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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
Cc i phn t sinh hc
I. Nucleic acid
Nucleic acid, vt cht mang thng tin di truyn ca cc h thng sng, l mt
polymer hnh thnh t cc monomer l nucleotide. Mi nucleotide gm ba thnh phn:
nhm phosphate, ng pentose (ng 5 carbon) v mt nitrogen base. Cc nitrogen
base thuc hai nhm: cc purine gm adenine (A) v guanine (G), cc pyrimidine gm
thymine (T), cytosine (C) v uracil (U). Cc nucleotide c ni vi nhau bng lin kt
phosphodiester to thnh chui di.
Nucleic acid gm hai loi phn t c cu to rt ging nhau l deoxyribonucleic
acid (DNA) v ribonucleic acid (RNA).
1. Deoxyribonucleic acid
Phn t DNA l mt chui xon kp gm hai si n. Mi si n l mt chui
nucleotide (Hnh 1.1). Mi nucleotide gm ba thnh phn: nhm phosphate, ng
deoxyribose v mt trong bn base (A, C, G v T) (Hnh 1.2). Hai si n kt hp vi
nhau nh cc lin kt hydrogen hnh thnh gia cc base b sung nm trn hai si: A b
sung cho T v C b sung cho G. Mi si n c mt trnh t nh hng vi mt u
5phosphate t do, u kia l 3 hydroxyl t do (quy c l 53). Hng ca hai si
n trong chui xon kp ngc nhau, nn c gi l hai si i song.
Nhng phn tch cu trc hin i cho thy cu trc ca DNA khng phi lun
lun tng ng vi dng c gi l B m Watson v Crick a ra. Do s tc ng
ca cc hp cht c khi lng nh hoc protein, dng B c th chuyn sang dng A (nn
nhiu hn) hoc l dng Z (xon tri). Chng c th t gp li hoc xon mnh, v d mt
si i DNA c di l 20 cm c nn trong mt chromosome c kch thc l 5 m.
ny c cu trc phc tp
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.
2. Ribonucleic acid
Phn t RNA c cu to tng t DNA ngoi tr ba im khc bit sau:
- Phn t RNA l chui n.
- ng pentose ca phn t RNA l ribose thay v deoxyribose.
- Thymine (T), mt trong bn loi base hnh thnh nn phn t DNA, c thay th
bng uracil (U) trong phn t RNA.
Cu trc v chc nng ca RNA c s bin i r rt. V c bn RNA ch l cht
mang thng tin di truyn virus, sau ngi ta chng minh rng n khng nhng ng
vai tr c bn vic chuyn thng tin di truyn m cn c vai tr cu trc khi to nn
phc h RNA-protein.
Theo mt l thuyt tin ha m i din l Eigen, RNA l cht mang thng tin di
truyn, thnh vin trung gian ca s biu hin gen, thnh phn cu to v l cht xc tc.
Nhm OH v tr th hai ca ribose cn thit cho a chc nng lm nhiu lon s to
thnh si i, qua lm tng khng bn vng ca lin kt phosphodieste.
Trong t bo c ba loi RNA chnh, c cc chc nng khc nhau:
E. coli, kch thc trung bnh ca mt phn t mRNA khong 1,2 kb.
II. Protein
1. Cu trc ca protein
Amino acid l n v c s (monomer) cu thnh protein. Tt c 20 amino acid c
mt trong protein u c xy dng theo mt kiu mu chung:
Trong , gc R (mch bn) cng l phn khc duy nht gia 20 loi amino acid,
quy nh tnh cht ca tng loi.
Nhm amine (NH2) nh nguyn t C2, theo tn c l nguyn t C. V vy,
ngi ta gi l nhm -amine. Cc amino acid tn ti ch yu trong t nhin c nhm
amine ng bn tri trc, c gi l amino acid dng L. Dng D-amino acid ch tn ti
ring bit, v d trong thnh t bo vi khun.
Cc amino acid ring bit c nhng c tnh khc nhau l do gc R ca chng.
Nhng amino acid trung tnh c mt nhm amine v mt nhm carboxyl. Nhng protein
cha nhiu amino acid trung tnh l nhng protein trung tnh. Khi chiu di gc R tng s
hnh thnh c tnh k nc. Nhng protein c cha nhiu amino acid nh valine,
leucine, isoleucine c tnh cht c trng l k nc. Nhng amino acid c tnh acid
trong phn gc c mt nhm carboxyl. Protein cha nhiu amino acid c tnh acid l
nhng protein acid. Tng t nh vy i vi protein ch yu c hnh thnh bi nhng
amino acid c tnh kim l nhng protein kim. Phn gc R ca amino acid c ngha
quyt nh i vi c tnh ca protein m chng to nn. iu ny khng nhng c
ngha i vi tnh cht ha hc m c cu trc ca protein.
