriction endonuclease, RE) trn mt phn t DNA. (genetic map).

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hay

. (restriction map). Xem bn ct hn ch. (analog base).

-C.

Baz nit (nitrogen base).

nn thng tin d

Bt cp b sung (complementary base pairing). S kt hp thnh tng i gia cc nitrogen base nm trn hai mch n ca chui xon kp DNA-DNA, DNA-RNA hoc RNA-RNA thng qua cc mi lin kt hydrogen. S bt cp mang tnh c hiu: guanine bt cp vi cytosine, cn adenine bt cp vi thymine trn DNA hoc uracil trn RNA. Bin i hu dch m (post-translational modification). S thay i cc lin kt ha tr xy ra trong chui polypeptide, sau khi chui polypeptide tch khi ribosome v trc khi tr thnh protein hot ng thc s. Bin i mRNA (mRNA processing). Qu trnh to thnh mRNA hon chnh (mature mRNA) hot ng chc nng t sn phm phin m s cp l mRNA tin thn cha hon chnh (pre-mRNA). Trong qu trnh ny cc on intron b loi b v cc on exon c ni li vi nhau.
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Bin np (transformation). L qu trnh truyn DNA ngoi lai vo mt t bo nhn, chng hn sphaeroplast hoc protoplast, v c th hp nht trong nhim sc th nh s ti t hp tng ng hoc c bin i trong mt n v sao chp t tr (autonomous replicon). S bin np c th xut hin trong cc iu kin t nhin mt s vi khun (v d: Bacillus, Haemophilus, Neisseria v Streptococcus), nhng nhiu vi khun (v d: E. coli) v cc c th sinh vt eukaryote s bin np ch c th xut hin nhng t bo thm c DNA bng cc phng php nhn to nh: ha bin np, in bin np... Bin tnh (denaturation). L hin tng chuyn t dng mch kp sang dng mch n ca DNA v RNA thng do nhit gy nn. Bin tnh ca protein l hin tng chuyn t cu hnh hot ng thnh dng khng hot ng. Biu hin ca gen (gene expression). L cc qu trnh phin m (transcription) v dch m (translation) ca mt gen to ra sn phm protein ca n. (backbone). .

Cm ng (induction). Lin quan n kh nng tng hp mt s enzyme (sn phm ca gen) vi khun ch khi c mt c cht (substrate) ca cc enzyme ny trong mi trng. Khi s dng cho khi nim iu ha biu hin ca gen, t ny c ngha l s khi ng qu trnh phin m do tng tc gia mt cht cm ng (inducer) vi protein iu ha. V d: E. coli, ). CAP (catabolite gene activator protein). Protein hot ha gen d ha. Mt protein iu ha c bit, kim sot s khi u phin m cc gen sn xut cc enzyme cn thit cho mt t bo, c th s dng nhng thc n khc khi khng c mt ca glucose. Cp baz (base pair, bp). L lin kt A-T hoc C-G trn mt phn t DNA mch kp, v l n v o chiu di ca mt phn t DNA. Cu trc bc mt (primary structure). Bao gm trnh t cc amino acid v nhng cu ni disulfide trong chui hoc gia cc chui
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polypeptide. trng hp nucleic acid, l trnh t ca cc nucleotide trn mt chui DNA hoc RNA c ni vi nhau nh lin kt phosphodiester. Cu trc bc hai (secondary structure). L s ni lin nhng amino acid t u n (u tn cng N) ti u kia (u tn cng C) ca mt phn t protein. Cng nh vy, trn mt trnh t nucleic acid l s ni lin nhng nucleotide t u n (u 5) ti u kia (u 3) ca mt chui DNA. Cu trc bc ba (tertiary structure). Cu trc ba chiu dng cun xon ca phn t protein hoc chui polynucleotide. Cu trc bc bn (quaternary structure). Cu trc ba chiu ca mt protein c nhiu tiu n v (subunit), theo kiu m nhng tiu n v kt hp khp vi nhau. Cu disulfide (disulfide bridge). Lin kt ng ha tr to thnh gia hai chui polypeptide qua trung gian ca mt gc cystine. Chc ba sao chp (replication fork). Cu trc hnh ch Y hnh thnh khi phn t DNA mch kp dn xon l ra hai si n lm khun mu cho tng hp DNA (s ti bn). Cht cm ng (inducer). Mt hp cht ha hc

. Cht d nhim sc (heterochromatin). Bao gm cc vng ca h gen thng xuyn trng thi c c v khng biu hin di truyn. Trng thi ny c th vnh vin hay tm thi. Cht nhim sc (chromatin). Phc hp DNA v protein trong nhn giai on ngh ca qu trnh phn bo. Trong giai on ny khng th phn bit tng nhim th ring l. Tn gi ny bt ngun t phn ng vi cc mu nhum c trng cho DNA ca phc hp. Cht nhim sc tht (euchromatin). Cht nhim sc cha nhng gen c phin m. Cht c ch (repressor). Sn phm protein ca mt gen c ch

.
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Chromosome walking. K thut ny dng lp bn nhim sc th t tp hp cc on DNA ct hn ch chng ln nhau (overlapping). Bt u t mt th vin trong cha cc on DNA ni trn c to dng. Mt on DNA mang mt gen bit c la chn v s dng nh mt probe nhn dng (v d: bng cch lai khun lc) cc on khc, l cc on chng ln nhau cha cng mt gen. Sau , trnh t nucleotide ca cc on ny s c phn tch v nh vy c th xc nh c ton b cc on ca nhim sc th. T , bn ca mt vng c bit s c xy dng dn dn. Chu trnh sinh tan (lylic cycle). Mt kiu chu trnh sng ca thc khun th (bacteriophage) khi n xm nhim vi khun, iu khin cc hot ng sinh sn v sinh trng bng cc gen ca n v sinh ra cc bacteriophage th h con . C (lysogenic cycle). L hin tng h gen ca bacteriophage hin din trng thi n nh v khng sinh tan trong t bo vt ch sng ca n. Cc t bo vt ch c th tip tc sinh trng v phn chia, v s sao chp ca h gen bacteriophage (prophage) c phi hp vi nhim sc th ca vt ch sao cho khi t bo phn chia th prophage cng c chuyn vo trong c hai t bo con. Prophage c duy tr bng cch hoc hp nht trong nhim sc th vt ch (v d: bacteriophage , bacteriophage 105) hoc nh l mt plasmid bn ngoi nhim sc th (v d: bacteriophage P1 v bacteriophage F116). T bo vt ch c th hoc khng th biu hin ra mt kiu hnh bin i. Chui contig (contiguous sequence). (overlapping). (concatemer). . Chui m ha (coding sequence). on phn t DNA mang m di truyn xc nh phin m thnh mRNA v sau dch m thnh chui polypeptide. (transgenic). (foreign DNA) bng cc k thut khc nhau (Agrobacterium, vi tim, bn gen, xung in...) vo mt c th vt ch (vi sinh vt, ng vt hoc thc vt).
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Cistron. Thut ng c, l mt n v ca DNA tng ng vi mt gen . Cordycepin. . Corepressor. . Cosmid. cos . Cytosine (C). Base pyrimidine c trong DNA. Trn DNA mch kp cytosine bt cp vi mt base purine l guanine (G). D-loop. . Deoxyribonucleotide triphosphate (dNTP). triphosphoryl ha ( ). Deoxyribonuclease (DNase). hy) DNA si i hoc DNA si n. Deoxyribonucleic acid (DNA). ,t (phn

nucleotide

(U), .

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. Nm 1962, Watson (M) v Crick (Anh) chia s Gii Nobel vi Wilkins (Anh) v pht minh ra cu trc khng gian ca DNA v ngha ca n trong vic truyn thng tin di truyn. iu ng tic l Franklin, ngi c nhng ng gp ng k cho pht minh ny mt trc . Theo qui nh th Gii Nobel khng dc php tng cho ngi mt. Deoxyribose. Phn t (heteropolymer). . (heterologous). . (nick translation). [ - P]dCTP nh enzyme DNA polymerase I ca E.
32

coli. (translation). rn khun mRNA. Qu trnh chuyn thng tin di truyn trong trnh t base ca mRNA sang trnh t amino acid ca chui polypeptide trong t bo cn gi l qu trnh sinh tng hp protein. (hybrid-arrested translation). trong s dch m ca mt phn t mRNA c bit (trong h thng dch m in vitro) b ngn chn do s hnh thnh on lai ca mRNA vi mt phn t RNA (antisense RNA) hoc DNA (v d: cDNA) b sung. Dch m ngc (reverse translation). L k thut phn lp cc gen nh kh nng ca chng trong vic lai vi mt on m oligonucleotide no , on ny c chun b bng cch d on on m nucleic acid t nhng on m ha ca protein bit trc. Dideoxyribonucleotide triphosphate (ddNTP). (sequencing). Dimer. . DNA (complementary DNA, cDNA). trn khun mu mRNA nh qu trnh
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transcription) : trn mRN

chu xy dng th vin cDNA (cDNA library). (foldback DNA). (inverted repe . DNA khun mu (template DNA).

. DNA polymerase. . Nm 1959, hai nh khoa hc ngi M l Kornberg v Ochoa c nhn Gii Nobel v nhng nghin cu lm sng t c ch c bn ca qu trnh sao chp DNA lin quan n DNA polymerase I. DNA replicase. Phc hp enzyme v protein c hiu, cn thit cho s ti bn DNA. DNA siu xon (supercoiled DNA). DNA xon li trn bn thn n, thng l kt qu ca s gp khc, m xon hoc xon li ca chui xon kp DNA. (satellite DNA). L nhng on DNA mang cc trnh t lp

. Dng (clone). Tp hp cc t bo hoc phn t ging ht nhau cng bt ngun t mt t bo hay phn t ban u.

