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Sinh Hoc Phan Tu
Sinh Hoc Phan Tu
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
Bin tp ni dung:
TS. Nguyn Th Mai Dung
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
Cc tc gi
Chng 1
I. Nucleic acid
(RNA).
1. Deoxyribonucleic acid
5
.
5 m.
Sinh hc phn t 5
Lin kt hydrogen
RNA
Hnh 1.1. Chui xon kp ca DNA
mch
o
20 A
o
100 A
o
300 A
1.3).
Sinh hc phn t 6
o
100 A
o o
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.
NH2
u 5
N N
O H Adenine
N N H
O P O CH2
O
O
Phosphate H H
Deoxyribose O
H H
O H N N H
H Guanine
O N N NH2
O P O CH2
O O
H H
NH2
H H
O H H N
Cytosine
O H N O
HOCH2 OH
O P O CH2
O O O
H H
H
O
H H CH H
HO OH O H 3 N Thymine
Ribose (RNA) (DNA)
O H N O
O O P O CH2
H O
H N O
H H
N O H H
H O H
Uracil (RNA)
u 3
Sinh hc phn t 7
DNA xon kp
Nhn ca
8 phn t histone 2 nm
DNA
11 nm
Histone H1
30 nm
300 nm
700 nm
1400 nm
:
- .
10-15% genome (h gen) -
Sinh hc phn t 8
).
- .
-
-
5S RNA.
- .
.
,
.
2. Ribonucleic acid
sau:
- .
- .
-
.
,s
-protein.
Sinh hc phn t 9
.
:
(mRNA)
2- .
:
Phin m Dch m
DNA RNA Protein
E. coli
1,2 kb.
(tRNA)
:
- .
-
.
n.
Sinh hc phn t 10
E. coli
5S.
. Ngi ta
cng thy ribosome trong ty th, c s tng hp mt s protein ty th.
E. coli
23 1,2 10 3 3700
rRNA 80
16 0 ,55 10 3 1700
5 3,6 101 120
mRNA 5
1
S (Svedberg): n v o vn tc lng. H s lng ca mt tiu n v ph thuc
khng nhng vo khi lng ca tiu n v m cn ph thuc vo hnh dng
v rn ca n, iu ny gii thch ti sao s kt hp ca hai tiu n v 50S v
30S li to ra mt ribosome 70S.
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.
5 -
.
Th .
.
E. coli
Sinh hc phn t 12
II. Protein
(monome
chung:
COOH
H2N CH
R
L- -amino acid
amino acid, qu .
(NH2 2
-
L D-
.
protein.
L- -
20 L-
-
:
Sinh hc phn t 13
- Amino acid t . Bao
.
- . Bao
threonine.
- . Bao
-
(-S-S-).
- . Bao
.
- . Bao .
- Iminoacid. Proline.
- . Bao
2O.
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
- 2. L
Sinh hc phn t 15
( -
- , cun xun
.
.
- 3. L
,l
chui polypeptide.
.
- 4. L
Sinh hc phn t 16
.
Sinh hc phn t 17
Protein vn chuyn Hemoglobin
Serum albumin
Glucose transporter
Protein d tr Ovalbumin
Casein
Zein
Phaseolin
Ferritin
Protein vn ng v co rt Actin
Myosin
Tubulin
Dynelin
Kinesin
Protein cu trc -Keratin
Collagen
Elastin
Fibroin
Proteoglycans
Protein cu trc tm thi Grb 2
(scaffold protein) crk
shc
stat
IRS-1
Protein bo v Immunoglobulins
Thrombin
Fibrinogen
Antifreeze proteins
Snake and bee venom proteins
Diphtheria toxin
Ricin
Protein l/ngoi lai Monellin
(exotic protein) Resilin
Glue proteins
Sinh hc phn t 18
1016
, tu
disulfite
).
albumin.
1.5).
Sinh hc phn t 19
.
(a) (b)
Bn ngoi Bn trong
Hemoglobin Hb(O2)4
(Hb) 4O2 Glucose
Tun hon
tnh mch Mng t bo
Tun hon
ng mch
Tim
4O2
Hemoglobin Hb(O2)4
(Hb)
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.
III. Lipid
Sinh hc phn t 22
i.
HOOC COOH
V tr kt hp vi cytophage
HOOC COOH
NH2 NH2
b
H2N NH2
Khng nguyn
c Khng th
1.7)
Sinh hc phn t 23
1.3.
Hai lp phospholipid
Lp tch ri
Li k nc
Phospholipid
Protein
Gc acid bo k nc
a nc
Protein xuyn mng u a nc
Protein ngoi bin
Glycoprotein
Phn t l ng
Hnh 1.7. S biu din mt on ct ca mng sinh hc
tnh
IV. Polysaccharide
a C1
.
Sinh hc phn t 24
.
nucleic
Sinh hc phn t 25
Chng 2
Cu trc genome
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.
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
1
DNA lp li m t cc chui hin din hn mt bn sao trong mi genome.
2
DNA khng lp li cha cc chui duy nht: ch c mt bn sao trong genome
n bi.
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
ND1
ND4L
ATPase 6
ND3 ND2
CO3
ATPase 8
CO2 CO1
Cc gen tRNA
Cc vng m ha
Hng ca gen, 5 n 3
CO: cytochrome oxidase
ND: NADH dehydrogenase
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.
ng vt 16-17 13 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.
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
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.
DNA si i
Bin tnh
DNA si n
Hi tnh
DNA c 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.
Tc phn ng
Tin phn ng
Thng s ti hn l C0t1/2
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.
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.
Sinh hc phn t 35
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.
Sinh hc phn t 36
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.
Sinh hc phn t 37
(gene family). Nh vy, ngoi cc gen c mt bn sao ging nh vi
khun, genome ca eukaryote cn cha cc h gen. Hu ht cc gen m ha
cho protein c phn lp u nm trong cc h gen khc nhau. Cc gen
trong mt h thng hot ng theo thi gian v khng gian. iu c
ngha, mi thnh vin trong h thng hot ng mt thi im nht nh
trong qu trnh hnh thnh v pht trin c th hoc hot ng trong cc m
chuyn bit. Khi mt thnh vin trong h b t bin (bt hot) th thnh
vin khc c th hot ng thay th.