2.4. Protein d tr
Cc protein l ngun cung cp cc cht cn thit c gi l protein d tr. Protein
l polymer ca cc amino acid v nitrogen thng l yu t hn ch cho sinh trng, nn
c th phi c protein d tr cung cp y nitrogen khi cn. Chng hn, ovalbumin
l protein d tr trong lng trng trng cung cp nitrogen cho phi pht trin. Casein
l protein sa cung cp nitrogen cho ng vt c v cn non. Ht thc vt bc cao cng
Hnh 1.5. Hai kiu vn chuyn c bn. (a): vn chuyn bn trong hoc gia cc t bo hoc m.
(b): vn chuyn vo hoc ra khi t bo.
Protein cng c th d tr cc cht khc ngoi thnh phn amino acid (N, C, H, O
v S), v d ferritin l protein tm thy trong m ng vt kt hp vi Fe. Mt phn t
ferritin (460 kDa) gn vi 4.500 nguyn t Fe (chim 35% khi lng). Protein c vai tr
2.5. Protein vn ng v co rt
Mt s protein mang li cho t bo kh nng vn ng, t bo phn chia v co c.
Cc protein ny c c im: chng dng si hoc dng polymer ha to si, chng
hn actin v myosin. Tubulin l thnh phn c bn ca thoi v sc (si xut hin khi phn
chia cc nhim sc th v cc cc).
2.7. Protein bo v
Trong vic gii c cc kim loi nng, phytochelatin c mt ngha quan trng,
y l nhng polypeptide n gin c ngun gc t glutation v c cng thc chung nh
sau:
(-glutamyl-cysteinyl)n-glycine
Do c nhiu nhm SH nn chng c kh nng kt hp cht vi cc kim loi nng,
lm cho nhng kim loi nng ny khng th gy ri lon trao i cht. S tng hp
phytochelatin c kch thch bi nhng kim loi nng nh Cd, Cu, Ag, Bi v Au.
Protein bo v c vai tr quan trng trong cc phn ng min dch. ng vt c
xng sng c mt c ch phc tp v pht trin cao ngn nga nhng tc nhn vi
sinh vt gy bnh. Chc nng ny c lin quan n c tnh ca chui polypeptide. Khi
mt protein l (c ngun gc virus, vi khun hoc nm) xm nhp vo mu hoc vo m
th phn ng t v ca c th s xut hin rt nhanh. Protein l c gi l khng nguyn
(antigen) cha mt vng c trt t xc nh cc nguyn t c th kt hp vi t bo
lympho v kch thch t bo ny sn sinh khng th. Nhng t bo lympho tn ti trong
h thng min dch vi s lng 109 v trn b mt ca n c nhng vng nhn bit ni
m khng nguyn s c kt hp (Hnh 1.6). Nhng vng nhn bit ny rt khc nhau
v c hiu cho tng loi khng nguyn. Trong c th lun c sn mt lng ln cc t
bo lympho khc nhau v chng c th tng hp rt nhanh cc khng th c hiu khi
III. Lipid
Mc d khng mang hot tnh sinh hc cao nh protein nhng lipid cng ng mt
vai tr c bit trong h thng sng. Chng l nhn t chnh to nn cc mng sinh hc
m nu thiu th mi hot ng ca protein s khng th phi hp nhp nhng.
n v cu trc ca lipid l cc acid bo. Mi acid bo c cu to t mt mch
carbohydrate (gm cc nguyn t C v H) gn vi mt nhm carboxyl c tnh acid. Cc
acid bo khc nhau bi di ca chng, bi s lng v v tr cc lin kt i. Cc acid
bo khng c lin kt i c gi l cc acid bo bo ha, cc acid bo khng bo ha
c t nht mt lin kt i.
Mng sinh hc c chc nng l gii hn nhng vng trao i cht v tham gia vo
vic vn chuyn cc cht. Mng sinh hc cng c kh nng chuyn i nhng tn hiu.
Protein mng cng c th l cc enzyme. Chc nng ny c th hin mng trong ca
ty th v lp th. Mng sinh hc bao gm lp kp lipid vi nhng protein phn b trong
(Hnh 1.7)
Cc lipid mng c hnh thnh t mt chui di acid bo ni vi nhng nhm c
c tnh a nc cao v c gi l nhng phn t lng cc v mt u tng tc vi
nc, cn u kia th k nc.
IV. Polysaccharide
Cc polysaccharide c nhiu chc nng quan trng trong t bo, chng tham gia
vo cu to t bo v l ngun d tr nng lng ch yu. Cc polysaccharide c hnh
thnh t nhiu monomer, l cc ng n gin (monosaccharide) ni vi nhau bng lin
kt glycoside. Lin kt ny c hnh thnh t s kt hp gia C1 ca mt phn t ng
vi nhm hydroxyl ca phn t k tip.
Ngun d tr tinh bt cc t bo ng vt l glycogen, trong khi thc vt l
tinh bt. Mt polymer khc ca glucose l cellulose th to nn thnh t bo thc vt v l
hp cht hu c hin dn nhiu nht trong sinh quyn.
