Professional Documents
Culture Documents
Sinh Hoc Phan Tu
Sinh Hoc Phan Tu
B Y T
SINH HC PHN T
(DNG CHO O TO DC S I HC)
M s: .20.X.06
NH XUT BN GIO DC
Ch o bin son:
V KHOA HC V O TO - B Y T
Ch bin:
GS.TS. NGUYN VN THANH
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 2 of 203
Li gii thiu
Thc hin mt s iu ca Lut Gio dc, B Gio dc & o to v B Y t ban hnh chng
trnh khung o to Dc s i hc. B Y t t chc bin son ti liu dy - hc cc mn c s v
chuyn mn theo chng trnh trn nhm tng bc xy dng b sch t chun chuyn mn trong
cng tc o to nhn lc y t.
Sch SINH HC PHN T c bin son da trn chng trnh gio dc ca Trng i hc Y
Dc Thnh ph H Ch Minh trn c s chng trnh khung c ph duyt. Sch c cc tc gi
GS.TS. Nguyn Vn Thanh, TS. Trn Thu Hoa, TS. Trn Ct ng, ThS. H Th Yn Linh bin son theo
phng chm: Kin thc c bn, h thng; ni dung chnh xc, khoa hc; cp nht cc tin b khoa
hc, k thut hin i v thc tin Vit Nam.
V KHOA HC V O TO - B Y T
LI NI U
Nm 1909, Johansen W. xut bn chuyn kho "Cc yu t ca hc thuyt ng n v bin d v di
truyn" (Element de exakten Erblichkeitslehre), trong ln u tin xut hin t gen. Nm 1953,
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 3 of 203
Watson v Crick khm ph ra m hnh xon kp ADN thc y nhanh chng s pht trin ca di
truyn hc mc phn t. Nm 1965, J. Watson xut bn sch "Sinh hc phn t ca gen" dy 494
trang v n nm 1976, trong ln ti bn ln th ba dy ln 739 trang. Tip sau , hng lot ti liu
v Sinh hc phn t ra i.
Cho n ngy 26 - 06 - 2000, ti Washington D.C, cng ty t nhn Celera Genomics (Anh) v D n
B gen Ngi (Human Genome Project) ca Vin nghin cu Quc gia v Sc khe ca Hoa K
(National Institute of Health) phc tho bn b gen ngi. Theo , b gen ngi c 3,12 t
nucleotid v 97% tng nucleotid c xc nh trnh t, trong c 85% s trnh t t ng v
tr. Khong 3% ADN c cha gen, 97% cn li l ADN "khng chc nng". Trong tng s 3% ADN
ny c khong 30 - 50000 gen.
Ngy 14 - 4 - 2003, T chc Quc t nh trnh t B gen Ngi (International Human Genome
Sequencing Consortium) tuyn b hon thnh nhng cng on cui cng ca bn gen ngi.
K bn gen, cc phng php tm gen c tnh cht tr liu s c thc hin quy m ln v
Dc l b gen (Pharmacogenomics) s khm ph su hn b gen ngi ng dng trong ngnh
Dc.
Sch "Sinh hc phn t" nhm gip cho sinh vin Dc khoa hiu c cu trc c bn v chc
nng ca gen. Sch gm cc bi: Nhp mn; Sao chp ADN; Cc loi ARN; S phin m v m di
truyn; Sinh tng hp protein; iu ho hot ng gen; B gen t bo nhn tht; t bin gen - Cc
phng php phn tch ADN.
CC TC GI
CC T VIT TT
A Adenine
ADN Acid desoxyribonucleic
AIDS Acquired immune deficiency syndrome
AP Apurinic or apyrimidinic
ARN Acid ribonucleic
bp Base pair
C Cytosine
cADN Complementary DNA
cds Coding sequence
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 4 of 203
CpG C phosphat G
CTF CCAAT binding Trascription Factor
DMD Duchenne muscular dystrophy
DNase Deoxyribonuclease
dNTP Desoxyribonucleozid triphosphat
dsADN Double stranded DNA
eEF Eukaryote elongation factor
EF Elongation factor
eIF Eukaryote initiation factor
eRF Eukaryote release factor
EGF Epidermal growth factor
FRET Fluorescence resonance energy transfer
G Guanine
GMO Genetic modified organism
Hfr High frequency recombination
Hft High frequency transduction
HIV Human immunodeficiency virus
hnARN Heterogenous nuclear RNA
Icr Insensitive to catabolite repression
IF Initiation factor
IGS Internal guide sequence
Kb Kilobase
LINE Long interspersed repetitive element
MALDI-TOF Matrix assisted lazer desorption ionization time of
fligt
mARN Messenger RNA
NAAAF N acetoxy 2 acetylaminofluorene
NAD Nicotinamide adenine dinucleotide
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 5 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 6 of 203
Bin np Transformation
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 7 of 203
B gen Genome
Cht cm ng Inducer
Cht c ch gc Aporepressor
C ch bn bo tn Semiconservative mechanism
C ch bo tn Conservative mechanism
D xc tc Heterocatalysis
a cistron Polycistron
u t Blunt end
in di trng xung
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 8 of 203
iu ho m Negative control
on d Probe
i m Anticodon
n cistron Monocistron
n v phin m Operon
Gen gi Pseudogene
Ht F F - prime
B gen hc Genomics
H protein Proteome
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 9 of 203
H protein hc Proteonomics
Hp t Zygote
Kh np Competence
Khun mu Template
Lc lp Chloroplast
Mi Primer
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 10 of 203
Nhim sc th Chromosome
Nt Loop
Nt kp tc Hairpin loop
Si sm Leading strand
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 11 of 203
S ct ni Splicing
S h bin Tautomerization
S phin m Transcription
S t ct ni Self - splicing
S t iu ho Autoregulation
Ti np Transduction
T bo cho Donor
T bo nhn Recipient
Th ct ni Spliceosome
Th a hnh Polymorphism
Th nhn Nucleoid
Th th Receptor
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 12 of 203
Tip hp Conjugation
Trnh t iu ho Operator
V tr AP AP site
V tr iu ho Regulatory site
V virus Capsid
Yu t ko di Elongation factor
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 13 of 203
Anticodon i m
AP site V tr AP
Aporepressor Cht c ch gc
Autoregulation S t iu ho
Blunt end u t
Cap Chp
Capsid V virus
Chloroplast Lc lp
Chromosome Nhim sc th
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 14 of 203
Cohesive Dnh
Competence Kh np
Conjugation Tip hp
Conservative mechanism C ch bo tn
Corepressor Cht ng c ch
De novo Mi
Donor T bo cho
Elongation factor Yu t ko di
F - prime Ht F
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 15 of 203
Genome B gen
Genomics B gen hc
Hairpin loop Nt kp tc
Heterocatalysis D xc tc
In situ Ti ch
Inducer Cht cm ng
Inhibitor Cht c ch
Leading strand Si sm
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 16 of 203
Si khun sm
Loop Nt
Monocistron n cistron
Nucleoid Th nhn
Operator Trnh t iu ho
Operon n v phin m
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 17 of 203
Polycistron a cistron
Polymorphism Th a hnh
Primer Mi
Probe on d
Proteome H protein
Proteonomics H protein hc
Pseudogene Gen gi
Receptor Th th
Recipient T bo nhn
Regulatory site V tr iu ho
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 18 of 203
Repressor Cht c ch
Self - splicing S t ct ni
Semiconservative mechanism C ch bn bo tn
Spliceosome Th ct ni
Splicing S ct ni
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 19 of 203
Tautomerization S h bin
Template Khun mu
Transcription S phin m
Transduction Ti np
Transformation Bin np
Zygote Hp t
Bi 1
NHP MN SINH HC PHN T
MC TIU
1.1. LC S
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 20 of 203
Gregor Mendel khm ph quy lut di truyn nm 1865, khi nghin cu s truyn tnh trng trn cy
u H Lan.
Nm 1944, Oswald Avery, Colin MacLeod, v Maclyn McCarty cng b bng chng thc
nghim u tin rng chnh ADN l vt liu di truyn. Avery v cc cng s c bi bo ca Griffith v
h c lp li th nghim vi nh tm kim cht chu trch nhim cho s chuyn i nhng khng
thnh cng. Sau nh mt phng php tch loi protein ra khi dung dch ca M. G. Sevag h
thnh cng trong vic chng minh ADN chnh l tc nhn truyn tnh trng.