Khi nim v gen c hnh thnh khi cc nh di truyn hc c in
nghin cu biu hin cc tnh trng mi do gy t bin DNA ca genome
vi khun hay thc khun th (bacteriophage). Mt gen c xem l mt
on DNA m bt c t bin no xy ra trn u dn n xut hin tnh
trng mi. iu ny d hiu i vi nhng genome ch cha cc gen c mt
bn sao (nh genome ca prokaryote). Tuy nhin, genome ca eukaryote,
khi c nhiu gen cng qui nh mt tnh trng hoc cc gen tng ng
trong mt h c th hot ng h tr thay th nhau th t bin trn mt gen
khng phi lc no cng quan st c mc phenotype. Mt khc, cc
on DNA tng ng vi trnh t m ha (coding sequence, exon) cho mt
protein thng b ngt qung bi cc on DNA khng cha thng tin di
truyn (non-coding sequence, intervening sequence, intron). Cc intron
c phin m cng vi cc exon sang phn t RNA gi l phn t tin thn
mRNA (pre-mature mRNA hay pre-mRNA) nhng sau chng b loi b
v cc exon c ni li vi nhau to thnh phn t mRNA hon chnh
(mature mRNA hay mRNA) c dng cho qu trnh sinh tng hp protein.
Qu trnh ct cc intron, ni exon khng tun theo mt trt t bt buc m
bin ha a dng to ra cc phn t mRNA khc nhau t mt phn t pre-
mRNA (Hnh 2.5). Bn cnh mRNA, cc phn t rRNA v tRNA cng
c hnh thnh t cc phn t tin thn cha intron. Ngoi ra, cn c hin
tng m di truyn ca gen ny nm xen k vi cc m di truyn ca gen
khc (cc gen nm gi ln nhau-overlapping) hoc trng hp dch chuyn
khung c ngay trn mt on DNA.
Chng ta bit n chc nng ca cc phn t RNA trong hot ng
sng ca t bo. Bn cnh ba loi RNA c nghin cu kh k (mRNA,
rRNA v tRNA), vai tr ca mt s loi RNA khc mi c pht hin vo
nhng nm cui th k 20. Chng kim sot s hot ng ca gen (hin
Sinh hc phn t 38
tng bt hot gen-gene silencing), tham gia phn ng c sa thng tin di
truyn trn phn t mRNA (hin tng RNA editing) hay quyt nh tnh
bn vng ca mRNA (cc ribonuclease) Thm ch c nhng phn t pre-
mRNA c tng hp khng phi m ha cho protein m vi mc ch
phn ct to ra nhng phn t RNA c kch thc nh hn tham gia qu
trnh kim sot hot ng ca cc gen khc. t bin nhng on DNA
m ha cho tt c cc loi RNA ny thng gn lin vi vic xut hin tnh
trng mi. Do , cn xem xt cc on nucleotide nh cc gen mc d
chng khng m ha cho protein.
Trnh t m ha RNA
DNA
Promoter Terminator
PHIN M
M ui Poly(A)
Exon Intron Exon Intron Exon
Pre-mRNA 5 AAAAAAA 3
Trnh t ch huy on ko di
Hon chnh RNA: loi b intron
Trnh t m ha protein
mRNA 5 AAAAAAAA 3
DCH M
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.
Sinh hc phn t 39
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.
Sinh hc phn t 40
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.
Bn sao mi
DNA ca genome Retroelement ca retroelement
(1) Phin m
(4) Tng hp si
DNA th hai
(
. sinh vt eukaryote, cc transposon cn c gi l
yu t kim sot (controlling elements).
. cy ng, ng
Sinh hc phn t 41
.
Khi di chuyn, cc transposon gy ra vic sp xp v t chc li
genome ca tng c th nh to cc on DNA mi v tr chng ghp vo
v tch ra. Chng c th di chuyn ti v tr bt k v hon ton khng cn
mt mi quan h no gia hai v tr mi v c. Khi tch ra transposon c th
mang theo cc on DNA bn cnh, gy ra s mt on ti v tr c. Ngc
li, khi ghp vo v tr mi, chng gy ra hin tng thm on hoc chuyn
on v tr mi. Do , transposon ging nh cc vector chuyn ch DNA
t ni ny sang ni khc trong mt genome hoc t genome ny sang
genome khc.
Ngoi ra, trao i cho gia cc transposon tng ng hai v tr
khc nhau trn mt hoc trn hai nhim sc th cng to ra nhng bin i
tng t. Nhng bin i dn n vic sp xp li genome, to ra tnh a
dng gia chng v tnh c th ring ca tng c th. c bit, s thay i
v tr ca cc transposon cn c th nh hng n hot ng ca cc gen
phn b xung quanh ngay khi chng khng lm thay i trt t nucleotide
nhng gen ny. Tn s ghp ca cc transposon vo genome khong 10-5-
10-7 sau mi th h. Ngc li, tn s tch ra khong 10-6 n 10-10.
Cc transposon c th chia lm hai nhm da vo kh nng di chuyn
c lp hay phi ph thuc vo s c mt ca transposon khc:
- Nhm th nht gm cc on DNA c kh nng di chuyn c lp.
Chng cha gen m ha cho cc protein iu khin qu trnh , v d nh
enzyme nhn bit hai u transposon tch chng ra khi v tr c v ghp
vo v tr mi. Do , qu trnh ny c thc hin hon ton c lp. Nh
kh nng ny, chng to ra cc t bin khng bn vng.
- Nhm th hai gm cc transposon khng c kh nng t hot ng,
tc l chng khng c kh nng di chuyn do khng mang thng tin di
truyn m ha cho cc enzyme cn thit. V vy, cc transposon loi ny to
ra nhng t bin gen mt cch t pht nhng l t bin bn vng. Vic di
chuyn ca transposon nhm ny ph thuc vo s c mt ca transposon
c kh nng hot ng c lp cng nhm. Hai transposon c th xp vo
cng nhm khi chng c cu trc tng ng nhau, c bit l cc on
Sinh hc phn t 42
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
Sinh hc phn t 43
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 987654321
123456789 987654321
Transposase gene
ATGCA
TACGT
DNA ch V tr ch DNA ch
ATGCA123456789 987654321ATGCA
TACGT123456789 987654321TACGT
Lp li Lp li Transposon Lp li Lp li
xui chiu ngc chiu ngc chiu xui chiu
Sinh hc phn t 44
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.
Sinh hc phn t 45
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
-
Sinh hc phn t 46
.
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
- A. rhizogenes
- A.
rhizogenes
.