Chng ta va im qua ring r tng thnh phn cu to t bo chnh. Trong thc
t, hot ng ca chng phi hp mt thit vi nhau. Cc nucleic acid trong t bo
thng kt hp cht ch vi cc protein to thnh nucleoprotein. DNA ca t bo
Chng 2
Cu trc genome
Genome (h gen, b gen) l thut ng c dng vi cc ngha khc nhau nh sau:
Hnh 2.1. DNA ty th ca ngi. Bao gm 22 gen tRNA, 2 gen rRNA, v 13 vng m ha
protein.
1.2. Genome ca lp th
DNA lp th (chloroplast DNA-ctDNA) cng l mt DNA genome c lp, thng
l mch vng, c tm thy trong lp th ca thc vt.
- Genome ca lp th rt khc nhau v kch thc, nhng ln m ha cho
khong 50-100 protein cng nh rRNA v tRNA.
- DNA lp th di t 120-190 kb. Cc genome ca lp th c phn tch trnh
t cho thy c khong 87-183 gen. Bng 2.2 m t cc chc nng c m ha bi
genome lp th cy trng.
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.
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.
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.
2. Cc transposon
Cc yu t di chuyn c th c chia lm hai loi da vo cch thc di chuyn ca
chng:
- Loi th nht c gi l retroelement, chng phi tri qua hnh thc trung gian
RNA trong qu trnh di chuyn (RNA genome c sao chp nh reverse transcriptase
to ra cDNA ghp vo v tr mi) (Hnh 2.6).
- Loi th hai l cc on DNA hoc b tch ra hoc c ti bn to thm bn sao
ghp vo v tr mi. Thng thng, loi th hai ny c gi l transposon.
Hnh 2.8. 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
3. Bin np gen
Mt phng thc tng kh nng di truyn l ng dng cc tng tc vt ch cng
sinh-k sinh, trong DNA l c chuyn vo t bo vt ch t mt vi khun. C ch
ny tng t vi s tip hp ca vi khun. S biu hin ca DNA vi khun trong vt ch
mi ca n s lm thay i kiu hnh ca t bo. V d in hnh l vi khun A.
tumefaciens cm ng to khi u t bo thc vt b chng xm nhim.
Khi DNA l c a vo t bo eukaryote, n c th tn ti ngoi nhim sc th
hoc c hp nht trong genome. Nu xy ra theo trng hp th hai, genome s mang
nhng t bin di truyn v nhiu khi DNA l vn tip tc hot ng lm xut hin cc
tnh trng mi. Vic a cc gen l vo t bo soma hoc t bo sinh dc m vn duy tr
hot ng ca nhng gen c gi l chuyn nhim (transfection). C th biu hin
tnh trng mi nh hot ng ca gen l a vo t bo sinh dc c gi l c th
chuyn gen. Qu trnh ny c th lm thay i s n nh ca genome. DNA sau khi
c tim vo trong cc t bo trng ng vt c th c hp nht trong genome v
truyn cho th h sau nh mt thnh phn di truyn bnh thng. Kh nng a mt gen
chc nng c trng c th tr thnh mt k thut y hc s dng cho vic cha bnh di
truyn.
Ti liu tham kho/c thm
1. V Th Thng Lan. 2002. Sinh hc phn t. NXB i hc Quc gia H Ni, H Ni.
8. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company Inc. New
York, USA.
Chng 3
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
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 acid 1,
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
Ch thch
Nhng n v m (codon) c c theo chiu 53.
STOP: codon kt thc (cn gi l v ngha).
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.
1. Cu trc gen
Mt gen thng c xem gm c hai phn, phn mang m di truyn c phin
m sang phn t mRNA v phn DNA lm nhim v iu khin hot ng ca gen (Hnh
3.5). C hai phn u c cu trc c bn kh ging nhau mi sinh vt prokaryote v
eukaryote. Tuy nhin, gen eukaryote cn c cu trc c th lin quan n cc c ch
kim sot khc nhau i vi hot ng ca gen.
Cu trc mt gen in hnh ni chung u c promoter, v tr RNA polymerase
hot ng khi u phin m. i khi nm rt xa gen, c th thy cc enhancer (vng
tng cng) hoc silencer (vng c ch) c vai tr lin quan n qu trnh phin m.
di ca mt gen thay i ty theo s lng v di ca cc intron cha trong n.
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
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.
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).
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
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.
Hnh 4.2. DNA dng mch vng si i. Chiu di thc t 1,6 mm (4,7106 bp).
2. Chc ba ti bn
Hnh 4.4 m t cu trc ca chc ba ti bn. Nh vo phng php phng x nh t
ghi, ngi ta nhn thy s ti bn thc hin theo hai hng (bidirectional synthesis).
ng thi cng chng minh c vi khun E. coli (prokaryote) c duy nht mt im
gc ti bn, l im m hai chui xon kp ca DNA m c tch ra, tng ng vi
hai chc ba ti bn pht trin ngc chiu nhau. Chui DNA sau khi tch ra c dng
lm khun mu cho s tng hp DNA mi (Hnh 4.5).