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(germ line). . Dot blot. L k on mi DNA c nh du ng v phng x. a cistron (polycistronic). . (restriction fragment length polymorphism, RFLP). Tnh a hnh chiu di cc on ct hn ch ch cc sai bit di truyn v tr nhn bit ca cc enzyme hn ch (chng hn nh do s thay i mt nucleotide) dn n s sai bit trong chiu di ca cc on hnh thnh t phn ng ct hn ch DNA vi cng mt enzyme. RFLP thng c dng thit lp bn di truyn vi mt s marker di truyn bit trc. i phn t (macromolecule). Mt phn t c khi lng khong t vi nghn ti nhiu triu. (end labelling). 4 polynucleotide kinase. li DN . u tn cng C (C terminus). Gc c chui polypeptide. u tn cng N (N terminus). Gc amine (NH2) v tr tn cng ca mt phn t protein hoc chui polypeptide. Tt c cc polypeptide u c tng hp bt u t u ny, n gc carboxyl tn cng. (nick). mt si n trn DNA si i. im nng (hot spot). V tr ca gen c xu hng b t bin nhiu hn hn so vi cc v tr khc trn phn t DNA. (blunt end). 5 (protruding ends). (cohesive ends hoc sticky ends).

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in di trn gel (gel electrophoresis). K thut dng phn tch cc phn t nucleic acid hoc protein da vo s dch chuyn ca chng trn gi th dng gel (agarose hoc polyacrylamide) di nh hng ca in trng. S dch chuyn ca cc phn t ny ph thuc vo in tch, cu hnh, kch thc v khi lng phn t ca nucleic acid hoc protein cng nh dung mi v nng ca cht dng lm gi th. iu ha hot ng gen (gene operation regulation). iu khin s hot ng ca cc gen trong qu trnh pht trin c th, m bo cho cc gen hot ng thi im cn thit, v do vy qu trnh pht trin c th din ra bnh thng. M hnh operon ca Monod v Jacob l c ch iu ha hot ng gen n gin nht. nh m (capping). Gn nucleotide guanine (G) c methyl ha (ci m) vo u 5 ca phn t mRNA tin thn (pre-mRNA) sinh vt eukaryote, ci m sau c gi li phn t mRNA hon chnh (mature mRNA). on duy nht (unique sequence). Cc on DNA ch c mt hoc mt vi bn sao trong h gen. (Klenow fragment). 76.000) ca E. coli 5 3.

on lp li o ngc (inverted repeats). . (direct repeats). . on Okazaki (Okazaki fragment). Cc on DNA si n tng i ngn ln lt c tng hp trong qu trnh sao chp (ti bn) DNA theo kiu gin on dc theo mt trong hai si khun, trong khi dc theo si khun kia th mch DNA mi c tng hp lin tc. Sau , cc on Okazaki c ni lin vi nhau bng cc mi lin kt ng ha tr nh enzyme ligase. (nested fragments). Mt lot cc on nucleic acid c chiu di ch khc nhau mt hoc mt vi nucleotide.
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ging nhau (palindrome). on DNA mch kp c trnh t sp xp cc base trn hai mch n ging ht nhau nu cng c c theo mt chiu (chng hn 5 3). V d: cc on nhn bit ca enzyme hn ch. ng du (replica plating). Phng php chuyn nguyn mu cc khun lc hoc vt tan t mt a thch gc sang a thch mi bng cch dng mng nylon (v d: mng Hybond-N+) va kht p ln mt thch ca a gc dnh ly cc t bo trong cc khun lc (colony) hoc vt tan (plaque) ca a gc, ri a mng ny p ln mt thch mi. n cistron (monocistron). Mi mRNA monocistron ch m ha cho mt protein eukaryote. , n c th di hn mt gen. n v sao chp (replicon). on DNA bt u t im khi u sao chp ko di v hai pha ti hai im kt thc sao chp. n v ti t hp (recon). on DNA ca gen c chiu di ngn s trao i cho khng th din ra bn trong n c na. Hin nay, c bit l mt cp nucleotide. (virulence). . i m (anticodon). L mt on gm ba nucleotide trn tRNA bt cp vi cm m (codon) trn mRNA theo nguyn tc b sung. ng polymer (homopolymer) (nucleic acid). (mutation). . t bin dch khung (frameshift mutation). t bin lm mt hoc thm mt cp base trong gen dn ti hu qu lm dch khung dc bnh thng ca mRNA i mt nucleotide. Do vy, lm thay i ton b thng tin di truyn on gen bt u t im xy ra t bin n cui gen. t bin ng hon (transition mutation). Kiu t bin thay th mt cp base purine-pyrimidine ny bng mt cp base purine-pyrimidine
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khc ti mt im trn DNA. V d: thay cp GC bng cp AT hoc ngc li. t bin gen (gene mutation). Cn gi l t bin im, xy ra do nhng bin i ca tng nucleotide nh: mt, thm hay thay i v tr ca nucleotide trn phn t DNA. L nguyn nhn ca s hnh thnh protein khng bnh thng. Phn ln t bin l c hi nhng chng vn thng c gi li trong qun th v thng l ln v do c th c duy tr trong kiu gen m khng nh hng n sc sng ca sinh vt. Tc t bin t nhin thp, nhng tn s t bin c th tng nh cc tc nhn nh phng x ion ha v cc ha cht gy t bin gy ra. t bin khuyt dng (auxotrophic mutation). Cn gi l t bin ha sinh. t bin lm mt kh nng tng hp mt cht cn thit cho s sinh trng ca t bo. t bin l r (leaky mutation). t bin ch lm gim hot tnh enzyme m khng lm mt hn hot tnh . t bin ngc (reverse mutation hay reversion). Cn gi l hi bin. t bin xy ra theo chiu t dng t bin tr v dng bnh thng, c tc dng phc hi trnh t nucleotide ban u. t bin nhm ngha (missense mutation). t bin gen khi mt cp base trong b ba (codon) b thay th bi mt base khc, dn n s thay th amino acid v tr tng ng trn chui polypeptide bng mt amino acid khc. t bin c ch (suppressor mutation). t bin xy ra mt v tr trn DNA c tc dng loi b hon ton hoc tng phn hu qu ca t bin ban u xy ra mt v tr khc. t bin v ngha (nonsense mutation). t bin gen khi mt base trn DNA b thay th bng mt base khc lm cho b ba tng ng trn mRNA t ch xc nh mt amino acid chuyn thnh b ba v ngha (khng xc nh amino acid no), mang tn hiu chm dt qu trnh dch m. V d : amber codon (t bin amber, amber mutation) hay ochre codon (t bin ochre, ochre mutation). Kt qu l chui polypeptide ch c tng hp n b ba xy ra t bin v ngha th dng li.