Nm 1951, Erwin Chargaff, chng minh trong ADN t l nucleotid Adenin lun bng Thymin;
Cytosin bng Guanin hay ni cch khc t l base purin bng t l base pyrimidin; trong khi t l A + T
G + C v thay i theo loi.
Nm 1953, m hnh cu trc ADN ca Watson - Crick ra i, c xem l khm ph ln nht trong
Sinh hc ca th k. M hnh xon kp ADN do James D. Watson v Francis H. C. Crick xy dng l s
kt hp ca cng trnh v t l nucleotid do Edwin Chargaff xng v cng trnh nghin cu si
ADN bng tn x tia X ca Rosalind Elsie Franklin v Maurice Wilkins.
Nm 1967, enzym ADN ligase c chit xut. Ngi ta xem enzym ny l keo dn phn t, c th
ni hai si ADN li vi nhau.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 21 of 203
Trong thp nin 70 ca th k XX, k thut di truyn ra i to nn cuc cch mng mi trong di
truyn v sinh hc phn t. Vic xc nh trnh t nucleotid (ADN sequencing) ca gen nhanh chng
dn n k thut mi: gy t bin im nh hng cho php to cc bin i tu trn gen.
Nm 1985, R.K. Saiki v K. B. Mullis cng b vic s dng PCR khuch i cc on gen
- globin in vitro. T y thao tc ADN (ADN manipulation), xc nh trnh t gen pht trin rng ri
v ng dng trong nhiu lnh vc khc nhau nh chn on, bin i di truyn, xc nh ph h,
Vic hon thnh bn gen ngi gip gii quyt nhng vn ln nh iu tr ung th, pht
trin phi, bit ho t bo.
Bng 1.1. Cc ct mc quan trng trong s hnh thnh v pht trin ca SHPT
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 22 of 203
1944 Oswald Avery v cng s chng minh ADN mang thng tin di truyn.
1946 Pht hin rng vt liu di truyn t cc virus khc nhau c th phi hp to
ra virus mi, mt th d v ti t hp di truyn.
1949 Pauling chng minh bnh hng cu li lim l mt "bnh phn t" do t
bin trong protein hemoglobin.
1953 James Watson v Francis Crick cng b cu trc xon kp ADN, m ra thi
i mi ca sinh hc.
1960 S dng tnh cht b sung cp base to phn t ARN - ADN lai.
1970 Pht hin enzym gii hn, m ng cho vic to dng gen.
1973 Stanley Cohen v Herbert Boyer ct v ni ADN thnh cng (dng enzym gii
hn v ligase) v to ra ADN mi vi khun.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 23 of 203
nh hng gen.
Ct ni ARN.
1982 Applied Biosystems, Inc., gii thiu my nh trnh t protein pha kh u tin,
gim thiu lng protein cn nh trnh t.
1995 Trnh t b gen hon chnh ca mt sinh vt khng phi virus c xc nh:
Hemophilus influenzae.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 24 of 203
2004 FDA ln u tin cng nhn h thng ADN microarray, Ampli Chip
Cytochrome P450 Genotyping Test, gip chn on bnh.
Gii m B gen G.
1.2.1. nh ngha
Hc thuyt trung tm (Central Dogma) ca Sinh hc phn t c pht biu ln u tin bi Francis
Crick nm 1958 v s lun chuyn thng tin ca sinh vt: "Thng tin khi chuyn sang protein th
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 25 of 203
khng th ly ra li c". Ngha l thng tin khng th chuyn ngc t protein n acid nucleic.
Thng tin y l trnh t chnh xc cc nucleotid ca acid nucleic quy nh trnh t acid amin ca
protein.
Theo thng tin c th c chuyn gia cc phn t ADN thng qua s sao chp hoc gia phn
t ADN v ARN thng qua s phin m hoc t acid nucleic n protein nh s dch m. Ngoi ra
thng tin cn c th di chuyn gia ARN n ARN trong qu trnh sinh sn ca cc ARN virus hay qu
trnh x l ct ni ca ARN hoc ngc li t ARN n ADN trong qu trnh phin m ngc ca
retrovirus. Cc qu trnh ny nghin cu chi tit trong Sinh hc phn t.
To dng biu hin c s dng nghin cu chc nng protein. on ADN cn quan tm c
to dng vo mt plasmid v a vo t bo biu hin. Bng cch thay i c cu biu hin trn
plasmid hoc tinh ch protein ngi ta c th hiu c cch thc iu ho hot ng ca gen, quan h
ca n vi hot ng ca cc gen hay protein khc cng nh cu trc v chc nng ca n.
1.2.3.2. PCR
PCR l mt k thut cho php khuch i mt on gen c hiu thu c nhiu bn sao phc v
nghin cu. Bn cnh , PCR cng cho php d dng a cc thay i, t bin vo ADN nghin
cu.
1.2.3.3. in di gel
y l mt cng c phn tch chnh trong Sinh hc phn t. N da vo vic phn tch cc phn t
sinh hc nh ADN, ARN, protein trong in trng, trong cc phn t ny s di chuyn di tc
ng ca in trng xuyn qua mt h gel, do chng c th c tch ra theo kch thc hay in
tch.
K thut lai vt Southern c s dng pht hin s hin din ca mt trnh t ADN c hiu
trong mu bng cch chuyn cc phn t ADN c phn tch bng in di gel sang mt mng rn
v cho lai pht hin nh mt on d c hiu c nh du. K thut lai vt Northern vi nguyn l
tng t nhng c p dng cho i tng ARN, c s dng nghin cu s biu hin ca cc
ARN.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 26 of 203
Protein cng c th c phn tch trn gel v lai vt trn mng rn (lai vt Western) v pht hin
bng k thut ho min dch. Nh c th nghin cu s hin din ca protein trong t bo v lin h
vi s biu hin ca gen tng ng.
C nhiu loi genomics tu theo kha cnh v lnh vc m n nghin cu p dng. Trong mi loi
genomics cng li gm nhiu lnh vc, v d nh plant genomics gm cng ngh sinh hc thc vt, sinh
hc phn t v cc k thut lin quan n thc vt (nng nghip, lm nghip, lm vn).
Kh nng gii trnh t y b gen sinh vt y mnh vic xc nh chc nng cc gen mc
ngha rng ca b gen. V cc gen c chc nng lin quan c iu ho cng nhau, cc k thut
nh gi s biu hin ton b gen s gip nhn nh c ch khi u v cm cc trnh t gen mi c
chc nng lin quan. Xp cc trnh t gen thnh cc nhm chc nng theo s biu hin ca gen s cho
hng dn c bn cc thc nghim b sung, nhm xc nh c im chc nng chnh xc ca sn phm
cui cng ca gen. Trong hai thp k va qua, cc k thut nh gi s biu hin gen c nhng
tin b, t cc phng php trn cc gen n l c hiu (v d lai vt Northern, slot v dot; phin m
ngc v PCR bn nh lng v nh lng; phng php bo v nuclease) n cc k thut tp trung
vo nhn nh tt c cc gen c biu hin khc nhau gia hoc trong cc mu th nghim.
Cc phng php nhn nh cc khc bit ca biu hin gen gm c lai loi tr (subtractive
hybridization), trnh din khc bit (differential display), phn tch hng lot biu hin gen (SAGE,
serial analysis of gene expression) v lai vi bn tri (microarray hybridization).
Trc y, theo phng php th cng, mi tun, mi ngi ch thc hin c mt vi phn ng
gii trnh t vi nng sut 300 bp/phn ng. Ngy nay vi h thng my mao mch c th xc nh t
ng ng thi 96 phn ng vi di trn 1000 bp/phn ng. Nh n gii trnh t b gen ngi
di 3,2 t nucleotid hon thnh vo thng 4/2003.
Proteomics l nghin cu quy m ln v protein nhm thit lp cc du hiu nhn dng, s lng
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 27 of 203
cu trc v chc nng sinh ho v t bo ca tt c protein trong c th, c quan hoc tiu c quan
cng nh s thay i cc c tnh ny theo khng gian, thi gian v trng thi sinh l. T "proteomics"
c t ra tng ng vi genomics. Proteome (b protein) ca mt sinh vt l tp hp cc protein
bi n trong thi gian sng ca n. Proteome ca mt t bo tp hp cc protein trong n. T
"proteome" xut pht t "proteins" v "genome", v genome (b gen) m ho cc protein.