Hnh 4.5. Hnh nh di knh hin vi in t. Phn t DNA mch vng nh ca E. coli c
chiu di thc t 0,01 mm (3.000 bp) ti bn bng kiu . Cc on DNA b m v con c
trnh by trong hnh v.
Hnh 4.6 so snh s khc nhau gia ti bn DNA khng nh hng v ti bn theo
hai hng. Trong ti bn khng nh hng, ch c mt chc ba ti bn. Trong khi ti bn
theo hai hng yu cu hai chc ba ti bn. Mi tn cong ch hng chuyn ng ca cc
chc ba. Hu ht DNA ti bn theo hai hng.
Hnh 4.7 v 4.8 m t phng thc hp nht cc vng ti bn DNA rui gim (D.
melanogaster). Qu trnh ti bn din ra ng thi trn hng chc ngn v tr khc nhau
ca phn t DNA v to thnh cc vng ti bn, cc vng ti bn sau s m rng theo
hai hng cui cng hp nht vi nhau to thnh hai phn t DNA.
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.
Hnh 4.11. Hai si DNA mi c tng hp theo hai kiu khc nhau
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.
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
C nhim v ni hai u 3-OH v 5-PO4 ca hai on DNA ri thnh mt on
lin tc. Mt mi hn nh vy cn mt nng lng l 2ATP (i vi eukaryote) hoc
NAD+ (i vi vi khun).
Hnh 4.17. Lin kt ca protein SSB ln DNA si n. Mt lng gii hn ca SSB lin kt
vi 4 trong 9 phn t DNA si n. Khi b sung thm protein SSB, n s lin kt vi cc protein
SSB lin kt t trc. Ch sau khi protein SSB bao bc hon ton cc phn t DNA si n
ban u, th n mi tip tc lin kt vi cc phn t DNA si n khc.
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
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 5.4. Cc tc nhn hot ha, phc hp trung gian v cc thnh phn bin i
nucleosome trong phin m
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).
4. Cc knh ca ribosome
l cc knh cho php mRNA i vo v i ra khi ribosome, v knh cho php
chui polypeptide mi sinh i ra khi ribosome.
mRNA i vo v i ra khi trung tm gii m ca ribosome thng qua hai knh hp
ti tiu n v nh. Trong , knh vo c chiu rng ch cho RNA khng bt cp i
qua. c im ny m bo cho mRNA c dui thng khi n i vo trung tm gii m,
bng cch loi b mi tng tc bt cp base b sung ni phn t.
Mt knh xuyn qua tiu n v ln to li thot cho chui polypeptide mi c
tng hp. Kch thc ca knh hn ch c s gp ca cc chui polypeptide ang
tng hp. V vy, protein ch c th hnh thnh cu trc bc ba sau khi n c gii
phng khi ribosome.
III. S hnh thnh aminoacyl-tRNA
1. Bn cht ca s gn amino acid vo tRNA
Qu trnh gn amino acid vo tRNA l qu trnh hnh thnh mt lin kt acyl gia
nhm carboxyl ca amino acid v nhm 2'- hoc 3'-OH ca adenine u 3' ca tRNA.
Lin kt ny c xem l mt lin kt giu nng lng. Nng lng gii phng ra khi
lin kt b ph v gip hnh thnh cu ni peptide lin kt amino acid vi chui
polypeptide ang c tng hp.
2. S nhn din v gn amino acid vo tRNA
S nhn din v gn amino acid vo tRNA tng ng c thc hin bi mt
enzyme gi l aminoacyl-tRNA synthetase.
Qu trnh ny din ra nh sau: u tin, amino acid c adenylyl ha bng cch
phn ng vi ATP, kt qu to thnh amino acid c gn adenylic acid qua cu ni ester
giu nng lng gia nhm COOH ca amino acid v nhm phosphoryl ca AMP, ng
thi gii phng ra pyrophosphate. Sau , amino acid c adenylyl ha ny (vn ang
gn vi synthetase) phn ng tip vi tRNA. Phn ng ny chuyn amino acid n u 3'
ca tRNA gn vi nhm OH, ng thi gii phng AMP.
Phn ng tng hp ca qu trnh ny nh sau:
1.2.3. Bc 3: Tiu n v nh tm thy codon khi u bng cch qut xui dng t u
5' ca mRNA v s hnh thnh phc hp khi u 80S
Mt khi c gn vo u 5' ca mRNA, tiu n v nh v cc yu t lin kt vi
n di chuyn dc theo mRNA theo hng 5' 3' cho n khi gp trnh t 5'-AUG-3' u
tin m n nhn dng l codon khi u. Codon c nhn dng bng s bt cp base b
sung gia anticodon (b ba i m) ca tRNA khi u v codon khi u. S bt cp
ny thc y phng thch eIF2 v eIF3 cho php tiu n v ln gn c vo tiu n v
nh. S gn ny dn n phng thch cc yu t khi u cn li thng qua s thy phn
GTP di tc dng ca eIF5B. Cui cng, 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).