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ui polyA (polyA tail). on trnh t di 50-200 nucleotide adenine c b sung vo u 3 ca hu ht cc mRNA eukaryote sau khi phin m. E. coli (Escherichia coli). Vi khun thng c trong rut non ca ng vt c xng sng. E. coli c coi nh sinh vt mu cho vic nghin cu hot ng ca t bo. y l vi khun Gram m c kch thc genome khong 4106 base-pair. Cc qu trnh biu hin gen (phin m v dch m) i i vi nhau, sinh ra si mRNA c tng hp mi v c s dng ngay cho qu trnh dch m. Khng c hin tng bin i sau dch m (post-translation). V th, E. coli c xem l mt trong nhng t bo vt ch n gin nht. Rt nhiu th nghim to dng gen ang c thc hin hng ngy ti cc phng th nghim u s dng E. coli lm vt ch vi nhiu chng khc nhau v mt di truyn v cho nhng ng dng c bit. Endopeptidase. Enzyme xc tc ct lin kt peptide trong chui polypeptide. Endonuclease. L e . Nuclease thy phn nhng lin kt phosphodiester gia cc nucleotide ca mt phn t nucleic acid. Cc nuclease c th c hiu i vi DNA (deoxyribonuclease) hoc c hiu i vi RNA (ribonuclease). Enzyme. . Enzyme ng phn ha (isomerase). Enzyme xc tc cho s chuyn ha mt hp cht thnh mt ng phn v tr. Enzyme gn DNA (DNA ligase). (restriction enzyme, RE). . Enzyme hn ch c pht hin vo nm 1970, chng tn ti trong t bo vi khun, c tc dng ct DNA ngoi lai (v d: DNA ca phage) ti nhng im xc nh, tiu dit DNA ny. Cho n nay hn 900 enzyme hn ch c tm thy. Cc enzyme hn ch c s dng rng ri trong cc
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phng th nghim thao tc gen nh nhng chic ko ct DNA ti nhng im c hiu. V tr ct ph thuc vo loi enzyme hn ch c la chn. Nm 1978, Arber (Thy S), Nathans (M) v Smith (M) c nhn Gii Nobel nh pht minh ra enzyme hn ch v nhng ng dng ca chng gii quyt nhiu vn quan trng ca di truyn hc phn t. Cc enzyme ny l nhng chic ko phn t c th ct DNA thnh nhng on xc nh, m ra mt thi k pht trin mi ca sinh hc hin iThi k thao tc gen. (reverse transcriptase). in vitro. Enzyme ton phn (holoenzyme). Enzyme hot ng gm tt c cc tiu n v cn thit, cc nhm ngoi v nhng cofactor. Episome. Nhn t di truyn c trong vi khun, c kh nng t sao chp trong t bo cht, ng thi li c th xen vo nhim sc th chnh ca vi khun v sao chp ng thi vi nhim sc th. Exon. Cc on DNA trong gen c chc nng phin m. Exon tn ti c sinh vt prokaryote v eukaryote. Ring sinh vt eukaryote cc exon nm xen k vi cc on intron. Cc intron chim ti 90% tng s DNA ca t bo eukaryote v khng c chc nng phin m. Eukaryote. Sinh vt c t bo mang nhn in hnh (nhn tht) ngha l nhn c bao bc bi mng nhn v tham gia vo hai c ch phn bo quan trng l nguyn phn v gim phn. Exonuclease. Loi enzyme nuclease ch tc ng vo u tn cng ca phn t nucleic acid, ct ra tng nucleotide mt theo thi gian. Chng c th chuyn ha theo u 5 hoc 3 ca si DNA. Ex vivo. Thut ng dng ch cc th nghim thc hin trn t bo nui cy, cc t bo ny sau s c a vo mt c th sng. -galactosidase. Enzyme c m ha bi gen lacZ. Enzyme ny thy phn lactose thnh glucose v galactose. Gy t bin nh hng bng oligonucleotide (oligonucleotidedirected mutagenesis). Cn gi l gy t bin nh hng im (sitedirected mutagenesis), l qu trnh to ra mt bin i xc nh trn DNA
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bng cch s dng mt oligonucleotide tng hp (primer) c mt vi bin i trong trnh t nucleotide. Gel chm (gel retardation). Phng php xc nh im bm ca protein trn cc on DNA, da vo di chuyn chm ca chng so vi DNA khng b protein bm trong cc th nghim in di trn gel. Gen (gene). L n v di truyn, yu t quyt nh mt tnh trng c th. Thng tin di truyn ca cc gen c m ha trong DNA quyt nh tnh bin d ca loi v ca c th. DNA l mt chui bao gm cc n v nucleotide, c bn loi nucleotide mang bn nitrogen base khc nhau l adenine (A), guanine (G), cytosine (C), v thymine (T). Trnh t cc nucleotide ca mt gen xc nh mt polypeptide hoc mt RNA. Gen c kh nng b t bin. Cc gen ch yu nm dc theo nhim sc th trong nhn t bo. Mi gen chim mt v tr xc nh trn nhim sc th gi l locus. Gen c th tn ti nhiu dng gi l cc allele. Cc gen biu hin thng qua cc phn t do chng sinh ra l RNA (trong qu trnh phin m) v protein (trong qu trnh dch m). Gen cu trc (structural gene) hay gen m ha (coding gene). L trnh t m ha sn phm protein. Gen ch th (reporter gene). L mt gen m ha m sn phm ca n c trc nghim mt cch d dng (v d chloramphenicol acetyltranferase), n c th c gn vi bt c mt promoter no sao cho s biu hin ca gen ny c dng th nghim chc nng ca promoter. Gen iu ha (regulatory gene). Mt gen m sn phm protein ca n tham gia vo s iu ha biu hin ca mt gen khc. V d: gen m ha mt protein km hm. Gen gy cht (death gene). Gen lm cht c th mt giai on no trong qu trnh pht trin ca chng. Gen nhy (transposon). L mt on DNA nh c cu trc c bit nn c kh nng di chuyn t mt v tr ny n mt v tr khc trn phn t DNA hay t mt nhim sc th ny sang mt nhim sc th khc trong t bo lng bi. Gen nhy ch chuyn n nhng v tr xc nh trong h gen. Trong nhiu trng hp gen nhy c th gy t bin ti v tr n di chuyn n. Gen nhy c th dng lm phng tin gy t bin nh hng. .
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Nm 1983, McClintock (M) nhn Gii Nobel nh khm ph ra cc nhn t di truyn vn ng (gen nhy) trn i tng cy ng. Gen c ch (repressor gene). C hai chc nng: (1) L mt gen iu ha (regulator) m sn phm ca n l mt protein c tc dng c ch hot ng phin m ca mt operon. (2) L mt gen phc hi (suppressor) kiu hnh ban u ca t bin trong mt gen khc. Ghp i lch (mismatch). S ghp i khng ng vi quy lut b sung gia cc nucleotide thuc hai si n DNA trong mch kp. Ghp exon hay splicing (RNA). Qu trnh ct b nhng intron v ni cc exon ca sn phm phin m ban u (tin thn mRNA) to thnh mRNA hon chnh (mature mRNA). Qu trnh bin i ny xy ra trong nhn t bo. Gi thuyt mt gen-mt enzyme (one gene-one enzyme hypothesis). Gi thuyt da trn cc cng trnh ca Beadle v Tatum v di truyn hc ha sinh, theo mi gen kim sot s tng hp mt enzyme. Gim phn (meiosis). S phn bo tri qua hai giai on ca mt t bo lng bi (2n) dn n s hnh thnh cc giao t n bi (1n) hoc cc bo t hu tnh mang mt na vt cht di truyn ca t bo lng bi ban u. Gim phn I (meiosis I). Ln phn chia th nht ca gim phn lm gim s lng nhim sc th i mt na v to ra cc t hp nhim sc th mi. Ln phn chia ny gm bn giai on: pha u, pha gia, pha sau v pha cui. Gim phn II (meiosis II). Ln phn chia th hai ca gim phn dn n s phn tch cc nhim sc t v hon thnh qu trnh gim phn ni chung. Gc ti bn (origin, ori). Trnh t nucleotide hoc v tr trn DNA m bt u s ti bn (sao chp). Guanine (G). Mt trong nm nitrogen base c trong thnh phn ca DNA v RNA, bt cp vi cytosine (C) trong chui xon kp nh ba lin kt hydrogen. Helicase. Enzyme xc tc vic tho xon ca chui xon kp DNA trong qu trnh sao chp E. coli.
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H gen hay b gen (genome). L tp hp cc gen c trong mt t bo n bi eukaryote, trong mt t bo prokaryote hoc trong mt virus.

. Hiu ng glucose (glucose effect). Hin tng kh hot tnh ca operon cm ng vi khun khi c mt glucose k c khi hin din cht cm ng operon. Histone. Nhm protein kim nm trong phc hp vi DNA cc nhim sc th eukaryote v ng vai tr quan trng trong vic xc nh cu trc ca nhim sc th. Hon chuyn gen (gene conversion). . Hot tnh c hiu (specific activity). L hot phng x trn mt n v nguyn liu, chng hn: mt mu d (probe) nh du phng x c th c hot tnh c hiu 106 ln m/pht trn microgram. Hot tnh c hiu cng c dng xc nh hot ca enzyme. Hi tnh (renaturation). S ti bt cp ca hai mch n DNA b sung ca mt phn t DNA mch kp. Hp CAAT (CAAT box). on DNA nm ngc hng cch im bt u phin m khong 75 nucleotide hin din sinh vt eukaryote. y l mt trong nhng on tng cng s bm ca RNA polymerase. Hp Goldberg-Hogness (Golderg-Hogness box) hay hp Hogness (Hogness box) hay hp TATA (TATA box). on DNA c trong cc promoter eukaryote tng t hp Pribnow c trong cc sinh vt prokaryote, nm cch v tr bt u phin m khong 30 nucleotide, c coi l operator sinh vt eukaryote, c trnh t ph bin ca si bn l TATAAAT.

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Hp Pribnow (Pribnow box). Mt phn ca promoter trong cc h gen prokaryote, nm cch pha trn im khi u phin m 10 nucleotide. Trnh t ca si tng ng vi hp Pribnow l TATAAT. In du DNA (DNA fingerprinting) hay in du di truyn (genetic fingerprinting). L phng php dng cc mu d phng x hoc dng k thut PCR nhn dng cc bng DNA c cc on lp li vi tn s cao. Bn mu hnh cc bng DNA l duy nht i vi mi c th, v do vy c th dng xc nh c trng c th hoc quan h huyt thng. In du chn DNA (DNA footprinting). Phng php nhn dng cc vng DNA m cc protein iu ha bm vo. Intron. Nhng on DNA nh sinh vt eukaryote khng mang thng tin m ha amino acid, phn b ri rc dc theo phn t DNA. Sau khi thng tin t DNA c phin m sang mRNA th cc intron trn mRNA b ct b, cc on mRNA cn li gm ton cc exon c ni li vi nhau v chuyn n ribosome dng lm khun mu cho qu trnh dch m. Intron khng thy c sinh vt prokaryote. In vitro v in vivo. (in vitro (in vivo in silico. Isozymes hay isoenzymes. Nhm cc enzyme cng xc tc mt phn ng nhng khc nhau v cu trc s cp hoc tc in di, allozymes . Ko di on mi (primer extension). S tng hp mt bn sao nucleic acid bt u t on mi. c s dng nh du phng x on DNA lm mu d hoc khuch i mt on DNA bng k thut PCR. Khong cch bn (map distance). S trao i cho trung bnh xy ra gia hai locus gen trn nhim sc th hoc trn si DNA. Khuch i (amplification). S sn xut nhiu bn sao ca mt trnh t DNA. Khung c m (open reading frame, ORF). L mt trnh t m ha chui polypeptide c bt u vi m khi u (initiator codon) v kt
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thc bng mt m dng (stop codon). Mt khung c m b ngn chn nu mt stop codon c nh v gn vi m khi u. Mc d v l thuyt , tuy nhin trong thc t khung c chnh xc c xc nh bi mt im bt u c nh. Khuyt on (deletion, deficiency). t bin nhim sc th dn n lm mt mt on vt cht di truyn v thng tin di truyn cha trong n ri khi nhim sc th. Kiu gen (genotype). Cu trc di truyn ca c th. Hin trng ca c th (kiu hnh) ph thuc vo quan h tri-ln gia cc allele trong kiu gen v vo mi tng tc gia kiu gen v mi trng. Kiu hnh (phenotype). Ngoi hnh ca mt sinh vt, l kt qu tng tc gia kiu gen vi mi trng. Nhiu gen c trong kiu gen nhng khng biu hin ra kiu hnh v chng b che khut bi cc allele tri. Nhng sinh vt c cng kiu gen c th c cc kiu hnh rt khc nhau trong nhng mi trng khc nhau. Kiu hnh ca c th ng hp t v allele ln gi l kiu hnh ln. Kiu hoang di (wild type). Dng thng thy nht ca mt gen trong qun th hoang di. Allele kiu hoang di c k hiu bng mt ch in hoa hoc thm du cng sau ch vit thng, v d: A hay a+. Allele kiu hoang di thng l tri v cho kiu hnh bnh thng. Kiu nhn (karyotype). B nhim sc th c trng ca mt loi vi s lng, kch thc v hnh thi xc nh ca cc nhim sc th. Mi loi c c trng bi kiu nhn ring ca n. Cc loi khc nhau c kiu nhn khc nhau. Kilobase (kb). 1000 base (hoc cp base), c dng nh n v o hoc xc nh chiu di ca cc phn t DNA hoc RNA. Kinase. Cc enzyme xc tc phn ng phosphoryl ha mt phn t nhn nh ATP. Lai (hybridization). Ghp cc phn t nucleic acid tch bit li vi nhau thng qua cc mi lin kt hydrogen gia cc base b sung.