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
Sinh hc phn t 47
quan n hnh thnh -1,2 glucan mch vng, trong khi gen chvA xc
nh mt protein vn chuyn, nh v mng trong ca t bo vi khun.
Protein vn chuyn gip vn chuyn -1,2 glucan vo khong gia thnh t
bo v mng sinh cht. -1,2 glucan gi vai tr quan trng vi khun
Agrobacterium tip xc vi thnh t bo thc vt. Nu khng c s tip xc
ny, s khng c s dn truyn T-DNA.
Cc sn phm protein ca vng vir c tc dng cho vic dn truyn T-
DNA t vi khun vo t bo thc vt. Cc loi protein rt cn thit cho
qu trnh ct T-DNA khi plasmid, cm ng thay i mng t bo thc vt
m chng tip xc, tham gia di chuyn phn T-DNA qua mng vi khun ti
t bo cht ca t bo thc vt, vn chuyn ti nhn ri cui cng xm nhp
vo genome ca cy ch.
Thc cht, ch ring T-DNA ca plasmid c chuyn vo genome t
bo thc vt, m khng cn phn no khc. Qu trnh dn truyn ch do sn
phm ca cc gen vir v gen chv quyt nh m khng lin quan n cc gen
khc trn T-DNA. Tuy nhin, chui DNA 25 bp (RB v LB ca T-DNA) c
vai tr l v tr cm ng cho cc sn phm ca t hp cc gen vng vir, c
bit l protein t gen virE mang chng dn truyn vo t bo thc vt.
Chng hot ng nh cc tn hiu nhn bit v khi ng qu trnh dn
truyn. Trc ht gen virA trong t hp gen vng vir c phosphoryl ha
nh tc ng ca cc hp cht phenol nh acetosyringone gii phng ra t
cc t bo thc vt tn thng. Sn phm ca qu trnh ny li tip tc
phosphoryl ha gen virG. Sn phm ca gen virG lin tip lm hot ha
ton b cc gen vir cn li, m hai gen cui cng c hot ha l gen virB
v virE. Trc , khi gen virD c hot ha, sn phm ca n cm ng
nhn bit RB v LB ca T-DNA v lm t phn 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.
Sinh hc phn t 48
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.
Sinh hc phn t 49
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.
Sinh hc phn t 50
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 Z HML
MATa
Ya Ct HO
Xm ln si
Ko di u 3
Y HML
Y 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
Sinh hc phn t 51
ra mt v tr c bit trong genome (v tr hot ng). Chuyn i gen m
ha khng nguyn b mt nhm mc ch hot ha gen m ha khng
nguyn b mt mi thay th cho khng nguyn tn ti trc . Khi mt gen
ang hot ng b thay th bi mt gen khc s tng ng vi vic xut
hin khng nguyn mi v loi b khng nguyn c.
- Cu trc ca mt VSG Trypanosome
Cu trc chung ca mt VSG c m t trn hnh 2.9 v 2.10. Mt
VSG va c tng hp di khong 500 amino acid gm tn hiu N-
terminus, tip theo l on peptide quyt nh tnh khng nguyn; on
peptide bo th gia cc VSG v ui k nc. Phn t ny c tng hp
di dng protein tin thn (pre-protein). Do , chng phi tri qua bin
i hai u NH2 v COOH tr thnh dng protein hon chnh (mature
form). Dng ny c nh vo mng t bo u COOH.
Mt loi Trypanosome c th to ra t nht khong 100 VSG t khi
nhim cho n khi gy cht vt ch. S gen m ha cho VSG c th nhiu
hn 1.000 gen, tt c cc gen ny u nm trong genome. Tuy nhin, ti mt
thi im bt k ch c mt gen hot ng tng hp nn mt loi VSG. Do
, s thay i khng nguyn tng ng vi s thay i hot ng ca gen.
Khi mt gen mi c m, gen hot ng trc n phi b c ch hon
ton. Lc , mt khng nguyn mi s thay th khng nguyn tn ti trc
n.
Hnh 2.9 cho thy chui polypeptide cha khong 500 amino acid. N-
terminus cha mt peptide tn hiu cho s vn chuyn qua ER (li ni sinh
cht, endoplasmic reticulum) v ti mng plasma, c tch ra khi protein
hon chnh. Vng bin thin l khc nhau mi VSG, iu cho thy cc
VSG c t hoc khng c tnh ng nht. Hng ti phn C-terminus, chui
polypeptide c bo ton tt hn v phn ny c gi l vng tng
ng. ui k nc cha mt tn hiu nhn bit gn vi m neo
glycolipid (glycolipid anchor) (Hnh 2.10). Khi m neo c gn th 20
amino acid cui cng s c tch ra.
Glycoprotein bin i b mt (VSG) c gn vi mng thng qua
mt m neo glycolipid cha ethanolamine, mt cu trc glycan mang mt
s gc mannose (mannose moiety), mt glucosamine v mt
phosphoinositol lin kt vi 1,2-dimyristoyglycerol c gai trong mng
plasma. Gc glycolipid l yu t quyt nh phn ng lai (cross-reacting)
Sinh hc phn t 52
(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 Vng tng ng Vng bin thin
H2N COOH
20 360 100 20
ui k nc
C-terminus
Protein
C-terminal
amino acid
Ethanolamine
Glycan Glucosamine
Inositol
Phospholipase C
Mng 1,2-Dimyristoylglycerol
Sinh hc phn t 53
+ 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:
Sinh hc phn t 54
- 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
Sinh hc phn t 55
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.
Sinh hc phn t 56
Chng 3
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
Sinh hc phn t 57
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...
Mc Nm Cc s kin chnh
thi gian
1961 M di truyn l b ba
Sinh hc phn t 58
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 .
Sinh hc phn t 59
Bng 3.2. Cc loi hemoglobin chui polypeptide
Tn amino acid Val Leu Lys Glu His Asp Glu Arg
Loi hemoglobin Hb I Asp
Hb Nocfolk Asp
Hb M Boston Tyr
Hb B Philadelphia Lys
Hb O Indonesia Lys
Tn amino acid Val His Leu Glu Glu Val Glu His
Loi hemoglobin Hb S Val
Hb C Lys
Hb E Lys
Hb M Minvauki Glu
Hb O Rp Lys
Sinh hc phn t 60
t bin c biu hin bi s thay th v tr ca mt amino acid ny
bng mt amino acid khc.
Cc v tr ca t
bin trn DNA
+
H3N COO-
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
Sinh hc phn t 61
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).