Proteomics thng c xem l bc tip theo trong nghin cu cc h thng sinh hc, sau
genomics. N phc tp hn genomics nhiu, ch yu l v b gen ca sinh vt n nh hn, proteome
khc nhau gia cc t bo v thay i lin tc qua cc tng tc sinh ho ca n vi b gen v mi
trng. Mt sinh vt c s biu hin protein khc nhau hon ton cc phn khc nhau trong c th ca
n. Mt kh khn chnh na l tnh phc tp ca cc protein lin quan n cc acid nucleic.
Kin thc v proteomics s gip hiu sinh vt tt hn nhiu so vi genomics. Cc phng php nh
phosphoproteomics v glycoproteomics c dng nghin cu cc bin i sau dch m (post -
translational modifications). Cc nghin cu proteomics bao gm nghin cu tng tc ca cc protein
v nhn din protein.
Nghin cu proteomics hin nay trc ht l protein phi c phn gii, i khi quy m ln. C
th tch protein bng in di hai chiu trn gel (two - dimensional gel electrophoresis). K thut ny u
tin tch protein theo im ng in v sau theo khi lng phn t. Cc im protein trn gel c
th nhn c bng cch dng cc cht nhum ho hc khc nhau hoc cc du hiu hunh quang.
Thng th cc protein c th nh lng da vo cng nhum mu ca chng. Cc protein c
nhn din khi c tch ring v nh lng. Cc m ring r c ct ra khi gel v ct thnh cc
peptid bng cc enzym thu phn gii protein. Sau cc peptid ny c nhn din bng khi ph, c
bit l khi ph MALDI - TOF. Trong quy trnh ny, peptid c t trn matrix lm peptid to cc tinh
th. Sau peptid c ion ho vi tia laser v s tng in th matrix c dng bn cc ion v
pha u d (detector) m thi gian ion n u d tu thuc vo khi lng ca n. Ion c khi lng
cng cao cng mt nhiu thi gian bay. Trong khi ph MALDI - TOF, cc ion cng c th b lch
hng vi gng phn x tnh in cng lm tp trung chm ion. Nh vy, c th xc nh vi chnh
xc cao s n u d th hai cc khi ca cc ion v cc khi ny c th pht hin chnh xc cc thnh
phn ca cc peptid, do nhn din chng.
Cc hn hp protein c th c phn tch khng cn tch trc. Quy trnh ny bt u bng phn
gii cc protein trong phc hn hp. Cc peptid to thnh thng c bm vo ct sc k lng hiu
nng cao (HPLC) lm tch cc peptid da vo tnh khng a nc. Cc peptid tch ra t ct c th
c nhn din bng khi ph song song (MS/MS). Giai on u tin ca khi ph song song l phn
lp cc ion peptid ring r, giai on th hai s ph v cc peptid thnh cc on v s dng khun mu
ca on (fragmentation pattern) xc nh trnh t acid amin ca chng. C th dng nh du vi
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 28 of 203
cc nhn ng v so snh nh lng nng cc protein trong hai hoc nhiu hn hai mu
protein.
- in di mt chiu v hai chiu trn gel (One - and two - dimensional gel electrophoresis).
- Tinh th tia X v cng hng t ht nhn (X - ray crystallography and nuclear magnetic
resonance).
- X - ray Tomography.
- Phn mm.
Pht minh v pht trin thuc l mt qu trnh di, sn phm ra th trng cn khong 15 nm t
khi c tng cho thuc c ch tim nng. Cc giai on c bn qu trnh c minh ha trong hnh
1.3. Di truyn hc c th tc ng n tng pha ca qu trnh ny. Tc ng ca di truyn hc v b gen
hc ln x hi s l d on di truyn v an ton v hiu qu ca thuc sm nht trong thng
trng.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 29 of 203
Genomics to ra s thay i c bn trong cch s dng thuc v qu trnh pht trin thuc. S thay
i quy m ny c l s xy ra trong 5 n 10 nm ti. Nguyn nhn ca s thay i l cuc cch mng
khoa hc v k thut trong lnh vc B gen Ngi (Human genome, bn a hnh n nucleotid
(single - nucleotide polymorphism, SNP), nh loi gen (genotyping) v tin sinh hc (bioinformatics).
SNP l nhng du hiu o lng v th nghim tm sot hiu nng cao. Cc tin b trong nh
gi biu hin gen cng l diu k. Trong vng nm nm, s lng cc gen, nhy ca cc th nghim,
lp li ca cc kt qu cng gia tng ng k kh nng phn tch cc s liu. o s biu hin gen
c dng trong di truyn ung th v dc hc b gen ung th (cancer pharmacogenomics). S biu
hin gen gip cch phn loi ung th v liu php thch hp tt hn.
Cc li ch m pharmacogenomics em li gm c:
- Cho cc thuc cng hiu hn. Cc thuc mi da trn cc phn t protein, enzym, v RNA lin
quan n cc gen v bnh c ch to. C th pht minh thuc v cho cc du hiu thuc (drug maker)
a ra liu php c ch chuyn bit. S chnh xc ny khng ch ti a ho hiu qu tr liu m cn
lm gim s ph hu cc t bo khe mnh gn .
- Cho cc thuc tt hn, an ton hn ngay t u. Thay v phng php chun th - v - sai (m
mm) cc bnh nhn ph hp vi cc thuc ng t ban u, cc bc s c th phn tch lc di
truyn ca bnh nhn v k toa thuc tt nht c. y khng ch l cng vic tm thi tm thuc
ng, m cn cho thi gian bnh phc nhanh v tng an ton v cc phn ng ph c th xy ra s
c hn ch.
- Xc nh liu thuc thch hp bng phng php chnh xc hn. Cc phng php hin nay
nh liu c s theo khi lng v tui s c thay th bng nh liu c s theo di truyn ca con
ngi c th x l thuc v thi gian chuyn ho n nh th no. iu ny s ti a ho gi tr ca
liu php v cn gim kh nng qu liu xy ra.
- Tin xa trong sng lc bnh. Kin thc m di truyn ngi s cho php con ngi to li sng
thch hp v cc thay i mi trng sm trnh hoc gim mc trm trng ca bnh di truyn.
Tng t nh vy, tin b trong hiu bit tnh nhy cm ca bnh, c bit s cho php a ra bin php
theo di v iu tr cn thn nhng giai on thch hp nht ti a ho liu php cho chng.
- Ci tin trong pht minh, pht trin thuc. Cc qu trnh thch hp lm d dng th nhm trn
nhm dn s c di truyn chuyn bit cho mc thnh cng ln hn. Gi thnh v nguy c ca cc
th nghim lm sng gim do ch nhm vo nhng ngi c th p ng vi thuc.
- Gim chi ph trong chm sc sc khe. Gim s lng cc phn ng ph ca thuc, s lng th
thuc tht bi, thi gian thuc c ph duyt, thi gian bnh nhn cn dng thuc, s lng thuc
m bnh nhn phi dng iu tr hiu qu, nh hng ca bnh ln c th (do pht hin sm) v tng
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 30 of 203
Cc thnh tu trong genomic v proteomic nhn din cc ch mi cho s can thip ca thuc l
cc c hi cha tng c cho pht minh cc tc nhn mi c cc kiu liu php tc ng mi.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 31 of 203
vng tip theo. Trong mi chu k, u th c gn cho cc ng vin tng hp bng cch s dng
cc qu trnh ti u ho a bin c thit k nhm m bo cc hp cht khng ch c ti u ho v
i lc gn vi ch m cn c cc c tnh ging thuc cho php chng c s dng trc tip nh l
cc hp cht mu trong cc m hnh sinh hc hay t bo thch hp (h).
Chip ADN c hnh dng ging nh mt con chip vi nhng chm ADN thay cho transitor. L mt
mnh mng lin kt cao c kch thc 20 40 mm c in trn cc on ADN. V d chip b gen
ngi c in 20000 - 50000 gen nhng im chm vung cc nh.
Khi lai chip ADN ny vi sn phm phin m ca b gen c th, cc chm ADN i mu tng ng
vi mc hot ng ca nhng gen tng ng trong c th trng thi v thi im nghin cu. T
kt lun c tnh trng bnh l ca bnh nhn.