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Lai khun lc (colony hybridization). .

in situ

Lai phn t (molecular hybridization). Qu trnh trong hai mch nucleic acid b sung (A-T, G-C) bt cp hnh thnh nn mt mch kp. y l mt k thut hu ch pht hin mt trnh t nucleotide chuyn bit. Lai ti ch (in situ hybridization). Qu trnh bt cp gia mu d (l mt trnh t DNA si n hay RNA) vi DNA ca t bo c c nh trn lam knh. Lp bn hn ch (restriction mapping). K thut dng xc nh v tr cc im ct hn ch trn phn t DNA. Lin kt peptide (peptide bond). Mi lin kt ng ha tr trong chui polypeptide, ni nhm -carboxyl ca mt amino acid vi nhm amine ca mt amino acid k tip. Lin kt phosphodiester (phosphodiester bond). Mt phn t cha hai alcol, ester ha vi mt phn t phosphoric acid lm cu ni gia chng vi nhau. V d: chui polynucleotide c ni bi lin kt 5 3 phosphodiester gia hai nucleotide cnh nhau. Linker. Mt oligonucleotide tng hp c hai u bng, trc E. coli to u bng. Cc linker sau khi gn vi hai u bng ca on cDNA nh DNA ligase s c ct hn ch to ra u so le tng ng vi hai u ca vector. Phn ng gn gia on cDNA c mang linker hai u vi vector cng c xc tc nh DNA ligase. Locus. V tr m gen nh v trn nhim sc th. LTR (long-terminated repeat). . Lysosome. Mt bo quan c mng bao bc trong t bo cht ca nhng t bo eukaryote. Lysosome cha nhiu enzyme thy phn. L thuyt trung tm (central dogma). Thuyt cho rng thng tin c truyn theo mt chiu t DNA n RNA v n protein.

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Mapping and sequencing gene. cc nucleotide. M di truyn (codon). Nhm ba nucleotide nm k nhau (b ba) trn phn t mRNA xc nh mt amino acid trn chui polypeptide, hoc l tn hiu kt thc vic tng hp polypeptide. M thoi bin (degenerate codon). M di truyn m mt amino acid c quy nh bi mt s b ba nitrogen base, ch khng phi ch bi mt b ba. Thoi bin l c im vn c ca m di truyn tn ti ph bin sinh gii. (nonsense codon) hay m dng (stop codon).

(UGA) Maturation. Qu trnh trong cc mRNA va c phin m tri qua mt s bin i ha hc tr thnh mRNA hon chnh sn sng lm khun mu cho vic tng hp protein. My m nhp nhy (scintillation counter). My dng xc nh hot tnh phng x trong mt mu th nghim. Mu d DNA (DNA probe). Mt on RNA hay DNA chuyn bit c nh du bng ng v phng x hay bng ha cht (cht pht hunh quang hoc enzyme), dng nh v mt trnh t nucleic acid nht nh thng qua k thut lai phn t (xem Northern blot, Southern blot...) Monomer. L cc phn t n v nh, c th lin kt vi cc phn t n v ging n hnh thnh nhng phn t ln hn (polymer). V d: cc nucleotide l cc monomer ca nucleic acid v cc amino acid l monomer ca protein. M hnh bo ton. M hnh sao chp DNA m theo hai si DNA b m vn c gi nguyn v lm khun tng hp chui xon kp mi (chui con). Mi (primer). Mt trnh t DNA hay RNA ngn, bt cp vi mt mch ca DNA khun mu v c mang mt u 3-OH t do gip DNA polymerase bt u tng hp mt chui DNA mi.

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

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Nhn t khi u (initiation factor). prokaryote vit tt l IF, cn eukaryote l eIF. L cc protein kt hp vi tiu n v ca ribosome vo giai on khi ng ca qu trnh dch m. Nhn t Rho (Rho factor). Mt protein c vai tr kt thc s tng hp mt s mRNA. Nhim sc th (chromosome). Mt thnh phn ca t bo, bao gm cc protein v mt phn t DNA c nht. DNA cha nhiu gen, c chc nng d tr v truyn thng tin di truyn. S lng nhim sc th trong mt t bo c th thay i t 1 ( vi khun) cho ti trn 100, nhng khng thay i mt loi nht nh (v d: 46 i vi ngi). Nhim sc th thng (autosome). Tt c nhim sc th tr nhng nhim sc th gii tnh hoc nhim sc th ca ty th. Nhim sc t (chromatide). Hai chui song song v ging nhau, ni vi nhau bi phn tm ca mt nhim sc th dng n c nhn i, thc th ny tn ti cho n khi bt u k sau (anaphase). Northern blot. K thut chuyn v c nh RNA t formaldehyde agarose gel (sau khi c phn on bng in di) ln mng lai bng nylon hoc nitrocellulose lai vi mu d (probe) c nh du ng v phng x [ -32P]dCTP hoc digoxigenin-dUTP. Tn hiu lai sau c pht hin trn phim X-quang (trng hp [ -32P]dCTP) hoc trn mng lai (trng hp digoxigenin-dUTP). Ni thc bo (endocytosis). S xm nhp ca cht ngoi t bo vo trong t bo. Nuclease S1. Enzyme thy phn DNA si n. Nucleic acids. Nhng polynucleotide sinh hc thin nhin, trong nhng n v nucleotide c kt hp vi nhau bi nhng lin kt phosphodieste thnh trnh t DNA hoc RNA ring bit. Nucleolar organizer. . Nucleolus. . Nucleoside. Mt hp cht gm mt base purine hoc pyrimidine kt hp ng ha tr vi mt phn t ng pentose.

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Nucleosome. n v cu trc ca nhim sc cht, gm mt on DNA c kch thc khong 200 bp qun bao quanh mt li tp hp 8 protein histone. Nucleotide. Mt nucleoside phosphoryl ha vi mt trong nhng hydroxyl ca pentose. Phn t ng vai tr cu trc c s ca DNA v RNA, gm ba phn: ng pentose (ribose trong RNA, deoxyribose trong DNA), nitrogen base v gc phosphate. Nucleolytic. . Nuclease Bal 31. Mt loi enzyme exonuclease thy phn c hai si ca phn t DNA cng mt lc. Oligo. Tip u ng c ngha l mt t, v d: oligonucleotide (polynucleotide) c mt t nucleotide hoc oligopeptide (polypeptide) c mt t peptide. Oligo(dT)-cellulose. Mt on ngn gm cc gc deoxythymidine lin kt vi c cht cellulose, c s dng tinh sch mRNA eukaryote. Oligomer. Thut ng chung ch mt on ngn cc monomer. Oligonucleotide. Mt on ngn cc monomer l nucleotide, thng t 20-30 nucleotide. Operon. Nhm cc gen vi khun chu s iu khin chung ca mt gen iu ha. Cu trc operon bao gm: 1) Nhm gen cu trc m hot ng ca chng chu s iu khin chung, 2) Gen iu ha sn sinh ra protein iu ha, 3) Vng ch huy (operator) v trnh t khi ng (promoter) chu tc ng ca protein iu ha. Protein iu ha c tc dng bm vo vng ch huy lm cho n b ng, do vy nhm gen cu trc ngng hot ng. Khi trong mi trng xut hin c cht ca cc gen cu trc th c cht ny bt hot ha protein iu ha khin n khng bm vo vng ch huy c na, vng ch huy c m v nhm gen cu trc li hot ng. Operon chu c ch (repressible operons). Cc operon sinh tng hp chui amino acid b c ch khi mt cht (v d: chui amino acid ) c a vo mi trng. Orche codon.
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Opal codon.