1
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).
Sinh hc phn t 62
t bin d nhin xy ra trn DNA v c sao li trn mRNA trong
phin m, ri trn protein trong dch m.
V tr V tr th hai V tr
th nht th ba
U C A G
Ch thch
Nhng n v m (codon) c c theo chiu 5 3.
STOP: codon kt thc (cn gi l v ngha).
Sinh hc phn t 63
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.
Sinh hc phn t 64
A AUG ACU CGG AAG UCA CUA ACG AUU AGG CUU UAC ...
Met Thr Arg Lys Ser Leu Thr Ileu Arg Leu Tyr ...
B AUG ACU AGG AAG UCA CUA ACG AUU AGG CUU UAC ...
Met Thr Pro Lys Ser Leu Thr Ileu Arg Leu Tyr ...
C AUG ACU CGG AAG UGA CUA ACG AUU AGG CUU UAC ...
D AUG ACU CGG ACA GUG ACU AAC GAU UAG GCU UUA ...
E AUG ACU CGG AGU GAC UAA CGA UUA GGC UUU AC ...
Sinh hc phn t 65
nhin, cn c vo hng lot tnh cht ha hc cc protein khng th lm
khun mu cho s tng hp ca chnh chng. V vy, khun mu tng
hp nn protein khng phi l protein.
- Sinh tng hp protein tch ri v khng gian vi ch cha DNA.
Nhiu nghin cu cho thy tng hp protein c th xy ra khi khng c mt
DNA. S kin ny th hin r rng nht nhng t bo eukaryote. Trong
nhng t bo ny, hu nh ton b DNA tp trung nhim sc th trong
nhn, cn tng hp protein ch yu din ra t bo cht. To xanh n bo
Acetabularia khi b ct mt phn cha nhn vn tng hp c protein v
sng vi thng nhng mt kh nng sinh sn. R rng, ni cha DNA mang
thng tin di truyn v ch sinh tng hp protein tch ri nhau v khng
gian.
- DNA khng phi l khun mu trc tip tng hp protein, do
phi c cht trung gian chuyn thng tin t DNA ra t bo cht v lm
khun tng hp protein. Cht phi c c trong nhn v t bo cht vi
s lng ph thuc vo mc tng hp protein.
- Cht trung gian c xem chnh l RNA nh cc c im sau:
+ RNA c tng hp ngay trong nhn c cha DNA, sau n i
vo t bo cht cho tng hp protein.
+ Nhng t bo giu RNA tng hp protein nhiu hn.
+ V phng din ha hc RNA gn ging DNA: chui polyribo-
nucleotide thng cng cha 4 loi ribonucleotide A, G, C v uracil (U). N
c th nhn c thng tin t DNA qua bt cp b sung.
Ni chung, trong t bo khng th tm thy cht no khc ngoi RNA
c th ng vai tr trung gian cho tng hp protein. Mi quan h ny chnh
l thng tin di truyn i t DNA qua RNA ri n protein v c biu din
hnh 3.3. Mi quan h ny cn c gi l l thuyt trung tm (central
dogma), c Crick a ra t 1956 n nay v cn bn vn ng.
Vo nhng nm 1970, ngi ta pht hin qu trnh phin m ngc
t RNA tng hp nn DNA nh enzyme reverse transcriptase. n nay, vic
sao chp (tng hp) RNA trn khun mu RNA cng c chng minh
nhiu loi virus. Ngoi ra, thng tin t protein cng c th c truyn sang
protein (prion ca bnh b in). Ring dng thng tin t protein ngc v
mRNA/DNA th cha c tm thy (Hnh 3.4).
Sinh hc phn t 66
Phin m Dch m
DNA mRNA Protein
Sao chp
Phin m Dch m
DNA mRNA Protein
Phin m ngc ?
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.
Sinh hc phn t 67
Nm 1964, Khorana tm ra phng php tng hp mRNA nhn to
vi trnh t lp li (nh AAG AAG AAG...) v nh n gii quyt xong cc
vn cn cha r rng.
Bng m di truyn (Bng 3.4) cho thy trong 64 codon, c 3 codon
UAA, UAG, UGA khng m ha cho amino acid c gi l v ngha (non-
sense), ng thi l codon kt thc (termination) tc du chm cu, chm
dt chui polypeptide.
M di truyn c tnh suy bin (degeneration) tc mt amino acid c
nhiu codon m ha, ch tr methionine v tryptophane ch c mt codon
(tng ng l ATG v TGG). Cc codon ng ngha tc m ha cho cng
mt amino acid thng c hai base u tin ging nhau, nhng khc nhau
ci th ba. V d: CCU, CCC, CCA v CCG tt c u m ha cho proline.
Trn thc t, U v C lun lun tng ng nhau v tr th ba, cn A v G
tng ng nhau trong 14 trn 16 trng hp.
Tr mt s ngoi l, m di truyn c tnh ph bin (universal) tc ton
b th gii sinh vt c chung b m di truyn.
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
Sinh hc phn t 68
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 Vng 3
n v phin m cng hng
ngc hng
Sinh hc phn t 69
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
Promoter
Sinh hc phn t 70
cho thy gen c cu to phc tp, s bin i ca gen c th dn n nhiu
trng thi allele khc nhau.
B m a1/a2 a1/a1
Giao t a1 v a2 a1
Cc con lai a1/a1 v a2/a1 - c hai u l kiu hnh t bin
Sinh hc phn t 71
Chng vi khun E. coli
B K
Dng hoang di
t bin rII
Sinh hc phn t 72
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.
Sinh hc phn t 73
cis trans
1 2
a1 a2 + a1 + +
I: Hai t bin
trn mt cistron
+ + + + a2 +
Gen A Gen B Gen A Gen B
3 4
a1 + b a1 + +
II: Hai t bin
trn hai cistron
+ + + + + b
Gen A Gen B Gen A Gen B
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.
Sinh hc phn t 74
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 B
I
ade4 ade2
III
ade6
IV
ade7
V
ade8 ade1
VI
Sinh hc phn t 75
Chng 4
Ti bn DNA
1.2. C ch ti bn eukaryote
S ti bn t bo eukaryote phc tp hn so vi t bo prokaryote
nhng c ch ca s ti bn eukaryote cng tng t nh prokaryote v
tin hnh theo cc nguyn tc:
- Hai hng.
- B sung, i song song, theo chiu 5 3.