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.
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
S nhn bit v sa cha cc sai lch do bt cp base sai trn DNA c pht hin
E. coli, nm men v t bo ng vt c v. vi khun E. coli, c ba h thng enzyme
khc nhau c s dng sa cha cc sai lch bng cch:
- Loi b ch sai trong ti bn (errors in replication).
- Loi b ghp i lch bn trong cc on trung gian ca ti t hp (mismatch
within recombinant intermediates).
- Loi b thymine ca cp GT trong trng hp 5-methylcytosine b mt nhm
amine (NH2) thnh thymine.
3.1. Ct b base
Vic loi b ch sai c thc hin nh mt hoc vi enzyme N-glycosylase. Nglycosylase nhn bit base bin i hay mt gc amine hoc bin dng cu trc xon do
sai lch to ra v thy phn lin kt N-glycosylic gia base vi ng vi pentose. L
hng va c to ra do ct b c DNA polymerase lm y li da vo khun mu
b sung i din v ligase ni lin chng (Hnh 7.4).
3.2. Ct b nucleotide
Vic ct b mt vng c nhiu thymine dimer (do UV) hoc vng lin kt cho
gia cc si, c thc hin nh incision nuclease (nuclease to khc trn DNA) nh
phc hp Uvr ABC ca E. coli, phc hp ny ct cc on 12-13 nucleotide t mt si
DNA. S thy phn c thc hin lin kt phosphodiester th tm u 5 n ch
hng v pha 3 l lin kt th t hay nm.
4. c sa i vi cc base bt cp sai
C ch c sa i vi cc base bt cp sai (proofreading for base-pair matching)
c thc hin trong ti bn DNA. S bt cp cn thn ca DNA polymerase m bo
cho s chnh xc ca ti bn. Trc khi thc hin phn ng polymer ha ni vi
nucleotide, nucleotide triphosphate mi phi bt cp vi base b sung trn si khun
mu. Nu s bt cp sai xy ra, DNA polymerase s loi b nucleotide bt cp sai.
Thm ch, trc khi nucleotide mi gn vo, enzyme d li cp base cui nu
chng khng bt cp th s polymer ha tip theo b dng. Cp nucleotide u cui 3
bt cp sai s b loi b bi hot tnh exonuclease 35 ca DNA polymerase. Khi s
bt cp ca cu trc si kp ng, qu trnh polymer ha mi c tip tc.
Hot tnh c sa i vi cc base bt cp sai l c tnh ca nhiu DNA
polymerase m bo cho s ko di chnh xc ca si ang c tng hp. Tuy nhin,
trong trng hp cn thit, DNA polymerase c th b qua ch sai v chp tip nh c
ch ti bn error prone (xem mc 6).
5. Cc h thng sa cha ti t hp
Cc v tr c s sai hng, xy ra trong mt phn t con (daughter molecule), s
c phc hi nh s ti t hp thu c mt bn sao khc ca trnh t t mt ngun
khng b sai hng. Bn sao ny sau c dng sa cha l hng (gap) trn si b
hng.
Khi DNA polymerase gp thymine dimer hay mt s sai lch khc trn si DNA
khun mu, c hai kh nng xy ra:
- Polymerase lp dn l hng bng ti bn khng theo khun mu do ti t hp v
vt qua ch sai.
6. H thng SOS
H thng SOS (cha khong 30 gen khng lin kt v thng b c ch bi protein
LexA) s hot ng khi t bo b cc tc nhn gy t bin to nhiu sai hng trn DNA.
Trong trng hp DNA b hng ngng ti bn, phn ng SOS s phc hi ti bn bng
phng thc ti bn error-prone.
vi khun E. coli ngi ta quan st thy s ph hy DNA lm m ra khong 20
gen ca h thng SOS, c kim sot m bi cht c ch LexA (LexA repressor).
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.
1. Cc methylase ca R-M
Methylase l enzyme c hot tnh xc tc gn mt nhm methyl vo mt phn t, v
d: DNA methyltransferase c chc nng methyl ha cc gc C trong DNA.
Enzyme methylase to ra mt cp adenine methyl ha (hoc cytosine) c v tr i
xng cho nhau. Cc methylase c lin quan cht ch vi cc kiu ct hn ch.
- Kiu I. Cc RE c th tin hnh c hai s bin i v ct hn ch. Mt RE kiu I
bao gm ba tiu n v khc nhau (R, M v S) chu trch nhim tng ng cho vic ct
hn ch, bin i v nhn bit trnh t. V d: EcoB v EcoK (t E. coli chng B v K),
cc trnh t nhn bit ca hai enzyme ny l TGA(N)8TGCT i vi EcoB v AAC(N)6GTGC i vi EcoK (trong N l mt trong bn loi nucleotide). Nu v tr nhn bit
khng c methyl ha trong mt si, th enzyme RE s methyl ha si , nhng nu c
hai si u khng c methyl ha th enzyme RE s ct DNA (cn c ATP). Phn ng
ct xut hin mt v tr khng c hiu >1000 bp tnh t trnh t nhn bit; r rng l
enzyme duy tr lin kt v tr nhn bit ca n, v DNA c di chuyn qua mt v tr
th hai trn enzyme hng ti mt v tr ct hn ch.