n ha (temperate). Trng thi tim tan ca cc bacteriophage t bo vt ch. -peptide. Mt phn ca protein -galactosidase, c m ha bi on gen lacZ. Periplasm. Phn ngoi bo tng nm gia lp mng ngoi v mng trong ca vi khun. Peroxisome. Bo quan c trong t bo cht eukaryote cha enzyme xc tc cho s to thnh v phn hy peroxide (H2O2). Phage. Vit tt ca bacteriophage (thc khun th), l loi virus xm nhim v sinh sn bn trong vi khun. Phage thng c v bc protein, phc hp bao gm phn u c hnh a din cha nucleic acid (DNA hoc RNA) v ui m qua nucleic acid xm nhp vo vi khun ch. Sau qu trnh nhn ln ca nucleic acid ca phage, t bo vi khun ch thng b tan bin. Loi phage lun lun lm tan t bo vi khun khi chng xm nhim vi khun gi l phage c. V d: phage T4. Ngc li, cn c phage n ha, khi xm nhim vi khun n gy nn phn ng tim tan, ngha l h gen ca phage gn vo nhim sc th vi khun v c sao chp cng vi nhim sc th . H gen ca phage trng thi gn nh vy vi nhim sc th vi khun gi l prophage. Phagemid. L mt loi plasmid vector c mang cc on trnh t ca phage. Phn ng chui polymerase (polymerase chain reaction, PCR). Phng php dng trong phng th nghim khuch i cc on DNA c bit ln hng triu ln trong vng vi gi thng qua 20-30 chu k nhit, mi chu k bao gm ba mc nhit : bin tnh 90-95oC, bt cp vi mi 40-65oC hoc hn v tng hp mch mi nh DNA polymerase chu nhit (Taq polymerase) 70-72oC. PCR c ng dng rng ri trong chn on y hc, phn tch s a dng sinh hc, chn ging v trong nhiu lnh vc khc. Nh khoa hc M (Tin s Mullis) ngi pht minh ra k thut PCR nhn gii Nobel nm 1993. Cng chia s Gii Nobel vi Mullis l Smith (Canada) do c nhng ng gp mang tnh nn tng cho vic gy t bin
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im nh hng, da trn cc oligonucleotide v vic pht trin chng trong cc nghin cu protein. (condensation reaction). . Phn ng thy phn (hydrolysis). Phn ng trong mt lin kt cng ha tr b ph v khi c s kt hp mt phn t nc. Pht sinh t bin (mutagenesis). Qu trnh to ra t bin trong DNA. Phn tch trnh t gen (gene sequencing). L k thut xc nh trnh t theo cu trc bc mt ca chui cc nucleotide trong mt phn t nucleic acid. Phn tch trnh t ca DNA c cc phng php ha hc ca MaxamGilbert v phng php enzyme ca Sanger. Trong nhng nm gn y, mt s phng php xc nh trnh t mi nh s h tr ca my tnh xut hin. Bn cnh k thut thng thng s dng cc polyacrylamide gel phn ly cc phn t DNA c di khc nhau, cc k thut mi lin quan n pht hin hunh quang ca cc nucleotide c nh du, phn tch trnh t DNA bng khi ph, in di mao dn hoc lai vi cc on oligonucleotide c tng hp nhn to cng ra i. Nm 1980, Sanger (Anh) v Gilbert (M) c trao gii Nobel do c nhng ng gp quan trn v phng php xc nh trnh t cc nucleotide trong phn t DNA. ng gp ny l mc lch s to ln trong sinh hc phn t, l nguyn l ca tt c cc my xc nh trnh t DNA t ng ang s dng hin nay trn khp th gii. Phn cui (telomere). on cui, phn cui ca mt nhim sc th thng ca eukaryote, bao gm nhng trnh t DNA ngn c lp li nhiu ln. Phn tm (centromere). Phn co tht c thy trn nhim sc th k gia, l ni hai nhim sc t nh vi nhau. Phin m (transcription). Qu trnh tng hp mRNA t khun mu DNA. Phin m ngc (reverse transcription). Qu trnh tng hp DNA t khun mu mRNA nh enzyme phin m ngc (reverse transcriptase).

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Phng x t ghi (autoradiography). K thut pht hin cc phn t c nh du phng x thng qua hiu ng to nh ca cc phn t ny trn phim X-quang. Phosphoryl ha (phosphorylation). Phn ng to thnh mt dn xut phosphate ca mt phn t sinh hc di tc dng xc tc ca mt enzyme. Phosphatase kim (alkaline phosphatase). Enzyme loi b cc nhm 5-phosphate t u ca cc phn t DNA v li cc nhm 5hydroxyl. Phi t (ligand). Mt phn t hoc mt ion kt hp vi mt protein, v d: hormone c th kt hp vi th th (receptor) c hiu ca n. Plasmid. c cu trc mch vng kp, nm trong t bo cht v ngoi nhn, c kh nng sao chp c lp i vi nhim sc th ca t bo. Tn ti c sinh vt prokaryote v eukaryote. Ngy nay, cc plasmid thit k nhn to c s dng rng ri nh l cc vector dng trong cc k thut to dng v biu hin gen. Polynucleotide. Trnh t nhng nucleotide ni ng ha tr vi nhau, trong v tr 3 ca pentose ca mt trong nhng nucleotide c ni vi mt lin kt phosphodieste v tr 5 ca pentose ca nucleotide tip theo. Polypeptide. Mt chui di nhng amino acid ni vi nhau bi nhng lin kt peptide. Polysome (polyribosome). Cu trc gm nhiu ribosome kt hp vi mRNA trong qu trnh dch m. Nhng polysome t do trong t bo cht, tng hp nhng protein ni bo. Nhng polysome kt hp vi mng ngoi ca li ni sinh cht, tng hp nhng protein dng cho s tng hp mng hoc c bi tit ra ngoi. Primase (protein Dna G). L mt dng ca RNA polymerase v l sn phm ca gen Dna G. Trong vi khun, primase lin kt vi DNA helicase to thnh mt phc hp gi l primosome. Primase c hot ha bi DNA helicase nh n s tng hp mt RNA primer ngn xp x 11 1 nucleotide, enzyme DNA polymerase s ko di RNA primer bng cch b sung cc nucleotide mi. V th, primase l mt nhn t quan trng trong ti bn DNA bi v nu khng c n DNA polymerase khng th khi u s tng hp mt si DNA mi.

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Prokaryote. Sinh vt n bo khng c nhn t bo in hnh, DNA nm trong t bo cht khng c mng bao bc, khng c nguyn phn v gim phn; i din in hnh l vi khun. Prophage. Phage n ha xen vo nhim sc th ca vi khun tim tan. N sao chp ng thi vi nhim sc th ca t bo vi khun ch. Protein. Mt phn t ln gm mt hoc nhiu chui polypeptide, mi chui c mt trnh t amino acid v mt khi lng phn t c trng. Protein l hp cht quan trng bc nht i vi c th sng. V cu trc, protein l phn t mch di gm cc n v cu trc nh l cc amino acid ni vi nhau qua mi lin kt peptide. Khi lng phn t ca protein t vi nghn n vi triu. C khong 20 loi amino acid. Cc loi protein phc tp hn c lin kt thm vi cc nhm b sung. Protein dung hp (fusion protein). L mt protein ti t hp lai c m ha bi mt gen lai (fusion gene) do s dung hp in vitro cc on gen khc nhau trn plasmid vector v sau bin np vo vi sinh vt ch (chng hn E. coli). V vy, protein dung hp s mang trnh t amino acid ca hai protein khc bit c tng hp t u N ca vector biu hin. Protein khng histone (non-histone). Protein acid lin kt vi DNA trong cc nhim sc th eukaryote. Cn gi l protein khng kim. Protein nguyn th (native protein). L mt protein ti t hp c m ha bi mt gen ngoi lai (foreign gene) trong vi sinh vt ch. Khc vi protein dung hp, protein nguyn th c tng hp t u N ca n ch khng phi t u N ca vector. Protein si (fibrous protein). Nhng protein khng tan, c chc nng bo v hoc cu trc, cha nhng chui polypeptide c cu trc bc hai. Purine. Mt hp cht d vng, kim, c nitrogen, l thnh phn ca nhng nucleotide v nucleic acid. Purine cha mt nhn pyrimidine kt hp vi mt nhn imidazol. Purmycin. Mt loi khng sinh c tc dng c ch s tng hp polypeptide bng cch gn vo chui polypeptide, cnh tranh vi aminoacyl-tRNA gn ln v tr ca ribosome, v nh vy to ra s kt thc sm tng hp chui polypeptide.

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Pyrimidine. Mt nitrogen base d vng c trong cc nucleotide v nucleic acid. Pyrophosphate. Phn t hnh thnh bi hai gc phosphate ni vi nhau bng lin kt anhydride. Pyrophosphatase. Enzyme thy phn mt pyrophosphate hu c thnh hai phn t orthophosphate. Qu trnh phn tch v tinh sch u ra (downstream processing). Giai (bioprocessing). Cc k thut thng c s dng trong b . Quang ti hot ha (photoreactivation, light repair). Mt cch sa cha cc pyrimidine dimer. Cc dimer hi bin trc tip tr li dng ban u di tc dng ca nh sng bnh thng bc sng 320-370 nm. Quang hp (photosynthesis). Qu trnh s dng nng lng ca nh sng to thnh carbohydrate bt u t CO2 v mt tc nhn kh. Replisome. Mt phc hp a enzyme c tc dng khi ng s ti bn DNA chc ba ti bn. Retroposon. Loi transposon hot ng di dng RNA; trc ht DNA c phin m thnh RNA, RNA ny sau c phin m ngc thnh DNA v gn xen vo mt v tr mi trn h gen. Retrovirus. L loi virus RNA cha enzyme reverse transcriptase v sinh sn di dng DNA mch kp. Chng c kh nng xm nhim t bo vt ch cao. Khi xm nhim n c kh nng gn h gen ca virus vi h gen ca t bo vt ch, l c s thit k cc vector liu php gen hiu qu. Ribonuclease. Enzyme xc tc c hiu vic phn hy RNA bng cch ct cc mi lin kt phosphodiester trn RNA. Ribonucleic acid (RNA). Thng l phn t a phn mch n gm cc n v cu trc c s l ribonucleotide. V mt ha hc RNA rt ging vi DNA. RNA l v .