Chip ADN ang dn dn tr thnh cng c chn on trong cng nghip ln men vi sinh, trong y
hc d phng, trong kim dch v an ton thc phm v trong kim sot mi trng.
Ngi ta thc hin chuyn vo cy trng cc loi gen tng cng kh nng khng su bnh nh gen
khng su nhm oxy/VIP, gen khng virus nhm CP/Nbi, gen khng thuc dit c nhm bar. Nhng
cng trnh chuyn gen l vo thuc l cho thy thc vt c biu hin gen l ging nh E.coli. Vo nhng
nm 1996, cc nh cng nghip (Monsanto) a ra th trng nhng sn phm cy chuyn gen u
tin nh bp (ng), u nnh v bng vi. Nm 1996 trn th gii mi c 0,5 ha cy trng chuyn gen
c a vo sn xut. n nay (2003) din tch trng cy chuyn gen trn quy m ton cu ln ti
67 triu ha. Ngy nay, ngi ta ang tm cch a gen sn xut vaccin, gen sn xut dc cht (cc
pepetid nh), cc cht dinh dng (vitamin A trong ht la), cy trng t lng thc thc phm tr
thnh cy sn xut dc liu c gi tr kinh t cao.
Trong s 50 loi cy trng mang gen l ang c th nghim th cc cy bng vi khng su, cy
u tng, cy ng khng su, khng thuc dit c chim tng s trn 90% din tch.
Cng ngh tin sinh hc bao gm cc phng php khai thc nhanh ngn hng d liu, phn tch
trnh t v cu trc ADN v protein. Tin sinh hc ang ci tin phng php x l phn tch s liu, ci
thin kh nng d on vng hot ng, vng ngng ngh ca b gen, ci tin kh nng phng on
phn ng t bo i vi tc nhn ngoi sinh, thit lp nn cc cu trc phn t c hot lc cao v nh
hng phn ho t bo mt cch hiu qu. Trn th gii hin c ba ngn hng d liu gen ln nht l
GenBank (NCBI Entrez Nucleotide - M), EMBL (European Molecular Biology Laboratory - Chu u)
v DDBJ (DNA Data Bank of Japan Nht Bn) lu tr trn 9 t d liu v gen.
Mt s thnh tu ni bt ca tin sinh hc l: chng trnh NMR a chiu thit lp cu trc khng
gian protein. Chng trnh FASTA so snh trnh t gen v protein ra i trc 1990 cho php so snh t
ng, min ph trnh t mt on gen di khong 1000 bp vi trnh t cng b trong vi pht.
Chng trnh BLAST, trung tm NCGR, chip ADN thit lp trc nm 2000 v gn y l n IBM
Blue Gene c bt u, h chng trnh trn gi EMBOSS c bn, chip ADN b gen ngi c
a ra th trng. n thi im ny c trn 60 cng ty ln chuyn dch v v kinh doanh trn lnh vc
sinh tin hc ang hot ng. DNAStar v GCG l hai trong nhng cng ty thnh cng nht trong lnh
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 32 of 203
y l lnh vc a ngnh, tp trung khai thc vt liu, thit b hoc phng php ca cc cht
phm vi kch thc ti hn nm gia chiu di phn t v bc sng nh sng kh kin t 0,1 n 500
nm. Cng ngh nano sinh hc l:
- Phng hng mi cho php thu nhn nhng thng tin v h thng sinh hc mc chm lng t,
to u d nano vi kch thc phn t dng trong chn on bnh.
- Vn chuyn v phn phi thuc hoc gen vo m v t bo thng qua vic khng ch kch thc
ht, hot ho v gii phng hot cht thuc thng qua c ch v thit b bm kch thc nano, van t bo
v c quan nhn to.
Trong Y hc, ngi ta hy vng phu thut gen, phu thut t bo, tr liu t bo, tr liu gen, tng
hp gen chn on i t bo, cc h thng c kh in t nano y hc - l cc "cng c nano" thng
minh, robot m kch thc nano, s c a vo thc tin trong vi chc nm ti.
CU HI
e) Watson v Crick
e) Watson v Crick
3. Bn gen ngi khi hon chnh cho thy c bao nhiu gen m ho cho protein?
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 33 of 203
e) 30 000 - 35 000
c) Thng tin ch lun chuyn gia cc dng acid nucleic khc nhau.
d) S sao chp, phin m, dch m l cc qu trnh chuyn thng tin trong t bo.
Bi 2
SAO CHP ADN
MC TIU
Mt t bo vi khun in hnh c th cha khong 2000 loi protein, cc t bo nhn tht cha
khong 50000. Thng tin v cc loi protein ny c m ho trong cc phn t acid nucleic. a s
loi, vt liu di truyn ch yu l ADN, mt s virus l ARN.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 34 of 203
Cc base ni vi nhau bng lin kt hydro theo tng cp b sung. Nh , khi chui xon kp tch
ra cho php cc base c sao chp to nn hai mch mi, c bn ging vi phn t ADN gc v y l
s sao chp c bn trong h thng sinh hc. Cc enzym xc tc qu trnh sao chp ca ADN l cc ADN
- polymerase. Mt s enzym khc cng c vai tr nht nh trong tin trnh ny.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 35 of 203
Trong th nghim ny, vi khun c cho pht trin vi th h trong mi trng N15 m bo cho
tt c ADN ca vi khun u l loi nng. Sau chuyn t bo vo mi trng c cha N14, nh l
ngun cung cp nit duy nht v cho phn chia trong mi trng N14, khi khi lng ADN tng ln
gp i, ADN c chit ra khi t bo v c ly tm trn thang nng cesium clorid. Nu c ch
bo tn c thc hin, hai bng ADN phi c thy r sau khi ly tm: mt bng nguyn thu (N15) v
mt bng cha N14 (hnh 2.2). Nu theo c ch bn bo tn th ch c mt bng c t trng trung gian
cha N14.5 xut hin. Nu ADN c un nng, ri lm ngui nhanh tch hai mch, ly tm trn thang
nng cesium clorid s thy hai bng tng ng vi ADN N15 v ADN N14.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 36 of 203
ADN polymerase I, II, III c tm thy t bo nhn nguyn thu. c tnh ca cc ADN
polymerase khc nhau c tm tt trong bng 2.1.
ADN polymerase I ca E. coli l mt chui polypeptid ln c khi lng phn t 109000. Trong
mi phn t c cha mt nguyn t km. Enzym polymerase I ch c th xc tc s polymer ho theo
hng 5 3 do s thm mt nucleotid t mt deoxyribonucleotid - 5 - triphosphat vo nhm 3 -
hydroxy ca chui ADN (hnh 2.3).
Tt c ADN polymerase ca t bo nhn nguyn thu cng c hot tnh exonuclease, v chng thu
phn mch n ADN ca chui t u 3 theo hng 3 5 (hot tnh exonuclease 3 5); hoc
chng c th thu phn chui ADN t u 5 (hot tnh exonuclease 5 3). ADN polymerase II ca
E. coli rt ging ADN polymerase I nhng khng c hot tnh exonuclease 5 3. ADN polymerase
III c cu to gm 7 n v nh: , , , , , , v . Tt c 7 n v nh ny u cn cho s sao
chp ca ADN ca E. coli. ADN polymerase III c th l enzym sao chp chnh ca E. coli v cc t
bin vi ADN polymerase III a nhit khng th sao chp c nhit 42oC. ADN polymerase I
chu trch nhim sa cha cc ADN h hng. Vai tr ca ADN polymerase II cha r.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 37 of 203
E. coli
Polymerase Virus
I II III
Khi lng phn t (Mr) 109 000 120 000 180 000 160 000
25 000 95 000
10 000
5 3 + - + -
3 5 + + + -
Polymerase
Khi lng 300000 45000 140000 180000 - 240000 290000
phn t (Mr)
n v nh 4 1 4 2 2
Hot tnh Sao chp Sa cha Sao chp Sao chp ADN Sao chp
polymer ho ADN nhim ADN ty th nhim sc th ADN
5 3 sc th (si sm) nhim sc
(si mun) th
ADN ca t bo nhn nguyn thu c dng vng, xon kp. Qu trnh sao chp ADN thng bt u
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 38 of 203
t mt bong bng sao chp, y l ch phnh khi u s tng hp ADN. Cc nt sao chp chnh l
cc chc ba sao chp (hnh 2.4.)