- Khng lin tc mt trong hai chui.
- Cn nhng RNA primer.
Tuy nhin, c mt s im khc nh sau:
- Trong khi prokaryote ch c mt im khi u, th s ti bn
eukaryote bt u cng mt lc nhiu im khi u. iu ny l cn thit
do DNA ca eukaryote c chiu di rt ln.
- Vn tc pht trin ca chc ba ti bn eukaryote (khong 50
nucleotide/s) ch bng 1/10 so vi E. coli.
Sinh hc phn t 76
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.
H H
Chui mi
Chui m
Cc phn t con
DNA lai (15N/14N) th h th nht
H L L H
DNA nh (14N)
Cc phn t con
DNA lai th h th hai
L L H L L H L L
Sinh hc phn t 77
- 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.
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 B
Chuyn ng
ca chc ba ti
bn
Gc
im t (nick) 1
im t 2
3 5 3
5 3 5
(a)
3
(b)
(c)
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
Gc ti bn Gc ti bn
Ti bn ng thi
Ti bn ch trong c hai hng
trong mt
hng Ti bn khng Ti bn theo
nh hng hai hng
Gc ti bn
Gc ti bn
Chc ba Chc ba Chc ba
ti bn ti bn ti bn
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 Gc ti bn
v theo hai hng
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.
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).
3-OH
5-P
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
Sinh hc phn t 82
(protein Dna G) s xc tc tng hp RNA primer gn vo khun mu
DNA.
Kp
Si ch Polymerase III dimer
Helicase
Chc ba ti bn
DNA b m
Khun mu si ch
Primase
RNA primer
Khun mu si th
Cc protein lin kt
DNA si n (SSB)
Sinh hc phn t 83
(
- .
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.
Sinh hc phn t 84
Si ch
DNA b m
Cc on Okazaki
DNA polymerase
Ligase
Si ch
Si th
Hng ti bn
Sinh hc phn t 85
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).
RNA primer
Si th RNA primer Primase
on Okazaki k tip
c tng hp
DNA polymerase I
DNA ligase
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)n+1 + PPi
DNA polymerase
3 4.13).
Sinh hc phn t 87
DNA khun mu
5 3
3 5
5 3 Mg2+
Primer
DNA polymerase I
4dNTP
4.13.
Sinh hc phn t 88
150 dNTP.
Sinh hc phn t 89
- 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
th . Phn
ng ny cn s c mt ca ATP (Hnh 4.16).
Sinh hc phn t 90
B phn ti bn
Ti bn
t gy DNA Topoisomerase II
Chuyn DNA i
qua ch t gy
Siu xon m ( )
(Hnh 4.17)
.
Sinh hc phn t 91
ATP
DNA helicase
ADP + P i
4. DNA ligase
- 5-PO4
2ATP
(i vi eukaryote) hoc NAD+ (i vi vi khun).
O O
Ligase
DNA-3-OH + O-P-O-5-DNA DNA-3-O-P-5-DNA
2ATP
O O
Sinh hc phn t 92
A B
Lin kt ca cc
SSB b sung
O-
-O-P-O -
HO O
Enzyme + ATP
hoc
Enzyme + NAD
Enzyme- AMP
O-
- O-P- O- Adenine-Ribose
O
- O-P-O-
HO O
-O O
P
O O
Sinh hc phn t 93
- -
Sinh hc phn t 94
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
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.
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.
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:
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.
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.
Sinh hc phn t 98
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.
Sinh hc phn t 99
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 5
Ti xon M xon
Ko di
RNA polymerase
RNA
Kt thc
5
Cu trc
kp tc
5 3
Prokaryote Eukaryote
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.
Hp TATA
TFIIA
RNA polymerase II
TFIIF
TFIIE
TFIIH
Tc nhn
hot ha
Phc hp
ti m hnh
Phc hp
chromatin
trung gian 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
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.
base base
3 HO 5 5 3
RNA triphosphatase
3 HO 5 3
Guanylyl transferase
5 5
3 HO 3
Methyl transferase
H3C
7 methyl
5 5
3 HO 3
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.
snRNP
5 3
snRNPs
5 3
5 3
5 3
5 3
tRNA
3
(1) 5 R U5 U3 R 3 B gen RNA ca
PBS retrovirus
DNA
tRNA
3 R U5
(2)
5 R U5 U3 R 3
PBS
tRNA
(3) 3 R U5
5 U3 R 3
PBS
tRNA
(4) 3 R U5
5 U3 R 3
PBS
PBS
tRNA
(5) 3 U3 R U5
5 U3 R 3
PBS
PBS
(6) tRNA
3 U3 R U5
5 3
PBS
tRNA
(7) 3 U3 R U5
5 U3 R U5 3
PBS
PBS
tRNA
(8) 3 U3 R U5
5 U3 R U5 3
PBS
PBS
(9) 3 U3 R U5 5
5 U3 R U5 3
PBS
Dch m
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:
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
Chnh xc l t 3-4 phn t RNA v t 55-82 protein.
NH3
Chui polypeptide
ang tng hp
tRNA c gii
phng t v tr E
Trung tm
peptidyltransferase
C
CC
A A A U CG
5 3
GG G U U U A G C
U
E P A
Trung tm gii m
Chuyn ng 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.
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:
Cc yu t khi u
IF2-fMet-tRNAi
+ mRNA
IF3
fMet
5 mRNA
IF1 + IF2
fMet
1.2. Eukaryote
1.2.1. Bc 1: S hnh thnh phc hp tin khi u 43S
Giai on khi u i hi s h tr ca hn 30 protein khc nhau,
mc d eukaryote cng c nhng yu t khi u tng ng vi prokaryote.
Cc yu t khi u ny c k hiu l eIF.
Khi ribosome ca eukaryote hon thnh mt chu trnh dch m, n
tch ri ra thnh tiu n v ln v tiu n v nh t do thng qua tc ng
ca cc yu t eIF3 v eIF1A (tng t vi IF3 prokaryote). Hai protein
gn GTP l eIF2 v eIF5B lm trung gian thu ht tRNA khi u gn
methionine (ch khng phi N-formyl methionine nh prokaryote) n
tiu n v nh. Chnh yu t eIF5B-GTP l tng ng vi IF2-GTP ca
prokaryote. Yu t ny lin kt vi tiu n v nh theo phng thc ph
thuc eIF1A. Ri eIF5B-GTP gip thu ht phc hp eIF2-GTP v Met-
tRNAiMet n tiu n v nh. Hai protein gn GTP ny cng nhau a Met-
tRNAiMet vo vng thuc v tr P ca tiu n v nh. Kt qu, hnh thnh
phc hp tin khi u 43S.