- Kiu II. Cc RE l dng chung nht trong vi khun; chng ch thc hin phn ng
ct hn ch-cn s bin i c tin hnh bi mt enzyme ring bit. Trnh t nhn bit
cho hu ht cc enzyme ny l cc trnh t palindrome ngn (v d: EcoRI nhn bit
GAATTC) v thc hin phn ng ct (khng cn ATP) trong hoc gn k vi v tr
nhn bit. Mt vi RE kiu II ct c hai si cng v tr to ra u bng , trong khi cc
RE khc li to ra u dnh. Cc RE kiu II l cng c hu ch trong cng ngh DNA ti
t hp.
- Kiu III. Cc RE ny tng t cc enzyme kiu I. Mi RE kiu III c hai tiu n
v; n nhn bit mt trnh t i xng ngn, v to ra mt u so le (cn c ATP) v tr
khong 24-26 bp so vi v tr nhn bit. V d enzyme EcoPI, c m ha bi prophage
ca bacteriophage P1.
5. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular
Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California,
USA.
6. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company, New
York, USA.
Chng 8
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).
5. Mc hu dch m
y c s iu ha hot tnh ca protein. Sau khi mch polypeptide c tng
hp, cc protein nhiu khi phi tri qua cc bin i th cp trc khi biu hin hot tnh
(chc nng). V d: trypsin l enzyme phn gii protein trong d dy ch c c hot
tnh sau khi cht tin thn ca n (pro-enzyme khng c hot tnh) b ct mt mt on
polypeptide.
Cc protein c th chu nhng bin i lp th nh s kt hp cc enzyme vi mt
s sn phm c bit c th lm thay i cu trc khng gian ca chng dn n mt hot
tnh.
- Cc qu trnh glycosylation, phosphorylation tc l gn thm cc nhm cht
nh ng, phosphor protein c hot tnh/chc nng sinh hc.
- Peptide tn hiu l on gm khong 20 amino acid nm gn pha u N ca
polypeptide, c vai tr gn polypeptide v ribosome ang tng hp mch ny vi mng
li ni sinh cht. Trong b my Golgi, polypeptide c phng thch ra ngoi.
- S phng thch ra protein c chc nng sinh hc t mt phc hp, nh t proinsulin thnh insulin.
III. iu ha biu hin gen prokaryote
Cc gen c phin m to RNA, c gi l cc gen cu trc. Cc protein c
dch m t mRNA c th l enzyme hoc khng phi enzyme. Trong s cc protein
khng phi enzyme c cc protein iu ha (regulatory protein), chng tng tc vi cc
trnh t DNA c hiu kim sot hot tnh phin m ca cc gen cu trc. Cc gen
tng hp cc protein iu ha c gi l cc gen iu ha (regulatory gen). Pha trc
mi gen cu trc (hoc mt nhm gen) c mt trnh t promoter, ni RNA polymerase
nhn bit (Hnh 8.3). C ch iu ha prokaryote ch yu c thc hin thng qua
operon. y l khi nim ch tn ti prokaryote.
1. Cu trc ca promoter
Thc cht ca khi s phin m l quan h trc tip gia RNA polymerase v
promoter. Khi RNA polymerase gn vo promoter, n s phin m to phn t RNA.
Phn ln promoter E. coli v cn bn c cng cu trc:
Nu base u tin c phin m thnh mRNA (lun l purine, thng l adenine)
c nh s +1, th tt c cc base pha 5 hay pha trc so vi n khng c phin
m l s tr (). Ngay pha trc +1 c 6 base thng vi trnh t TATAAT xung
quanh 10, v trnh t TTGACA (trnh t lin ng-consensus sequence) xung quanh
35. C hai trnh t phi hp nhau cho php RNA polymerase gn vo v khi s dch
m, trnh t 35 to iu kin u tin cho vic gn vo.
2. Cu trc ca operon
Operon l n v phin m gm t nht mt promoter v mRNA bc tip theo
m ha cho cc trnh t ca mt hay nhiu chui polypeptide. Tuy nhin, operon c th
c mt hay nhiu im iu ha khc vi promoter. Cc gen khng chu s iu ha do
tc ng mi trng, to sn phm thng xuyn, c gi l cc gen cu trc. S lng
sn phm ca cc gen ny c th dao ng ph thuc vo i lc tng i ca cc
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
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).