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Ribonucleotide. n v cu trc c s ca RNA, gm ba thnh phn: ng ribose, nitrogen base v nhm phosphate. Ribosome.

. Ngi ta cng thy ribosome trong ty th, c s tng hp mt s protein ty th. Ribozyme . RNA b sung (complementary RNA). . RNA kch thc nh ca nhn (small nuclear RNA, snRNA). Ngoi . RNA ligase. . RNA polymerase. . RNA ribosome (ribosomal RNA, rRNA). L

E. coli

. RNase. Enzyme thy phn RNA. RNA thng tin (messenger RNA, mRNA). Mt loi RNA c phin m t mt trnh t DNA. mRNA truyn thng tin di truyn t nhim
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sc th ti ribosome

otein trn ribosome. (transfer RNA, tRNA)

. 10 sequence. . 35 sequence. . Sn phm phin m s cp (primary transcript). Sn phm phin m ban u ca cc gen eukaryote cn c gi l mRNA tin thn (premRNA), thng c kch thc rt ln. N phi c x l (splicing) to ra phn t mRNA hon chnh (mature mRNA) dng cho dch m. Sng lc (screening). K thut nhn dng mt dng DNA trong mt th vin h gen (genomic library) hoc th vin cDNA (cDNA library) bng mt phng php lai mu d c nh du [ -32P]dCTP vi cc vt tan (trng hp dng bacteriophage lm vector to dng v cho xm nhim vo vi khun E. coli) hoc khun lc (dng plasmid lm vector to dng) ca cc th vin trn mng nylon hoc nitrocellulose. Tn hiu lai c pht hin bng phng x t ghi trn phim X-quang. Sinh hc phn t (molecular biology). Khoa hc nghin cu cc hin tng sng mc phn t. Lnh vc khoa hc tr tui ny l im gp nhau ca cc khoa hc kinh in nh di truyn hc, ha sinh hc, t bo hc, vt l hc, ha hc hu c v ha l. Theo cch hiu ph bin hin nay, sinh hc phn t l khoa hc nghin cu cc gen v hot ng ca chng

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mc phn t, bao gm phin m, dch m, sao chp, iu ha biu hin gen, ti t hp v chuyn gen... Sinh tng hp protein (protein synthesis). Phn ng ha hc din ra trn ribosome to nn cc phn t protein t cc amino acid trn c s thng tin di truyn nhn c t trong nhn t bo thng qua mRNA. Somatotropin. y l loi hormone c bit, thng c tng hp trong no ng vt v ngi vi mt hm lng v cng thp. Somatostatin c vai tr iu ha hormone s

somatotropin. Southern blot. K thut chuyn v c nh DNA bin tnh t agarose gel (sau khi c phn on bng in di) ln mng lai bng nylon hay nitrocellulose lai vi mu d (probe) c nh du ng v phng x [ -32P]dCTP hoc digoxigenin-dUTP. Tn hiu lai sau c pht hin trn phim X-quang (trng hp [ -32P]dCTP) hoc trn mng lai (trng hp digoxigenin-dUTP). S bn sao (copy number). (1) S cc phn t plasmid c trong mt t bo vi khun. (2) S lng cc bn sao ca mt gen trong h gen ca mt sinh vt. S phng x t ghi (autoradiogram). Hnh nh sinh ra trn phim X-quang do s pht x ca cc ht phng x. Si ch (leading strand). Chui DNA trong qu trnh ti bn c tng hp cng chiu vi hng di chuyn ca chc ba ti bn. Si c ngha (sense strand). Si n trong chui xon kp DNA c dng lm khun mu tng hp mRNA trong qu trnh phin m. Si n DNA th hai khng c dng lm khun mu phin m gi l si i ngha (antisense strand). Si th (lagging strand). Chui DNA trong qu trnh ti bn c tng hp ngc chiu vi hng di chuyn ca chc ba ti bn. Suppressor. C hai loi: (1) Extragenic-thng l mt gen m ha mt tRNA t bin c trn codon t bin, theo ngha ca codon u tin. (2) Intragenic-l t bin c tnh cht b p phc hi khung c m u tin sau khi c s chuyn dch khung.
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Sa cha bng ct b (excision repair, dark repair). Qu trnh sa cha cc pyrimidine dimer trn DNA (do tia t ngoi sinh ra) bng cch ct b cc dimer v tng hp on DNA mi thay th no di tc dng ca cc enzyme c hiu m khng cn n nh sng. Tc nhn t bin (mutation factor). Bt k tc nhn vt l hoc ha hc no lm tng ng k tn s t bin so vi tn s t bin t nhin th u c xem l tc nhn t bin. Cc tc nhn t bin thng dng l cc tia phng x nh tia X, tia gamma, tia t ngoi; cc ha cht nh cc ng ng ca cc nitrogen base, nitrogen acid, hydroxylamine, acridin... Ti bn (replication). S sao chp vt cht di truyn trong chu trnh phn bo hoc s tng hp DNA ca phage khi phage sinh sn trong t bo vi khun. Ti bn DNA kiu bn bo ton (semiconservative DNA replication). Qu trnh sao chp DNA m hai si cha m dn xon v c dng lm khun tng hp hai si mi. Khi sao chp xong th mi chui xon kp con gm mt si c v mt si mi. Ti bn DNA kiu gin on (discontinuos DNA replication). S tng hp DNA thnh nhng on ngn, sau ni li vi nhau to thnh chui polynucleotide di. Ti bn DNA kiu na gin on (semidiscontinuos DNA replication). S tng hp DNA lin tc trn mt si khun mu trong hai si ca DNA mch kp; cn trn si khun mu kia, DNA c tng hp thnh nhng on ngn gi l on Okazaki, sau tt c chng c ni li vi nhau nh ligase to thnh chui polynucleotide di. Ti bn hai hng (bidirectional replication). S ti bn theo hai hng ngc chiu nhau. Ti t hp (recombination). Qu trnh m trong nhim sc th hay phn t DNA t ra ri cc phn t c ni li theo mt t hp mi. Qu trnh ny c th xy ra trong t bo sng (qua s trao i cho trong phn bo gim nhim) hay trong ng nghim nh cc enzyme ct v ni DNA. To dng (cloning). Cn gi l nhn dng, tch dng hay dng ha, l s sn sinh nhiu bn sao ca mt phn t DNA, thng l phn t DNA
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ti t hp trong plasmid vector, bng cch sao chp phn t trong mt vt ch thch hp chng hn vi khun E. coli. Tn s lp li (repetition frequency).

DNA. Terminal transferase. Enzyme b sung cc gc nucleotide vo u 3 ca oligonucleotide hoc polynucleotide. (B lymphocyte). . T bo Hfr (high frequency recombination cell). T bo gii tnh c E. coli, c mang nhn t F gn lin vi nhim sc th vi khun. Khi nhn t F thc y s tip hp ca t bo Hfr vi t bo ci (F ) th cc gen ca vi khun c truyn sang t bo ci vi tn s cao. T bo mm phi (embryonic stem cell). T bo phi cha bit ha, a th chut, c th c nui cy trong mt thi gian di m vn gi c tnh a th, ngha l kh nng bit ha theo nhiu hng pht trin thnh cc t bo khc nhau nh: t bo c tim, t bo thn kinh, t bo gan... Th bin np (transformant). T bo hoc sinh vt nhn c gen ca mt sinh vt khc trong qu trnh bin np v biu hin chc nng ca gen ra kiu hnh. (polymorphism).

(restriction patterns). Th t bin (mutant). Sinh vt (hoc gen) mang t bin di truyn. Th khm (mosaic). Phi hoc c th c cc t bo mang cc h gen khng ging nhau. Th nhn (nucleosome). Cu trc c s ca cc nhim sc th eukaryote gm DNA bao quanh mt li histone. Th nhn c dng ht, quan st thy di knh hin vi in t. Th ti t hp (recombinant). Cc c th hoc t bo mang cc t hp gen khc vi cha m ca chng do cc qu trnh ti t hp di truyn sinh ra.
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Thng tin di truyn (genetic information). Thng tin c lu tr trong cc phn t DNA ca sinh vt dng trnh t sp xp ca bn nucleotide k hiu l A, T, C v G ng vai tr nh nhng ch ci ca ngn ng di truyn. Trong ngn ng ny, mi t ch c ba ch ci gi l mt b ba. Ngha ca mi t l mt amino acid c mt trn phn t protein tng ng. Mi cu ca ngn ng di truyn l mt gen cha ng thng tin di truyn m nhim mt chc nng trn vn. Mi chc nng l mt c tnh sinh l, hnh thi hay cu trc ca s sng. Do c ch sao chp theo kiu na bo ton ca DNA m thng tin di truyn c truyn chnh xc t th h n sang th h kia hu nh khng thay i. Th th (receptor). Protein mng, mt phn nm trong mng t bo, c kh nng gn mt phi t (ligand) mt ngoi ca mng kt qu l gy ra mt bin i hot ng trong t bo cht. (orphan drug).