ATP
ADP + Pi
Met
Tiu n v 60 S
Cc yu t khi u
Met
2. Giai on ko di
2.1. Bc 1: Aminoacyl-tRNA c a n v tr A nh yu t ko di EF-
Tu
Mt khi tRNA gn amino acid th EF-Tu n gn vo u 3' ca
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.
Phc hp
bc ba
Pi
Aminoacyl-
tRNA gn
vo v tr A
tRNA gii
phng v tr E
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.
RF1
5 3
Phn tch
peptidyl-tRNA RF1
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
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.
Ni u khng tng ng
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
Hp th nh sng
Li ti bn to ra s ghp
i lch A-C
GATC A
CTAG C
A
GATC
CTAG
Si khng c methyl
ha b ct gy
GATC A
CTAG T
Tng hp DNA
Methyl ha sa cha li
GATC A
CTAG T
3. Sa cha ct b
C hai loi h thng sa cha ct b, l:
- H thng sa cha ct b base (base excision repair) loi b trc tip
base sai hng v thay th n trong DNA. V d in hnh l enzyme DNA
uracil glycolase, loi cc uracil khng c bt cp (mispaired) vi cc
guanine.
- H thng sa cha ct b nucleotide (nucleotide excision repair) ct
b mt trnh t bao gm cc base sai hng; sau mt on DNA mi c
tng hp thay th cc nguyn liu b ct b. Cc h thng ny ph bin
hu ht sinh vt.
3.1. Ct b base
Vic loi b ch sai c thc hin nh mt hoc vi enzyme N-
glycosylase. N-glycosylase nhn bit base bin i hay mt gc amine hoc
bin dng cu trc xon do sai lch to ra v thy phn lin kt N-glycosylic
gia base vi ng vi pentose. L hng va c to ra do ct b c
DNA polymerase lm y li da vo khun mu b sung i din v ligase
ni lin chng (Hnh 7.4).
3.2. Ct b nucleotide
Vic ct b mt vng c nhiu thymine dimer (do UV) hoc vng lin
kt cho gia cc si, c thc hin nh incision nuclease (nuclease to
khc trn DNA) nh phc hp Uvr ABC ca E. coli, phc hp ny ct cc
on 12-13 nucleotide t mt si DNA. S thy phn c thc hin lin
kt phosphodiester th tm u 5 n ch hng v pha 3 l lin kt
th t hay nm.
DNA
glycosylase
V tr AP
AP
endonuclease
NTPs
DNA
polymerase Deoxyribose phosphate + dNMPs
DNA ligase
DNA mi
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
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.
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
VNG IU HA CC GEN CH
CM NG CA RecA
RecA c hot ha
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.
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.
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
RNA
polymerase
DNA
mRNA
Polyribosome
0,25 m
Chiu dch m
DNA RNA
polymerase
Polyribosome
Ribosome
mRNA Polypeptide
Tn hiu
NHN
Cht nhim sc
PHIN M
RNA Exon
Th phin m s cp
Intron
Hon chnh RNA
ui
mRNA trong nhn
M
Chuyn vo t bo cht
T BO CHT
mRNA trong t bo cht
S t gy
ca mRNA Dch m
Polypeptide
Nhng bin i ha hc hu
dch m v vn chuyn ti
t bo
S t gy ca protein
Protein b t gy
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.
4. Mc dch m
S bin i ca cc nhn t khi u IF (inititation factor). L cc
protein kt hp vi tiu n v ca ribosome vo giai on khi ng ca
qu trnh dch m.
5. Mc hu dch m
y c s iu ha hot tnh ca protein. Sau khi mch polypeptide
c tng hp, cc protein nhiu khi phi tri qua cc bin i th cp trc
khi biu hin hot tnh (chc nng). V d: trypsin l enzyme phn gii
protein trong d dy ch c c hot tnh sau khi cht tin thn ca n (pro-
enzyme khng c hot tnh) b ct mt mt on polypeptide.
Cc protein c th chu nhng bin i lp th nh s kt hp cc
enzyme vi mt s sn phm c bit c th lm thay i cu trc khng
gian ca chng dn n mt hot tnh.
- Cc qu trnh glycosylation, phosphorylation tc l gn thm cc
nhm cht nh ng, phosphor protein c hot tnh/chc nng sinh
hc.
- Peptide tn hiu l on gm khong 20 amino acid nm gn pha
u N ca polypeptide, c vai tr gn polypeptide v ribosome ang tng
Operon
Operator
Gen cu trc
Gen iu ha RNA Gen a Gen b Gen c
Promoter polymerase Promoter
Phin m
Phin m
mRNA
mRNA
Dch m
Dch m
Protein iu ha
Cc protein (enzyme) A B C
Cc con ng ha sinh
Tin cht X Cc sn phm Sn phm Y
trung gian
35 bp 10 bp +1
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.
Operon
(a) Khng c lactose
RNA Operator lacO
polymerase Gen cu trc
Gen iu ha
lacI Khng th lin kt X lacZ lacY lacA
pI lacP
Protein iu
ha hot ng
(protein km
hm)
X
Phin m v
Phin m v
dch m
dch m
Protein iu
ha hot ng
Enzymes
Protein iu -Galactosidase Permease Transacetylase
ha bt hot
Glucose
Allolactose
Lactose Galactose
-Galactosidase
mRNA
Protein iu ha Enzymes
bt hot
Chorismate
(trp repressor) Tryptophan
Operator
(b) Nng tryptophan cao
PR
Protein iu ha
bt hot
dn ti to thnh cu trc
stem-and-loop c ch RNA
polymerase v kt thc phin
m
L-Arabinose
Gen c ch
(bn ngoi)
araC mRNA Cc v tr
iu chnh
Cc gen cu trc
araC araO2 araO1 CAP araI araB araA araD
Gen iu ha
araBAD mRNA
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).
Promoter
Activators
Gen
DNA
Enhancers
Hp TATA
2 Cc nhn t phin m
RNA polymerase
3 RNA polymerase
A. Xon-nt-xon B. Xon-vng-xon
Khung
Cp base
ng-phosphate
DNA COOH
NH2
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
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.