5.1. S phong ph
Cc gen m ha cho rRNA v tRNA c nghin cu k. Phn ng lai n gin
DNA-RNA cho thy nhim sc th ca Xenopus c vng t chc hch nhn (nucleolus
organizer) cha 450 bn sao ca DNA m ha cho 18S v 28S rRNA. Ngc li, trong
mi nhn c 20.000 bn sao ca cc gen m ha cho 5S rRNA v cc gen ny khng nm
vng t chc hch nhn.
Cc gen m ha cho histone cng c vi nhiu bn sao c lp li hng trm ln.
S phong ph ca cc gen nu trn m bo s lng cn thit cho dch m khi
phi tng hp hng triu phn t protein vo lc cn thit.
V. S bit ha t bo
8. Watson JD, Hopkins NH, Roberts JW and Weiner AM. 2004. Molecular
Biology of the Gene. The Benjamin/Cummings Publishing Company, Inc. California,
USA.
9. Weaver RF. 2003. Molecular Biology. 2nd ed. McGraw-Hill Company, New
York, USA
Chng 9
2. Lm vic mc phn t
K thut gen cho thy mt lot c hi, m ra cc phng thc cn thit (m trc
y c th khng c) gn nh l hin nhin. Vn c bn l cc gen c kch thc
qu nh v c hng ngn gen trong mi t bo. Thm ch, khi quan st trn knh hin vi
mnh nht, th DNA xut hin nh l mt si dy b xu, cc nucleotide ring r khng
th thy, v khng c mt du hiu no v cc ng nt vt l ch bt u v kt thc
ca mt gen.
minh ha vn ny, chng ta hy xem xt mt v d c trng v di truyn
phn t nh sau: Gi thit rng chng ta mun phn lp mt gen c bit ca ngi v
t n vo trong vi khun sn xut mt lng ln cc protein ngi c m ha.
Vn u tin l tm c gen mong mun. Genome n bi ca ngi cha khong
3,3 t cp base ca DNA. Gi s gen m chng ta mun phn lp di 3.000 bp. Nh vy,
gen ch ca chng ta ch chim mt phn triu ca genome; v th tm kim gen ca
chng ta trong mt genome s nh th l kh khn hn rt nhiu so vi vic tm kim
II. Endonuclease hn ch
Trong t nhin, cc enzyme endonuclease hn ch (restriction endonuclease, RE),
gi tt l enzyme hn ch, hin din trong hu ht cc t bo vi khun ngn cn DNA
ngoi lai tip qun b my tng hp protein ca t bo. DNA ca chnh chng s c
bo v khi tc dng ca enzyme hn ch nh s c mt ca cc enzyme ni bo c th
methyl ha (methylation) cc nucleotide c bit, v th cc nucleotide ny khng c
nhn bit bi cc enzyme hn ch.
Vic pht hin ra cc enzyme hn ch ca vi khun ct DNA nhng trnh t c
bit, gip cho vic thao tc gen d dng hn, do n c th gim chiu di ca cc phn
t DNA thnh mt tp hp bao gm cc on ngn hn. Hin nay, cc enzyme hn ch
c phn lp t cc sinh vt prokaryote.
Mi enzyme hn ch ch nhn bit v ct mt trnh t DNA c bit thng cha
bn hoc su cp nucleotide. V d enzyme EcoRI tch chit t E. coli ct trnh t
GAATTC, enzyme BalI ca Brevibacterium albidum ct trnh t TGGCCA. C hn 900
Hnh 9.1. Hai kiu ct ca enzyme hn ch. (a) to ra u so le, v (b) to ra u bng.
1. Gn cc u b ct bi enzyme hn ch
C hai kiu gn khc nhau: gn u bng v gn u dnh, bng cch dng enzyme
DNA ligase ca bacteriophage T4 c th gn cc u bng hoc u dnh li vi nhau,
tuy nhin trng hp u bng thng cho hiu qu thp hn u dnh.
V d: Hnh 9.2 minh ha vic gn cc u dnh c ct bng enzyme HindIII.
2. Isochizomer
Ni chung, cc RE khc nhau nhn bit cc trnh t khc nhau (Bng 9.1), tuy
nhin cng c mt s trng hp cho thy c nhng enzyme c phn lp t nhiu
ngun khc nhau nhng li ct trong mt trnh t, cc enzyme c gi l
1. Plasmid vector
Plasmid l cc thng tin di truyn ngoi nhn c trong nhiu loi vi khun khc
nhau. Chng l nhng phn t DNA mch vng, si i, kch thc t 1- 200 kb, c
kh nng t sao chp tn ti c lp trong t bo. Cc plasmid c th mang mt s gen
ca vi khun v cc gen ny c th biu hin ra protein.
DNA ca plasmid c th c phn lp t nui cy vi khun cha plasmid bng
cch b sung cht ty (nh l sodium dodecyl sulfate-SDS) v ly tm s sinh tan
(lysate)1. Phc hp nhim sc th vi khun, ln hn plasmid nhiu, s lng xung y ca
tube ly tm, plasmid siu xon v cc on nhim sc th mch thng gi li trong th
ni. Plasmid siu xon mt ln na c phn tch bng ly tm sau khi x l vi CsCl v
EtBr.