Hnh 2.4. Chc ba sao chp trong qu trnh sao chp ADN
ADN ty th c dng vng v c hai chc ba sao chp. S tng hp ADN ca nhim sc th vi khun
lun lun bt u cng mt im. im ny c gi l v tr Origin hay v tr Ori, l mt vng gm
254 cp base. Dng cc chng vi khun t bin, ngi ta chng minh c rng ch c mt s nh
trong s cc base ny l cn cho s khi u sao chp. E. coli, qu trnh sao chp bt u khi protein
B nhn bit c im khi s sao chp ny (OriC).
Cc si ADN c tch ra v mt si ADN c hng 3' 5' s c sao chp trc tip bi ADN
polymerase III. Si con b sung va to ra c gi l si sm (leading strand). Si gc cn li c
hng 5' 3', khng c sao chp cho n khi mt phn ca si gc c tho xon. Bn sao ny
c gi l si mun (lagging strand). Si mun c tng hp bng mt qu trnh sao chp khng lin
tc. S sinh tng hp si mun v si sm c iu ho bi s to nt tht (loop) ca si mun, nh
si mun cng c tng hp cng hng vi si sm.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 39 of 203
OH t do bt u s tng hp v gn c
desoxyribonucleotid vo mch con, b sung c vi
ADN khun.
S sao chp ADN vng to ra cu trc theta, hnh thnh bi hai chc ba sao chp xut pht t mt v
tr Origin. S tng hp ADN c tin hnh theo c hai chiu thun v ngc kim ng h cng mt
lc. to ra cc si n ADN, hai si ADN gc phi vn xon (quay). C mi 10 base c sao chp
th phn t ADN phi vn xon 1 vng.
Chc ba sao chp ca E. coli cn phi di chuyn vi tc 800 base/giy, i hi ADN gc phi
tho xon vi tc 80 vng/giy. S tho xon ADN gy ra hin tng siu xon (supercoiling). Vic
tho xon si kp dn n siu xon m (xon ngc chiu v lm gim s vng), trong khi , s xon
thm s dn n siu xon dng (xon kp cng chiu, s vng tng) (hnh 2.6).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 40 of 203
Hnh 2.6. S hnh thnh cu trc theta do s sao chp hai hng ca ADN kp
Hai si ADN lng vo nhau c gi l vng lng ghp (catena) hay ADN lng ghp v thng
c to ra khi sao chp ADN vng. Loi topoisomerase II thng lm mt i vng catena, ct si ADN
v phn t ADN si kp i xuyn qua im ct, to phn t ADN kp mi (hnh 2.7 - B).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 41 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 42 of 203
Cc enzym helicase cn thit h tr cho s tho xon ADN gc v hai si n ADN khng th t
nhin tch ra c (hnh 2.4).
Helicase cn ATP nh mt cofactor. Helicase dng nng lng t do khi thu phn ATP i dc
theo si ADN, lm tng tc tch hai si ADN.
C hai loi helicase khc nhau: mt l Rep - protein gn vi ADN gc, trc tip tng hp si sm v
di chuyn theo hng 3' 5'. Helicase th hai gn vi si khun kia tng hp si mun, enzym ny
to phc hp vi primase to mi ARN.
Ba protein khc h tr cho vic gn ADN polymerase v hip ng tc ng nh ATPase, thu phn
ATP to nng lng t do cho helicase hot ng. Tc ng hip ng ca cc protein ny trong hnh
2.4 v hnh 2.9 v chc nng ca chng c tm tt trong bng 2.3.
Bng 2.3. Chc nng ca cc protein sao chp quan trng ca E. coli
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 43 of 203
hnh thnh.
ADN ligase lin quan ti vic tng hp ADN khng lin tc bng cch ni cc on Okazaki sau khi
thay th cc on mi ARN. Chng cng cn thit cho vic sa cha cc ADN h hng. C ch tc
ng ca cc enzym ny khc vi cc ADN polymerase l do lin kt pyrophosphat ca NAD+ tham gia
(hnh 2.8).
C ch tng hp ADN t bo nhn tht cng tng t nh t bo nhn nguyn thu. im khc
bit ch yu l ADN t bo nhn tht ng cun trong nhiu nhim sc th v di hn. Tc di
chuyn ADN polymerase t bo nhn tht chm hn rt nhiu so vi t bo nhn nguyn thu (khong
50 nucleotid/giy). Nhng t bo nhn tht cha ti 20 000 phn t enzym. Do vy, nhim sc th t
bo nhn tht hnh thnh mt lng ln chc ba sao chp, khong 2000 hay nhiu hn. Cc on
Okazaki nh hn, di khong 40 - 300 base. Do , tc sao chp ADN t bo nhn tht nhanh hn
rt nhiu so vi E. coli. im khc bit c bn l ADN t bo nhn tht c nhiu replicon, v d
Saccharomyces cerevisiae c ti 500 replicon tc l c 500 im Ori.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 44 of 203
Hai loi virus, l Adenovirus v SV40 (virus linh trng) c dng lm cc m hnh ny. C ch
sao chp ca nhim sc th vng ca SV40 cng tng t vi c ch sao chp t mt v tr khi u
(Ori) ca nhim sc th t bo nhn tht.
M hnh to chc ba sao chp t bo nhn tht da trn cc nghin cu ny c trnh by hnh
2.9.
C hai loi polymerase tham gia vo qu trnh sao chp: Polymerase tham gia tng hp si sm v
polymerase tng hp si mun. Si mun c tht nt xung quanh polymerase , cho php enzym
di chuyn theo hng chc ba sao chp, ging nh s sao chp t bo nhn nguyn thu.
2.2.5.2. Chu k t bo
Pha G2 ngn hn pha G1, trong pha ny ngi ta bit c t nhng iu g xy ra t bo. Pha G2
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 45 of 203
Pha M hay pha phn bo dn n s ho tan mng nhn, s tch nhim sc th v s phn chia t
bo.
Nu c dng thng, b gen virus s tr nn ngn hn sau mi ln c sao chp, tr khi virus c c
ch c bit khc phc iu ny. Do ADN polymerase cn c mi ARN v ch hot ng tng hp
ADN theo chiu 5' 3' nn n khng th tng hp chnh xc cc ADN thng (hnh 2.13).
trnh hin tng ny, cc phage s dng mt trong hai cch sau: phage vng ho b gen ca
n nh cc trnh t cos, cn phage T7 thnh lp cc phc ni.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 46 of 203
Phn ssADN pha phi (3') ca mt phn t con c th gn vi phn pha tri (cng l 3') ca phn
t khc. Khong trng c lp nucleotid bi ADN polymerase v c ni li bi ADN ligase. Phn t
nh trng thu c c th c ct ra tr li, nhng khi s to ra u dnh 5 thay v 3, u dnh 5 c
th dng lm khun mu hon chnh ADN si i (hnh 2.15).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 47 of 203
T7 c gen m ho cho ADN ligase (gen 1.3), SSB (gen 2.5) v ADN polymerase (gen 5) ca ring
n.
S sao chp theo kiu theta khng phi l cch duy nht sao chp cc phn t ADN vng. Mt c
ch khc sao chp ADN theo kiu ln vng mc d sn phm to ra khng phi l ADN vng m l
ADN dng thng.
Phn t ADN vng c ct kha mt hay hai lin kt phosphodiester, to ra u 3' OH t do, u
ny c th dng nh khun mu sao chp bi ADN polymerase. Khi u 3' OH c ko di, u 5'
b y ra khi vng di dng si n, trong khi vng ADN ln tip tc ko di u 3. Kiu tng
hp trn vng tng t nh tng hp si sm. Trong lc u 5 b y ra c th dng lm khun mu
tng hp si i theo kiu tng t si mun nu c s hin din ca on mi thch hp (hnh 2.16).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 48 of 203
Kiu ny gp phage lambda v to ra nhiu bn sao ni tip, trong khi phage M13 ch to mt
bn sao.
Phage M13 (v cc phage dng si khc) c b gen l mt phn t ADN si n vng. Khi nhim t
bo E. coli, phn t ny c bm vo v c bao bi cc SSB protein. V phage M13 khng c gen
m ho cho ADN polymerase ring nn phi l thuc hon ton vo b my ca t bo ch sao chp.
Mc d b gen l ADN si n ca n rt thch hp lm khun mu tng hp ADN, nhng li khng
thch hp vi vic tng hp ARN bi ARN polymerase hay primase.