erythropoetin . Th vin cDNA (cDNA library). Tp hp cc dng DNA c to ra t mRNA ca mt t bo hoc mt m c th trong bacteriophage vector, i din cho thng tin di truyn m cc t bo biu hin. Th vin h gen (genomic library). Tp hp tt c cc on DNA c to ra t phn ng ct hn ch genome trong bacteriophage vector, i din c cho ton b cho thng tin di truyn ca mt h gen. Thymine (T). Base loi pyrimidine c trong DNA nhng khng c trong RNA. Trong mch kp DNA thymine bt cp vi adenine (A). Tnh trng (trait). Mt c im hoc tnh cht bn ngoi ca cc c th sinh vt. Tnh trng a gen (polygenic trait). Tnh trng c xc nh bi mi tng tc gia nhiu gen, chng hn mu mt ngi. Topoisomerases. Nhng enzyme xc tc s thay i nhng dng siu xon m hoc dng ca chui DNA xon kp vng. Trao i cht (metabolism). Cn gi l qu trnh chuyn ha, l ton b nhng thay i trong t bo sng ca nhng phn t hu c nh xc tc
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bi nhng enzyme. S chuyn ha bao gm c qu trnh tng hp ln thoi ha. Trnh t dn u (leader sequence). Mt trong ba phn ch yu ca mt phn t mRNA. Trnh t ny nm u 5 ca mRNA v mang thng tin ribosome v cc protein c hiu nhn bit bt u qu trnh tng hp polypeptide, trnh t dn u khng c dch m thnh trnh t cc amino acid. Trnh t iu ha (regulatory sequence). Mt trnh t ca DNA tham gia vo qu trnh iu ha ca gen. V d: trnh t promoter hoc operator. Trnh t chn (insertion sequence, IS). C vi khun, kch thc nh v tng ng vi transposon, ch mang gen cn cho s gn chn ca chnh n. Trnh t ch huy (operator). on DNA ngn nm u operon, k st promoter ni m protein c ch c th bm vo, c tc dng m hoc ng operon cho cc gen cu trc trong operon hot ng hoc ngng hot ng. Trnh t khi ng (promoter). Trnh t nucleotide c hiu nm trong thnh phn operon, c chc nng iu ha hot ng ca operon, ni RNA polymerase bm vo bt u qu trnh phin m. Trnh t c trng ca promoter c khong 20-200 nitrogen base. Trnh t lp li (repetitive sequence). Cc trnh t base c nhiu bn sao cng mt lc trn phn t DNA. C bn loi trnh t lp li: trnh t lp li n (c mt bn sao), trnh t lp li va (c t 1 n 10 bn sao), trnh t lp li trung bnh (c t 10 n vi trm bn sao) v trnh t lp li cao (c t vi trm n vi triu bn sao). Trnh t lp li ngc chiu (inverted repeat sequence). Mt on ngn ca DNA c lp li, thng cc u ca mt on di hn, c hng ngc chiu. Trnh t lp li xui chiu (direct repeat sequence). on DNA gm mt dy cc trnh t nucleotide c lp li theo cng mt chiu. Trnh t ph bin (consensus sequence). on trnh t thy c trong hu ht cc mu ca mt yu t di truyn c th v c bn vng cao, v d hp CAAT.
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Trnh t Shine-Dalgarno (Shine Dalgarno sequence, SD). Cn gi l vng lin kt ribosome (RBS), l mt phn ca trnh t nucleotide u 5 ca mt mRNA prokaryote c th kt hp b sung cp base vi u 3 ca 16S rRNA, dng lm tn hiu cho s khi u dch m. Trnh t tng cng (enhancer). Trnh t nucleotide dng cis lm tng cng phin m ca promoter trong gen eukaryote. N c th nm cch promoter hng ngn cp base v hot ng theo c hai hng bt k v tr no so vi promoter. Tng ng (homologous). (1) Ni v cc nhim sc th cp i trong cc sinh vt lng bi. (2) Dng m t cc trnh t DNA ging ht nhau mt cch tuyt i; tuy nhin, phn trm tng ng gia cc on lin quan i khi ch tng i. Ty th (mitochondria). Bo quan c mng bao bc, nm trong t bo cht ca cc sinh vt eukaryote. Ty th cha nhng enzyme cn thit cho chu trnh citric acid, vn chuyn electron v qu trnh phosphoryl oxy ha. gn mi (annealing). cch s dng cc dNTP c trong mi trng ko di primer nh s xc tc ca enzyme Taq DNA polymerase (trong khuch i PCR) hoc DNA polymerase I (trong tng hp cDNA). c ch amber (amber suppresser). . Vt ch (host). T bo dng nhn cc phn t DNA ln nhiu ln. Vector. L cc phn t DNA v biu hin gen, (E. coli hoc nm men). C ba nhm vector chnh gm: (1) Nhm plasmid, (2) Nhm phage/phagemid, v (3) Nh

.
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Vector biu hin (expression vector). , v d: E. coli. biu hin cc gen ngoi lai trong E. coli phi bt u bng vic gn n vo trong vector biu hin (thng l plasmid). Vector ny phi c cc cu trc cn thit sau: (1) Cc trnh t m ha gen ch th chn lc (selectable marker) m bo duy tr vector trong t bo. (2) Mt promoter kim sot phin m cho php sn xut mt lng ln mRNA t cc gen c to dng. (3) Cc trnh t kim sot dch m nh vng lin kt ribosome c b tr thch hp v codon khi u ATG. (4) Mt polylinker a gen ngoi lai vo trong mt hng chnh xc vi promoter. Vector to dng (cloning vector). Phn t DNA mch kp c kh nng t sao chp trong t bo vt ch. C th gn vo phn t ny mt on hoc mt vi on DNA khc ngun to nn phn t DNA ti t hp dng nhn dng. Vector thay th (replacement vector hay substitution vector). L mt bacteriophage vector m cc im to dng sp xp thnh cc cp, do vy phn ca h gen nm gia cc im ny c th c thay th bng on DNA chn. Vt tan (plaque). Vng trn trong sut xut hin trn thm c ca cc vi khun mc trn mi trng thch c, do s tan v lp li nhiu chu k ca cc t bo vi khun b bacteriophage xm nhim v sinh tan. V tr cos (cos site). phage . Virus. Phc hp cha nucleic acid (DNA hoc RNA) nm trong mt v bc protein, c kh nng gy nhim v ti bn bn trong t bo vt ch c hiu, to ra nhiu virus, lan truyn t t bo ny sang t bo khc. Virus l dng sng khng c cu trc t bo, c kh nng xm nhp vo cc t bo sng xc nh v ch sinh sn bn trong cc t bo . Ging nh tt c c sinh vt khc, virus c b my di truyn ca ring mnh, m ha vic tng hp cc ht virus t cc cht c trong t bo vt ch. Nh vy, virus l nhng vt k sinh ni bo. Virus phn b khp ni trong t nhin, xm nhp vo tt c cc nhm sinh vt. Ngi ta bit khong 500 loi virus xm nhp ng vt mu nng, 300 loi xm nhp thc vt bc cao. Mt s khi u ung th ng vt v ngi c th do virus. Virus tn ti hai
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dng: dng ngh hay ngoi bo v dng sinh sn hay ni bo. Kch thc ca cc ht virus t 15-350 nm, chiu di ca mt s loi virus c th t ti 2000 nm. Phn ln virus ch nhn thy c qua knh hin vi in t. Cht mang thng tin di truyn ca virus l nucleic acid: DNA hoc RNA. V vy, c th phn virus thnh hai loi: loi mang DNA v loi mang RNA. Vi tim (microinjection). K thut a DNA vo nhn hoc vo t bo cht ca t bo bng kim mao dn v bm p lc. y l (animal cell biotechnology), n i hi thit b vi thao tc (micromanipulator) cc nhy, k nng thc hnh v s kin nhn cao ca k thut vin. VNTR (variable number of tandem repeats). Cn gi l tiu v tinh, ch cc vng ca h gen c trng bi s lp li ca cng mt trnh t DNA. S lng ln lp li thay i theo c th nn mang tnh a allele rt cao. Vng kp tc (hairpin loop). Vng chui n b sung to np gp cha cc cp base to thnh xon kp, . Vn gen (gene pool). Ton b thng tin di truyn c trong tt c cc gen ca mt qun th ti mt thi im xc nh. Vng cng hng (downtream). cp n v tr ca mt on trnh t no nm pha u 3 ca gen hoc mt on gen quan tm. Vng a ni (polylinker hay polycloning sites). Mt trnh t DNA mch kp c tng hp nhn to c mang mt lot cc v tr nhn bit ca cc enzyme hn ch. Trnh t ny c gn vo cc vector dng trong k thut to dng gen (nh vng to dng). Vng lin kt ribosome (ribosome binding sites, RBS). (xem trnh t Shine-Dalgarno). Vng nhn (nucleoid). Mt vng trong t bo vi khun tp trung vt cht di truyn. Vng ngc hng (upstream region). V tr ca mt trnh t nucleotide no nm pha u 5 ca phn t DNA so vi gen quan tm.