Plasmid mang cc gen m ha cho cc enzyme thng c li cho vi khun vt ch.
Cc plasmid c th mang cc kiu hnh khc nhau nh: khng khng sinh, sn xut khng
sinh, phn hy cc hp cht hu c phc tp, sn xut cc enzyme hn ch v enzyme
bin i (modification enzymes).
Hnh 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.
4. Th vin cDNA
Th vin cDNA (complementary DNA) l tp hp cc on DNA b sung (cDNA)
c tng hp t mRNA ca mt b phn trong c th sinh vt. S dng th vin cDNA
c hai u im sau:
- Cc dng cDNA cha trnh t m ha lin tc ca mt gen. Nhiu gen
eukaryote l gin on, c cha nhiu on intron. Sau khi ct mRNA tin thn (premRNA) v ni li, cc on intron b loi v mRNA hon chnh (mature mRNA) c
trnh t m ha lin tc c to thnh. Do cDNA c to thnh t khun mu mRNA
hon chnh nn cc dng cDNA c th tng hp protein cn thit vi s lng ln nh
mong mun.
- Nhiu protein c tng hp vi s lng ln do nhng t bo chuyn ha v nh
vy trong cc t bo ny mRNA ca protein s c t l cao v th vin cDNA c
to ra t cc t bo ny s c nhiu cDNA m ha cho cc protein tng ng. S di do
cDNA ca mt vi loi no lm gim nh ng k vic xc nh ng dng mong
mun t th vin gen.
Phng php tng hp gen t mRNA ngy cng c pht trin, n kt hp vi cc
phng php khc ca sinh hc phn t cho php ng dng trong nhiu lnh vc.
Xy dng mt th vin cDNA bao gm nm bc chnh sau:
- Tinh sch mRNA t RNA tng s ca mt phn c th sinh vt.
1. Cc protein nguyn th ti t hp
Cc protein nguyn th (native protein) c th c sn xut trong E. coli bng
cch s dng promoter mnh v mt vng lin kt ribosome (ribosome binding sitesRBS) hiu qu. biu hin gen prokaryote c RBS mnh ch cn cung cp mt
promoter l . Trong khi , biu hin mt gen eukaryote (hoc mt gen prokaryote
vi mt RBS yu) cn phi cung cp c promoter ln RBS.
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.
Hnh 9.9. Vector pAS1. Vector pAS1 l mt plasmid di khong 5,8 kb mang promoter
PL ca bacteriophage v v tr ct hn ch duy nht BamHI nh v codon khi u
ATG ca gen cII ca bacteriophage .
2. Cc protein dung hp ti t hp
2.1. Protein dung hp
Protein dung hp cn gi l protein lai c m ha bi mt gen lai (fusion gene)
do s dung hp in vitro cc on gen khc nhau. V vy, protein dung hp s mang trnh
t amino acid ca hai protein khc bit. Cc protein dung hp c xy dng cho cc
Hnh 9.11. K thut Western blot v ELISA da trn phn ng lin kt khng nguynkhng th
Hnh 9.13. S ca k thut lai Southern blot. Cc mu DNA ch v genomic DNA (A) v
(B) trong v d ny c ct bng EcoRI v c phn on bng in di trn agarose gel. DNA
ca plasmid mang on chn DNA dng lm mu d (C) cng c ct bng EcoRI v in di
nh l mt i chng dng tnh.
Cc th nghim lai trn mng thng c tin hnh bng cch nh du probe
bng P (mc d cc ng v phng x khc nh 35S cng c th c s dng). Phn
ng lai ca Southern i hi hot tnh c hiu ca probe ti thiu phi l 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: digoxigenindUTP) 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.
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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.
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.
Ph lc
Mt s thut ng c bn
Adenine (A). Mt trong bn nitrogen base c trong thnh phn ca DNA v RNA,
bt cp vi thymine (T) trn DNA v vi uracil (U) trn RNA. Adenine cng c trong
thnh phn ca mt s coenzyme.
Adenosine diphosphate (ADP). Mt ribonucleoside 5-diphosphate c cu to
t adenine, ng ribose (5C) v hai gc phosphate. ADP c tc dng nhn phosphate
trong chu trnh nng lng ca t bo.
Adenosine triphosphate (ATP). Mt ribonucleoside 5-triphosphate c cu to
t adenine, ng ribose (5C) v ba gc phosphate. ATP l phn t cha nng lng ha
hc chnh ca t bo, ch yu c tp hp trong ty th (mitochondria) v lp th
(chloroplast). Cc gc phosphate ca ATP c mang cc lin kt khi b thy phn s
phng thch mt nng lng t do ln. Nng lng ca qu trnh h hp hoc quang hp
c s dng to thnh ATP t ADP. Sau , ATP c bin i ngc tr li thnh