Vic sao chp tip ca RFI khng thc hin theo kiu theta m l ln vng. gp2 endonuclease, c
m ho bi gene 2 ca phage, ct kha ADN ca RFI ti v tr c hiu (im gc trn si dng) v s
sao chp ln vng din ra nhng khng to phn t phc m gp2 endonuclease ct phn t mt ln na
hon tt bn sao. Sn phm thu c gm mt phn t ADN vng n (si b th ra c ni li
thnh vng) v mt phn t ADN si i l RFI. Phn t ssADN vng lc ny c th c sao chp
bng cch lp li t u quy trnh trn.
Sau khi phage nhim t bo E. coli v tim ADN vo, nhim sc th ca n vng ho nh cc v tr
cos. Nh n trnh b ct bi cc exonuclease ca vi khun v c th c sao chp theo chu trnh tiu
gii tim n. Qu trnh sao chp ca phage lambda gm hai giai on.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 49 of 203
Giai on sm
Phage lambda khi u sao chp theo m hnh theta. im gc sao chp (ori) nm gen O, c sn
phm tng t DnaA. Phn t ny gn trnh t lp im gc v khi u vic tch hai si ADN ti
ch. Mt gen khc, gen P, c sn phm vi chc nng tng t DnaC. Sn phm ny gip DnaB gn
vo ADN tch mch. Sau , cc thnh phn khc ca b my sao chp ca vi khun gn vo v bt
u vic sao chp. Kiu sao chp ny ko di khong 5 - 15 pht.
Giai on mun
Sau 15 pht, phage lambda chuyn sang kiu sao chp ln vng. Hin cha bit nguyn nhn v c
ch ca s chuyn i ny.
ADN c sao chp bi ADN polymerase vi chnh xc cao (c khong 108 - 1012 base th c
mt base sai c gn vo mi ln sao chp). S sao chp chnh xc ca phn t ADN c tm quan
trng sng cn i vi hot ng bnh thng ca t bo.
Ngi ta dng cc nucleotid v ADN polymerase tng hp ADN in vitro. Sai st trong trng
hp ny l 105 tc l trong 100 000 nucleotid c mt sai st. E. coli c 3.106 cp base, vy mi ln
sao chp theo tnh ton nh trong thc nghim phi c ti 30 sai st. Tuy nhin, trn thc t sai st trong
t nhin thp hn nhiu.
Bng cch nh gi tn s cc t bin mi xut hin trong qun th ln v theo di bin i enzym
no trong nui cy m t bo, ngi ta tnh c sai st trong c th sinh vt khi sao chp in vivo l
109 tc l mt sai st trn mt t base. Nh vy t bo c 3.109 cp base mi ln sao chp ch c 3 sai
st.
t bo nhn nguyn thu ADN polymerase I v III va polymer ho, va c hot tnh exonuclease
5 3 v 3 5. Nu trn ng di chuyn polymer ho gp base sai, ADN - polymerase s li
li ct b theo hng 3 5 (hot tnh 3 5).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 50 of 203
Phn t ADN c th b bin i ngay c khi khng sao chp. Cc bin i t bin ny xy ra vi
tn s kh cao. Nh c ch sa sai nn tn s t bin c duy tr mc thp.
Hng lot enzym c hiu lm nhim v d tm v sa sai. C khong 20 enzym r sot dc ADN
d tm cc base b bin i ho hc, mi enzym c mt chc nng chuyn bit cho mt loi sai hng.
Khong 5 enzym khc c hiu cho cc lin kt cng ho tr sai gia base vi cc cht ho hc khc
hoc gia cc base k nhau trn mt mch. Mt s enzym khc pht hin s bt cp sai, nh trng hp
mt purin. Tng cng c khong 50 enzym chuyn bit pht hin v sa cc sai hng trn ADN.
Tm tt
S sao chp chnh xc ca ADN nhim sc th l mt s khi u cn thit cho s sinh sn. S sao
chp theo c ch bn bo tn v mi phn t ADN mi bao gm mt mch ADN gc v mt mch mi
c tng hp. S tng hp ADN c thc hin bi cc ADN polymerase chuyn bit, chng dng
ADN c sn lm khun mu. Ch c loi ADN polymerase III l cn thit cho s sao chp ADN ca t
bo nhn nguyn thu. Tuy nhin, cc t bo nhn tht c mt s polymerase tham gia vo qu trnh
sao chp. Mt s protein khc cng tham gia vo qu trnh tng hp ADN c t bo nhn tht ln t
bo nhn nguyn thu.
CU HI
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 51 of 203
2. Chn t hp sai
a) I b) II
c) d)
e) b v d
c) V tr Origin d) V tr Okazaki
e) Tt c u sai
c) Ni vi nhau to thnh si sm
d) Cn gi l loop
e) a v b
6. V tr Origin
c) Gm 254 cp base d) a v b
e) a, b v c
7. Primase l enzym
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 52 of 203
b) Gm nhiu N - protein gn
c) Cn gi l primosome
e) a v c
e) To phc hp vi N - protein
a) Topoisomerase I b) Topoisomerase II
e) Gc phosphat t do ca Topoisomerase
Bi 3
CC LOI ARN
MC TIU
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 53 of 203
- Trnh by c cc cch thc bin i cc bn sao nguyn thu ARN v cc chc nng ca s
bin i ny.
ADN mang thng tin di truyn ca t bo nhng bn thn n khng trc tip ch huy qu trnh tng
hp protein, v ADN nhim sc th ch mang mt bn sao duy nht cho mi gen, trong khi t bo c
hng nghn phn t protein cng loi tn ti. Mt khc, khng phi tt c cc gen u c biu hin
thnh protein cng lc m cc gen khc nhau s biu hin khc nhau ti mi thi im trong vng i
ca t bo v tu theo iu kin mi trng. Do vy cn c c ch trung gian khuch i thng tin di
truyn t ADN n protein, ng thi kim sot s biu hin ca gen theo nhu cu ca t bo. ARN
ng vai tr trung gian quan trng ny. T mi gen c th c nhiu bn sao ARN ch huy qu trnh
tng hp protein, ng thi vng i ca ARN ngn nn s biu hin ca gen qua trung gian ARN d
dng c kim sot bi nhu cu ca t bo v iu kin mi trng.
Ngoi vai tr trung gian trong qu trnh biu hin gen, nhiu ARN cn c vai tr cu trc hay xc
tc.
t bo nhn tht, ARN c tng hp ngay trong nhn (cha ADN), sau i vo t bo cht
tham gia tng hp protein. Nhng t bo giu ARN tng hp protein nhiu hn. V d: cc t bo tng
hp nhiu protein nh gan, l lch, tuyn t ca tm cha ARN nhiu hn so vi t bo t tng hp
protein nh thn, tim, phi.
V cu trc, ARN ging ADN: mch polyribonucleotid thng ca ARN cng cha 4 loi
ribonucleotid: Adenin (A), Guanin (G), Cytosin (C) v Uracil (U) thay cho Thymin (T).
- Mch n polyribonucleotid
- ng ribose (5C)
ARN gm cc loi:
- pre - rARN (tin rARN) l ARN c tng hp t ADN, sau khi chng c ct ni s tr thnh
rARN.
- pre - tARN (tin tARN) l ARN tng hp t ADN, sau khi c ct ni s tr thnh tARN.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 54 of 203
C ti 7 loi ARN nh khc nhau, trong c mt s snARN c lin quan n vic x l hnARN
thnh mARN chc nng. Gn y ngi ta mi tm thy mt vi loi ARN c c t bo nhn nguyn
thu v t bo nhn tht l thnh phn ca enzym c chc nng xc tc trc tip.
Trong dch nui cy Escherichia coli ang giai on phn chia nhanh, ribosom chim ti 1/3 khi
lng t bo. Khong 1/2 khi lng ribosom l ARN nn rARN chim n 75% ca tng s ARN.
Mi mt ribosom c Mr = 2.5 - 4.5 106. C 3 loi ribosom khc nhau, thng c phn bit da
vo h s lng ca chng khi siu ly tm. Ribosom ca Eubacteria v lc lp c ch s lng khong 70S.
Ribosom ca t bo nhn tht l 80S, ca ty th ng vt c v khong 50S.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 55 of 203
Hnh 3.1. Thnh phn ca ribosom 70S (t bo nhn nguyn thu) v 80S (t bo nhn tht)
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 56 of 203
- n v ln 60S cha 3 loi rARN khc nhau: 1 rARN 28S, 1 rARN 5,8S, 1 rARN 5S v 45
protein.