Glucocorticoid receptor
trong s hin din ca
glucocorticoid hormone Glucocorticoid hormone
gen 1 gen 1
gen 2 gen 2
gen 3 gen 3
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.
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.
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
a HindIII
u dnh
b PvuII
u bng
5-G GATCC-3
BamHI Bacillus amyloliquefaciens Dnh
3-CCTAG G-5
5-A GATCT-3
BglII Bacillus globigii Dnh
3-TCTAG A-5
5-G CGC-3
CofI Clostridium formicoaceticum Dnh
3-CGC G-5
5-TTT AAA-3
DraI Deinococcus radiophilus Bng
3-AAA TTT-5
5-G AATTC-3
EcoRI Escherichia coli Dnh
3-CTTAA C-5
5-GG CC-3
HaeIII Haemophilus aegypticus Bng
3-CC GG-5
2. Isochizomer
.
:
- Mbo Sau :
5 GATC 3
3 CTAG 5
Ligase
- Bam :
5 GGATCC 3
3 CCTAGG 5
Sal XhoI:
5G TCGAG3 5GTCGAG3
3CAGCT + C5 3CAGCTC5
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).
1
Cht ty lm bin i b mt t bo gii phng cc thnh phn t bo ra mi
trng bn ngoi.
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.
RE
Ct DNA ngoi lai v RE RE
plasmid vector bng mt
Plasmid vector
loi RE ging nhau
Gn DNA
DNA c to dng
Plasmid ti t hp
Bin np
T bo 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
45 kb Nhim sc th ca
EcoRI EcoRI Bacteriophage
20 kb 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
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.
Tet r
ori
Scal
BamHI
cos ori Tetr cos
T4 DNA ligase
50 kb
ng gi phage in vitro
u cos
DNA vector c u
ng gi 50 kb
ui
Si ui
Xm nhim
ori
cos
Tet r
Plasmid
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
:
-
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.
) trnh t
nucleotide quan tm
(physical mapping).
2
YAC (yeast artificial chromosome): Nhim sc th nhn to ca nm men.
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.
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 lac- lac
lac (lac repressor).
- -
E. coli
16S
rRNA.
Vng to dng
tac
5S promoter
T1
Ptac
T2
pKK177-3 ori
(2,9 kb)
r
Amp
2000
1000
Vng to dng
SmaI 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 :
- 16S rRNA.
-
AUG.
EcoRI 0
HindIII
1000
5000
r
Amp PL PL
nuL
pAS1
nuR
(5,8 kb) tR
cII
ori RBS
BamHI
2000
4000
3000
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 .
EcoRI
5000 Vng to dng
Pst I
r
Amp
1000
4000
pUR278
(5,2 kb) lacZ
ori
Plac
lac
3000 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
1. Gn khng nguyn ln
b mt
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
Hnh 9.11. K thut Western blot v ELISA da trn phn ng lin kt khng
nguyn-khng th
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
, s lng
DNA 3- -
5-
- ).
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.
Lm lnh cc primer
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
ia
in vitro
.
- .
.
- .
in vitro
3
bin
3
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.
gel
9.13).
DNA + enzyme Cc on
ct hn ch Mng ny lon Giy thm
hn ch
(hoc nitrocellulose)
I Gel
1
II
Vt xp
III thm nc
Dung dch kim
Giy thm c
tch ra
Phim
Ra tri cc probe I
khng c gn 1II
DNA c nh III
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.
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.
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
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.
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
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
.
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).
.
(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.
.
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
D-loop.
.
Deoxyribonucleotide triphosphate (dNTP).
triphosphoryl ha (
).
Deoxyribonuclease (DNase). (phn
hy) DNA si i hoc DNA si n.
Deoxyribonucleic acid (DNA).
,t
nucleotide
(U),
.
(sequencing).
Dimer.
.
DNA (complementary DNA, cDNA).
trn khun mu mRNA nh qu trnh (reverse
chu
xy dng th vin cDNA (cDNA library).
(foldback DNA).
(inverted repe
.
DNA khun mu (template DNA).
.
DNA polymerase.
.
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.
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.
(blunt end).
li 5 (protruding ends).
(cohesive ends hoc sticky ends).
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.
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.
, 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
.
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.
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 .
.
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.
(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.
.
Phn ng thy phn (hydrolysis). Phn ng trong mt lin kt
cng ha tr b ph v khi c s kt hp mt phn t nc.
Pht sinh t bin (mutagenesis). Qu trnh to ra t bin trong
DNA.
Phn tch trnh t gen (gene sequencing). L k thut xc nh trnh
t theo cu trc bc mt ca chui cc nucleotide trong mt phn t nucleic
acid. Phn tch trnh t ca DNA c cc phng php ha hc ca Maxam-
Gilbert v phng php enzyme ca Sanger. Trong nhng nm gn y,
mt s phng php xc nh trnh t mi nh s h tr ca my tnh
xut hin. Bn cnh k thut thng thng s dng cc polyacrylamide gel
phn ly cc phn t DNA c di khc nhau, cc k thut mi lin
quan n pht hin hunh quang ca cc nucleotide c nh du, phn
tch trnh t DNA bng khi ph, in di mao dn hoc lai vi cc on
oligonucleotide c tng hp nhn to cng ra i. Nm 1980, Sanger
(Anh) v Gilbert (M) c trao gii Nobel do c nhng ng gp
quan trn v phng php xc nh trnh t cc nucleotide trong phn t
DNA. ng gp ny l mc lch s to ln trong sinh hc phn t, l nguyn
l ca tt c cc my xc nh trnh t DNA t ng ang s dng hin nay
trn khp th gii.
Phn cui (telomere). on cui, phn cui ca mt nhim sc th
thng ca eukaryote, bao gm nhng trnh t DNA ngn c lp li nhiu
ln.
Phn tm (centromere). Phn co tht c thy trn nhim sc th
k gia, l ni hai nhim sc t nh vi nhau.
Phin m (transcription). Qu trnh tng hp mRNA t khun mu
DNA.
Phin m ngc (reverse transcription). Qu trnh tng hp DNA
t khun mu mRNA nh enzyme phin m ngc (reverse transcriptase).
.
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
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 (pre-
mRNA), thng c kch thc rt ln. N phi c x l (splicing) to
ra phn t mRNA hon chnh (mature mRNA) dng cho dch m.