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Vng siu bin (hypervariable region, HVR). Vng trong h gen bao gm mt s lng bin ng cc trnh t lp li v c dng xc nh c trng c th (xem in du di truyn). Vng to dng (multiple cloning sites, MCS).

thng dng, c thit k chn on DNA ngoi lai vo y (xem vng a ni). Western blot. K thut phn tch protein da trn nguyn l lin kt khng nguyn-khng th pht hin protein c hiu c bn cht khng nguyn (thng c thm tch ln mng nitrocellulose sau khi chy in di SDS-PAGE, v c nh ). Sau khi protein trn mng lai gn vi khng th th nht c hiu v tip n l khng th th hai c nh du enzyme (alkaline phosphatase hoc horse-radish peroxidase) th phc hp ny s c lin kt vi c cht to mu. S hin din ca protein ngoi lai (sn phm dch m ca gen ngoi lai c chuyn vo t bo vt ch) s c pht hin nh s xut hin mu ca phn ng lai. Xon alpha (helix ). Cu trc xon ca chui polypeptide, thng l quay phi, vi s lng ti a lin kt trong chui, mt trong nhng cu trc bc hai ph bin ca protein c tm thy keratin. Xon kp (double helix). Cu trc ba chiu t nhin ca hai chui DNA b sung, i song v xon vi nhau. YAC (Yeast artificial chromosome). Nhim sc th nhn to ca nm men, c dng lm vector to dng nhng on DNA c kch thc rt ln trong nm men. Yu t ngoi nhim sc th (extrachromosomal element). Phn t DNA khng phi l thnh phn ca nhim sc th t bo vt ch. Yu t tc ng cis (cis-acting element). on trnh t DNA ch biu hin hiu qu trn chnh phn t DNA m n tc ng. V d: hp CAAT l mt phn t tc ng cis i vi qu trnh phin m cc sinh vt eukaryote.
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Yu t tc ng trans (trans-acting element). Yu t di truyn c th biu hin hiu qu m khng cn nm trn cng mt phn t vi on trnh t ch. Thng yu t m ha cho mt sn phm protein (c th l mt enzyme hay mt protein iu ha) v sn phm ny c th khuch tn n im tc ng.

Ti liu tham kho/c thm

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

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MC LC Trang LI NI U Chng 1. CC I PHN T SINH HC I. Nucleic acid 1. Deoxyribonucleic acid 2. Ribonucleic acid II. Protein 1. Cu trc ca protein 2. Chc nng ca protein III. Lipid IV. Polysaccharide Ti liu tham kho/c thm Chng 2. CU TRC GENOME I. Cc thnh phn v c im ca genome 1. Genome ca c quan t 2. ng hc ca phn ng lai DNA 3. Kch thc ca genome 4. Tng s gen c bit mt s loi eukaryote II. Tnh phc tp ca genome III. Thay i trt t ca cc on DNA trong genomeTransposons. 1. S thay i trt t ca cc on DNA trong genome 2. Cc transposons IV. Tng tc ca T-DNA vi genome thc vt 1. Ti-plasmid v Ri-plasmid 2. T-DNA 5 5 5 9 13 13 19 22 24 25 26 27 28 32 34 35 37 40 40 41 45 46 46

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3. Vng vir 4. Qu trnh chuyn T-DNA vo t bo thc vt V. Sp xp v khuch i cc gen trong genome 1. Sp xp li cc gen 2. Khuch i cc gen 3. Bin np gen Ti liu tham kho/c thm Chng 3. CU TRC V CHC NNG CA GEN I. nh ngha gen II. L thuyt trung tm 1. S xc nh di truyn cu trc bc 1 ca protein 2. Cc enzyme mt hot tnh do t bin 3. Bn cht cc bin i di truyn ca protein 4. S tng quan ng tuyn tnh gen-polypeptide 5. L thuyt trung tm ca sinh hc phn t 6. DNA v m di truyn III. Cu trc v chc nng ca gen 1. Cu trc gen 2. S phn chia nh ca gen 3. Th nghim chc nng allele 4. Gen l n v chc nng nh nht Ti liu tham kho/c thm Chng 4. TI BN DNA I. Chng minh ti bn DNA theo c ch bn bo th 1. C ch ti bn bn bo th 2. Th nghim ca Meselson v Stahl II. M hnh ti bn DNA-chc ba ti bn

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1. M hnh ti bn 2. Chc ba ti bn 3. Ti bn DNA theo vng trn quay III. Bn cht xon ca DNA-Cc giai on ca s ti bn 1. M xon 2. Ko di-Tng hp chui Okazaki 3. Kt thc IV. Khi nim mi V. Enzyme ti bn 1. DNA polymerase 2. Cc topoisomer v DNA topoisomerase 3. Helicase v protein SSB 4. DNA ligase Ti liu tham kho/c thm Chng 5. PHIN M I. Cc c im c bn ca qu trnh phin m 1. S phin m to ra RNA b sung vi mt si DNA 2. S phin m l mt phn ng enzyme 3. S phin m ch sao chp chn lc mt s phn ca genome v to ra nhiu bn sao 4. Ch mt trong hai si n ca phn t DNA c dng lm khun mu 5. S phin m c khi pht khng cn mi II. Cc giai on ca qu trnh phin m 1. Giai on khi u 2. Giai on ko di 3. Giai on kt thc III. Phin m prokaryote

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1. Enzyme RNA polymerase prokaryote 2. Promoter ca gen prokaryote 3. Vai tr ca enzyme RNA polymerase v promoter trong qu trnh phin m 4. Tn hiu kt thc IV. Qu trnh phin m eukaryote 1. Cu trc gen eukaryote 2. Enzyme RNA polymerase ca eukaryote 3. Cc yu t gip RNA polymerase khi u phin m 4. Cc yu t kch thch RNA polymerase II hot ng trong giai on ko di 5. Qu trnh bin i cc RNA mi c tng hp V. Phin m ngc Ti liu tham kho/c thm Chng 6. DCH M I. M di truyn 1. Cc codon 2. Cc quy tc chi phi m di truyn II. Cc ribosome 1. Thnh phn cu to ca ribosome 2. Khi nim polyribosome 3. Cc v tr gn tRNA trn ribosome 4. Cc knh ca ribosome III. S hnh thnh aminoacyl-tRNA 1. Bn cht ca s gn amino acid vo tRNA 2. S nhn din v gn amino acid vo tRNA 3. Tnh c hiu ca aminoacyl-tRNA synthetase 4. Phn loi aminoacyl-tRNA synthetase IV. Cc giai on ca qu trnh dch m

98 99 99 101 102 102 103 104 107 108 110 114 115 115 115 116 116 116 117 118 118 119 119 119 119 120 120
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1. Giai on khi u 2. Giai on ko di 3. Giai on kt thc V. Cc nhn t c ch dch m Ti liu tham kho/c thm Chng 7. SA CHA V BO V DNA I. Khi qut v cc c ch sa sai 1. Cc bin i xy ra trn phn t DNA 2. Khi qut cc c ch sa cha mc phn t 3. Bin i lm tng tn s t bin II. Cc kiu sa cha 1. Quang ti hot ha 2. Sa cha ghp i lch 3. Sa cha ct b 4. c sa i vi cc base bt cp sai 5. Cc h thng sa cha ti t hp 6. H thng SOS III. Bo v DNA: H thng ct hn ch (R)-bin i (M) 1. Cc methylase ca R-M 2. Adenine v cytosine methyltransferase E.coli 3. S methyl ha DNA ca eukaryote Ti liu tham kho/c thm Chng 8. IU HA BIU HIN GEN I. Cc hin tng iu ha 1. iu ha thch nghi 2. Hot ng ni tip ca cc gen 3. Bit ha t bo

120 125 128 130 132 134 135 135 137 137 138 138 138 141 143 143 146 150 151 152 153 153 155 156 156 156 156
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4. Khi qut v iu ha prokaryote v eukaryote II. Cc mc iu ha 1. Mc cht nhim sc 2. Mc phin m 3. Mc hu phin m 4. Mc dch m 5. Mc hu dch m III. iu ha biu hin gen prokaryote 1. Cu trc ca promoter 2. Cu trc ca operon 3. iu ha thoi dng: Kim sot m-cm ng 4. iu ha bin dng: Kim sot m-c ch 5. Kim sot dng v cm ng IV. iu ha hot tnh ca eukaryote 1. Cc promoter 2. Cc enhancer 3. Cc protein l nhn t c tc ng trans 4. Hormone 5. Kim sot cc cht thng gp trong nhn V. S bit ha t bo 1. Cc t bo bit ha mang thng tin ging nhau 2. Cc t bo bit ha tng hp cc nhm protein khc nhau 3. S iu ha mc phin m l ngun gc cn bn ca cc sai khc gia nhng t bo bit ha Ti liu tham kho/c thm Chng 9. CNG NGH DNA TI T HP I. M u 1. Tc ng ca cng ngh DNA ti t hp

157 160 160 160 161 161 161 162 163 163 164 165 170 171 172 172 172 175 175 176 176 177 179 180 181 181 181

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2. Lm vic mc phn t II. Endonuclease hn ch 1. Gn cc u b ct bi enzyme hn ch 2. Isochizomer III. Phng thc to dng 1. Plasmid vector 2. Bacteriophage 3. Cosmid vector 4. Th vin cDNA 5. Th vin genomic DNA IV. Biu hin gen ngoi lai trong vi khun 1. Cc protein nguyn th ti t hp 2. Cc protein dung hp ti t hp 3. Xc nh mc biu hin ca gen c to dng V. Phng php pht hin dng vi khun c DNA ti t hp 1. Lai khun lc v vt tan 2. Sng lc th vin gen bng PCR 3. Sng lc cc th vin cDNA bng cc probe khc nhau 4. Phn tch genomic DNA bng phng php lai Southern VI. Cc ng dng ca cng ngh DNA ti t hp 1. ng dng trong dc phm 2. Cc vi khun c bit 3. Cc sn phm nng nghip 4. Thuc oligonucleotide 5. Liu php gen 6. Chn bnh can thip sm 7. DNA fingerprinting 8. Lp bn gen Ti liu tham kho/c thm vector

182 183 186 186 188 188 192 193 195 196 197 198 201 203 204 204 206 208 209 211 211 212 212 213 214 214 215 216 217
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Ph lc. MT S THUT NG C BN Ti liu tham kho MC LC

218 259 260

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