Hu ht r - protein ca ribosom E.coli c cc trnh t bc 1 khc nhau, ngoi tr L7, L12 v L8. Cc
protein L7 v L12 c nhng trnh t c xc nh, L7 l mt dng aminoacetyl ho L12 (s acetyl ho
cn thit cho hot tnh GTPase trong qu trnh dch m). L8 l phc hp ca mt phn t L10 lin kt vi
L7 v L12. i khi trong qu trnh tinh ch, phc hp ny khng tch c.
3.2.1.2. S lp rp ribosom
Cc thng tin cho vic t lp rp ca ribosom c cha ng trong cc phn t thnh phn rARN
v protein. Cc tiu n v ribosom c th ti hp li in vitro c s dng rng ri cho vic nghin
cu qu trnh lp rp ca ribosom. Cc tiu n v c treo trong mt dung dch c nng mui cao
(v d LiCl 4 M v urea 8 M) ngn cn i lc yu khng ho tr gia protein v cc phn t ARN, lm
cho cc protein tr nn khng bn vng vi ribosom. ARN v cc protein khng lin kt c tch ra
khi nhau. Chng c thm phn loi b mui v urea, sau vi nhau. Di nhng iu kin
thch hp, cc tiu n v c t hp li theo chc nng ca chng v khng th phn bit c vi s
t hp cc tiu n v t nhin.
Khi dng s t hp khc nhau gia cc thnh phn ring l ca ribosom, ngi ta c th xc nh
c cc trnh t lin kt trong qu trnh lp rp.
T bo cht nm men
3392 1787
(Saccharomyces cerevisae)
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 57 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 58 of 203
Pre - rARN 45S (gm khong 12000 nucleotid) l bn sao ca cc gen ARN - ribosom. Cc pre -
ARN c vi trm bn sao v tp hp li to thnh phn u trong mt vng nhim sc th v c xem
nh vng thit lp nhn. rARN 45S c bin i do a vo khong 110 nhm methyl cc nucleotid
c hiu. Hu ht nhng phn c methyl ho u c bo tn rARN trng thnh. Hng lot din
bin trong v ngoi nhn to ra c cc tin th trung gian 32S v 20S t pre - rARN 45S. Sau ,
pre rARN 32S ct ni thnh 28S v 5,8S, cn 20S thnh 18S.
Qu trnh sa i ca pre - ARN khc nhau v chi tit gia t bo nhn tht v t bo nhn nguyn
thu. t bo nhn nguyn thu, chui rARN 5S l thnh phn ca n v phin m ca c rARN 16S
v rARN 23S. Hn na, gen rARN t bo nhn nguyn thu li c cha 2 chui tARN hay nhiu hn.
Nhng s phn ct u tin ca pre - rARN c xc tc bi ARNse III c hiu cho ARN mch kp
(hnh 3.3).
3.2.2.1. Cu trc
Nm 1950, F. Crick nu ra gi thit v cht ni. ng cho rng trc khi gn thnh polypeptid, cc
amino acid phi gn qua cht ni trung gian, cht ny s bt cp c hiu vi cc base trn mARN. Vo
nm 1957, M. Hoagland v cc cng s tm ra tARN v chng minh c rng mi phn t tARN
gn vi mt phn t amino acid v mang chng n ribosom.
t nht, mi loi tARN c hiu cho mt loi amino acid. V d: Arginine ch gn c hiu vi ARN
vn chuyn arginine. Tuy nhin, tt c cc tARN c mt s c tnh cu trc chung: chiu di khong 73
- 93 nucleotid, cu trc gm mt mch cun li nh hnh l ch ba (hnh 3.4). u mt 3 c trnh t kt
thc CCA.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 59 of 203
T = Ribothymidine
= pseudouridine
m22G = dimethylguanosine
m1I = methylinosine
D = dihydrouridine
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 60 of 203
t bo nhn nguyn thu v t bo nhn tht, cc tARN u c tch ra t nhng phn t ban
u di hn nh mt s phn ng x l. E. coli, cc gen ca tARN thng c pht hin trong cc
operon hn hp, cng lc m ho cho 4 tARN (tARNTyr, tARNThr, tARNGly, tARNThr) v mARN
cho s tng hp protein - yu t ni di Tu.
RNse P l mt enzym, gm 1 ARN c 375 nucleotid v mt protein c trng lng phn t 20 000.
Trong cc iu kin sinh l, c 2 thnh phn protein v ARN u cn nhng yu t hot ng, nhng
khi nng Mg++ cao th ch c thnh phn ARN c hot tnh xc tc. Theo sau hot ng ca RNse P,
mt s phn ng trong nhn v bin i base s to nn tARN hon chnh.
E. coli, tARN c trnh t cui l CCA - OH, phin m t cc base b sung c trong ADN ca
nhim sc th. Trong khi t bo nhn tht nhng base ny c thm vo sau khi phin m (hnh
3.5).
- Aminoacyl ho
- Gn nhng yu t ni di
- Gn ribosom
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 61 of 203
tARN c nhiu im nhn din enzym. Mt s im nhn din c th cho tng tARN ring bit hay
cho mt s loi ng ng (nh aminoacyl ho, formyl ho, nhn din codon - anticodon). Trong khi ,
c nhng im nhn din c hiu cho tt c tARN ( l, s tng tc vi nhng yu t ni di I, II v
s gn kt ribosom).
mARN c tng hp khi enzym ARN polymerase II tip xc vi trnh t promoter trn phn t
ADN. mARN nguyn vn ca t bo vi khun v ca t bo nhn tht cha trnh t nucleotid nhiu hn
s c dng m ho protein.
3.2.3.1. Cu trc
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 62 of 203
1. on 5 khng m ho
2. Vng m ho
3. on 3 khng m ho
Cc mARN ca t bo nhn nguyn thu c thi gian bn hu ngn: trung bnh 2 pht, ca t bo
nhn tht t 30 pht n 24 gi.
t bo nhn tht, sau khi mARN c tng hp ti nhn, chng phi tri qua 2 giai on "hu
phin m":
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 63 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 64 of 203
mARN ngc c th ngn cn qu trnh dch m nhiu t bo nhn tht khi chng c trnh t b
sung vi trnh t ca mARN. iu ny c ngha l mt gen khng biu hin c thnh protein nu
trong t bo c mARN ngc. y c th l mt c ch chng li hin tng gen nhy ngc hay virus;
do c 2 trng hp u dng si i mARN lm trung gian. Trong sinh ho, hin tng ny c dng
nghin cu chc nng ca gen bng cch thm vo mARN ngc.
T bo nhn tht c cha nhiu phn t ARN nh khc ngoi tARN v 5S v 5,8S rARN. In vivo,
nhng phn t nh ny phc hp vi cc protein c hiu to nn cc ht ribonucleoprotein (RNP) c
gi l snRNP hoc scRNP tng ng cho nhn hay cho t bo cht. Chng di khong 90 - 300
nucleotid.
Vic bin i tin ARN thnh ARN c thc hin vi s tham gia ca nhng phn t nh l cc
spliceosom, chnh l cc snRNP. Ngy nay tm thy c 7 loi snRNP t U1 n U7. U1 v U2 c vai
tr trong vic sa i hnARN thnh mARN hon chnh, cn U3 c vai tr trong sa i pre - rARN
thnh rARN t bo cht. Cc ARN c kh nng xc tc c gi l ribozyme. Ngi b bnh t min
hng ban Lupus (systemic lupus erythematosus), do bnh nhn to ra khng th gn vi mt trong cc
protein U1 - U7 snRNP.
scRNP c vai tr bit r nht l 7SL ARN, mt thnh phn ca phc hp nhn din tn hiu xut
SRP. SRP cu to bi mt scARN c cha 294 nucleotid v 6 polypeptid c Mr t 9000 - 72000. Khi
mARN ca protein xut bt u c dch m, SRP tng tc c hiu vi on peptid tn hiu xut ca
protein mi sinh v vi ribosom, ngn chn s dch m cho n khi ribosom tip xc vi li ni cht,
khi n ri khi ribosom v tn hiu xut ca protein mi sinh gn vo b my chuyn v protein trn
mng li ni cht v s tng hp protein c tip tc, protein sinh ra c chuyn vo trong li ni
cht.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 65 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 66 of 203
B gen t bo nhn nguyn thu l b gen gin on. Trnh t khng m ho xen k cc trnh t m
ho. Qu trnh loi b cc trnh t khng m ho gi l qu trnh ct ni ARN.