Sng lc (screening). K thut nhn dng mt dng DNA trong mt
th vin h gen (genomic library) hoc th vin cDNA (cDNA library) bng
mt phng php lai mu d c nh du [ -32P]dCTP vi cc vt tan
(trng hp dng bacteriophage lm vector to dng v cho xm nhim
vo vi khun E. coli) hoc khun lc (dng plasmid lm vector to dng)
ca cc th vin trn mng nylon hoc nitrocellulose. Tn hiu lai c
pht hin bng phng x t ghi trn phim X-quang.
Sinh hc phn t (molecular biology). Khoa hc nghin cu cc
hin tng sng mc phn t. Lnh vc khoa hc tr tui ny l im
gp nhau ca cc khoa hc kinh in nh di truyn hc, ha sinh hc, t bo
hc, vt l hc, ha hc hu c v ha l. Theo cch hiu ph bin hin nay,
sinh hc phn t l khoa hc nghin cu cc gen v hot ng ca chng
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.
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.
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
gn mi (annealing).
, 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
Trang
LI NI U
Chng 1. CC I PHN T SINH HC 5
I. Nucleic acid 5
1. Deoxyribonucleic acid 5
2. Ribonucleic acid 9
II. Protein 13
1. Cu trc ca protein 13
2. Chc nng ca protein 19
III. Lipid 22
IV. Polysaccharide 24
Ti liu tham kho/c thm 25
260
3. Vng vir 46
4. Qu trnh chuyn T-DNA vo t bo thc vt 47
V. Sp xp v khuch i cc gen trong genome 49
1. Sp xp li cc gen 49
2. Khuch i cc gen 54
3. Bin np gen 55
Ti liu tham kho/c thm 56
Chng 4. TI BN DNA 76
I. Chng minh ti bn DNA theo c ch bn bo th 76
1. C ch ti bn bn bo th 76
2. Th nghim ca Meselson v Stahl 77
II. M hnh ti bn DNA-chc ba ti bn 78
261
1. M hnh ti bn 78
2. Chc ba ti bn 78
3. Ti bn DNA theo vng trn quay 81
III. Bn cht xon ca DNA-Cc giai on ca s ti bn 82
1. M xon 82
2. Ko di-Tng hp chui Okazaki 83
3. Kt thc 84
IV. Khi nim mi 84
V. Enzyme ti bn 87
1. DNA polymerase 87
2. Cc topoisomer v DNA topoisomerase 89
3. Helicase v protein SSB 90
4. DNA ligase 92
Ti liu tham kho/c thm 94
Chng 5. PHIN M 95
I. Cc c im c bn ca qu trnh phin m 95
1. S phin m to ra RNA b sung vi mt si DNA 95
2. S phin m l mt phn ng enzyme 96
3. S phin m ch sao chp chn lc mt s phn ca genome 96
v to ra nhiu bn sao
4. Ch mt trong hai si n ca phn t DNA c dng lm 96
khun mu
5. S phin m c khi pht khng cn mi 97
II. Cc giai on ca qu trnh phin m 97
1. Giai on khi u 97
2. Giai on ko di 98
3. Giai on kt thc 98
III. Phin m prokaryote 98
262
1. Enzyme RNA polymerase prokaryote 98
2. Promoter ca gen prokaryote 99
3. Vai tr ca enzyme RNA polymerase v promoter trong qu 99
trnh phin m
4. Tn hiu kt thc 101
IV. Qu trnh phin m eukaryote 102
1. Cu trc gen eukaryote 102
2. Enzyme RNA polymerase ca eukaryote 103
3. Cc yu t gip RNA polymerase khi u phin m 104
4. Cc yu t kch thch RNA polymerase II hot ng trong 107
giai on ko di
5. Qu trnh bin i cc RNA mi c tng hp 108
V. Phin m ngc 110
Ti liu tham kho/c thm 114
263
1. Giai on khi u 120
2. Giai on ko di 125
3. Giai on kt thc 128
V. Cc nhn t c ch dch m 130
Ti liu tham kho/c thm 132
264
4. Khi qut v iu ha prokaryote v eukaryote 157
II. Cc mc iu ha 160
1. Mc cht nhim sc 160
2. Mc phin m 160
3. Mc hu phin m 161
4. Mc dch m 161
5. Mc hu dch m 161
III. iu ha biu hin gen prokaryote 162
1. Cu trc ca promoter 163
2. Cu trc ca operon 163
3. iu ha thoi dng: Kim sot m-cm ng 164
4. iu ha bin dng: Kim sot m-c ch 165
5. Kim sot dng v cm ng 170
IV. iu ha hot tnh ca eukaryote 171
1. Cc promoter 172
2. Cc enhancer 172
3. Cc protein l nhn t c tc ng trans 172
4. Hormone 175
5. Kim sot cc cht thng gp trong nhn 175
V. S bit ha t bo 176
1. Cc t bo bit ha mang thng tin ging nhau 176
2. Cc t bo bit ha tng hp cc nhm protein khc nhau 177
3. S iu ha mc phin m l ngun gc cn bn ca cc 179
sai khc gia nhng t bo bit ha
Ti liu tham kho/c thm 180
265
2. Lm vic mc phn t 182
II. Endonuclease hn ch 183
1. Gn cc u b ct bi enzyme hn ch 186
2. Isochizomer 186
III. Phng thc to dng 188
1. Plasmid vector 188
2. Bacteriophage vector 192
3. Cosmid vector 193
4. Th vin cDNA 195
5. Th vin genomic DNA 196
IV. Biu hin gen ngoi lai trong vi khun 197
1. Cc protein nguyn th ti t hp 198
2. Cc protein dung hp ti t hp 201
3. Xc nh mc biu hin ca gen c to dng 203
V. Phng php pht hin dng vi khun c DNA ti t hp 204
1. Lai khun lc v vt tan 204
2. Sng lc th vin gen bng PCR 206
3. Sng lc cc th vin cDNA bng cc probe khc nhau 208
4. Phn tch genomic DNA bng phng php lai Southern 209
VI. Cc ng dng ca cng ngh DNA ti t hp 211
1. ng dng trong dc phm 211
2. Cc vi khun c bit 212
3. Cc sn phm nng nghip 212
4. Thuc oligonucleotide 213
5. Liu php gen 214
6. Chn bnh can thip sm 214
7. DNA fingerprinting 215
8. Lp bn gen 216
Ti liu tham kho/c thm 217
266
Ph lc. MT S THUT NG C BN 218
Ti liu tham kho 259
MC LC 260
267