Exon l trnh t biu hin, c mARN hon chnh. Tuy nhin, u tn cng 5 v 3 c th khng
dch m thnh trnh t protein, do vy trnh t exon khng hon ton l trnh t m ho.
Intron l trnh t thng khng cha gen, xen k cc exon, hin din hu ht cc phn t ADN ca
t bo nhn tht bc cao. Chng thng c kch thc ln hn cc exon. Intron c rt t t bo nhn
tht bc thp, nm v hon ton khng c vi khun tr mt vi ngoi l.
3.2.5.2. Ct ni ARN
Qu trnh ct ni xy ra cc phc hp bn sao nguyn thu pre - ARN hay hnARN vi protein
nhn gi l hnRNP. Qu trnh ny din ra trong nhn t bo trc khi di chuyn vo t bo cht tham
gia dch m.
Nguyn tc GU - AG
1. T ct ni
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 67 of 203
2. Intron c loi b khi tin tARN nhn ca nm men tng t qu trnh x l tin tARN.
3. Ct ni nh spliceosom
Spliceosom l phc hp cha nhiu loi ribonucleopotein. Chng nhn din cc trnh t exon
intron loi b intron v ni cc exon. Qu trnh ny thng xy ra ARN nhn ca t bo nhn tht
bc cao
Spliceosom
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 68 of 203
- snRNP U1 gn vo v tr cho.
- snRNP U2 to lin kt hydro vi v tr nhnh ca intron. Giai on ny s dng ATP thu phn v
"yu t tr gip" snRNP (U2AF).
ATP cung cp nng lng cho mi giai on. U4 tng tc vi U6 bng lin kt hydro.
- U5 di chuyn t phn intron sang phn exon ca v tr cho, U2 thay th U4 lin kt vi U6 nh lin
kt hidro.
- Spliceosom c tho ri, gii phng U1, U2, U4, U5, U6.
Ct ni cho
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 69 of 203
3.2.5.4. X l tARN
Ct ni tARN nm men
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 70 of 203
3.2.5.5. X l rARN
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 71 of 203
- T ct ni
- Cu trc bc 2 ca vng 9
Ct ni loi tetrahymena
Tin rARN 35S c x l thnh 18S, 5,8S, 26SrARN. mt vi chng Tetrahymena, 26S c mt
intron gm 400 base. Intron ny c th t ct ni nhng i hi s c mt ca cc ion v nucleotid G
(GTP, GDP, GMP)
Phn ng t ct ni: gm 3 bc
1. G 3 - OH gn u 5 ca intron.
7. Nc thu phn vng intron ti cng v tr, to L - 19ARN. L - 19ARN c hot tnh enzym xc
tc phn ng ct v ni cc oligonucleotid.
8. Nhnh P1 (exon 1 v IGS InteARNl Guide Sequence) ng vai tr quan trng trong bc 1 ca
phn ng ct ni.
Vai tr xc tc ca L - 19ARN
Tm tt
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 72 of 203
C 3 loi ARN chnh: mARN, rARN v tARN. Tt c u tham gia vo qu trnh dch m, nhng
ch c mARN mang thng tin m ho cho cu trc c bn ca phn t protein. rARN c vai tr trong
vic cung cp ni tng hp protein, ribosom. tARN lm nhim v vn chuyn acid amin n ribosom,
tng tc vi aminoacyl - tARN synthetase c hiu v kt hp vi ribosom.
CU HI
a) Mch n polynucleotid
a) 50S b) 30S
c) 60S d) 40S
e) 70S
3. Cu to t 45 phn t protein, 1 rARN 28S, 1 phn t rARN 5.8S, 1 phn t rARN 5S l tiu n
v:
a) 60S b) 40S
c) 50S d) 30S
e) 70S
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 73 of 203
c) tARN d) scARN
e) snARN
d) u mt 5 kt thc G
a) Aminoacyl ho
b) Formyl ho tARN m u
c) Gn nhng yu t kt thc
d) Gn ribosom
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 74 of 203
9. Loi snRNP no tham gia vo vic sa i hnARN thnh mARN hon chnh?
a) U1, U3 b) U4, U5
c) U6, U7 d) U1, U2
e) U1, U4
10. t bo nhn tht mARN sau khi c phin m phi tri qua:
a) Gn chp b) Gn ui polyA
c) Ct ni loi intron d) a, b
e) a, b v c.
Bi 4
S PHIN M V M DI TRUYN
MC TIU
- M t c s phin m c iu ho.
4.1. M U
ADN trong nhim sc th ng vai tr lu tr thng tin di truyn ca t bo. N quy nh trnh t
acid amin ca hng ngn protein khc nhau. Tnh ng nht v s lng cc protein ny quyt nh tnh
cht sinh ho cn bn ca t bo. ADN khng trc tip sp xp cc acid amin cho s polymer ho thnh
protein m l mARN, l bn sao b sung vi mt mch polynucleotid ca gen. Qu trnh to ra cc ARN
c gi l s phin m. Tn hiu c m ho trong ADN hng dn enzym thc hin phin m, ni
m u, ni kt thc.
S iu ho mc phin m l s kim sot quan trng hng u trong biu hin gen. ARN c
phin m t gen ph thuc vo mt vng nht nh c gi l promoter (vng khi ng). Bn cnh
vng khi ng, cn c mt vi trnh t khc trn ADN cng c vai tr quan trng trong vic iu ho
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 75 of 203
S nhn din cc acid amin lin kt chng vi nhau thnh chui polypeptid c xc nh bi cc
trnh t ribonucleotid trong mARN. l mARN c dch m t "ngn ng" nucleotid ca ADN. M
di truyn m t mi quan h gia 2 ngn ng ny.
Qu trnh truyn thng tin di truyn t ADN sang ARN c gi l s phin m. Tham gia vo qu
trnh phin m c h thng enzym ARN polymerase, ng ra, tn chnh xc l ARN polymerase ph
thuc ADN. ADN th hin kh nng d xc tc tc l lm khun tng hp nn mt phn t khc.
- Ch 1 trong 2 mch ca phn t ADN dng lm khun tng hp ARN (hnh 4.1)
- ARN polymerase bm vo ADN lm tch mch v di chuyn theo hng 3 5 trn ADN cho
"ARN" c tng hp theo hng 5 3.
- ARN polymerase gn vo ADN gy ra hin tng "chy" ti ch bn trong chui xon kp ADN.
Kt qu lm t cc lin kt hydro gia cc i base b sung. C cht cho ARN polymerase l cc
ribonucleotid 5 - triphosphat ATP, GTP, CTP v UTP. Mt ion kim loi ho tr 2 hoc l Mg2+ hoc l
Mn2+ ng vai tr co - factor.
Cu trc ca phn t ARN thng bt u bng 1 nucleotid purin (ATP hay GTP) b sung cho
nucleotid im bt u phin m ca mch khun mu ADN. NTP th 2 c a vo khun mu
ADN (li theo nguyn tc b sung cp base) v lin kt 3 - 5 phosphodiester c hnh thnh gia
nhm 3 - OH ca NTP u tin v nhm phosphat ( ) ca NTP th 2.
Phn ng xy ra theo pha thu phn pyrophosphat va c gii phng. Bng cch ny ARN c
tip tc ko di v mt t hp lai ADN - ARN c hnh thnh. Chui ARN ko di theo hng 5 - 3
v ARN polymerase cng vi vng si kp ADN dui xon (cn gi l bong bng phin m) chuyn
ng dc theo si khun mu theo hng 3 5. Khi t hp lai ADN - ARN t c chiu di 12
base, u tn 5 ca chui ARN mi hnh thnh c tch ra t mch ADN b sung vi n v 2 mch
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 76 of 203
kp ca ADN ban u phc hi li. ARN polymerase tip tc di chuyn dc theo mch ADN khun
mu n khi gp im kt thc, l tn hiu c m ho trong mt trnh t ADN quy nh chm dt
s ni di ca mch. ARN polymerase v chui ARN hon chnh c tch ra khi khun mu ADN.
ARN polymerase thc hin s lin kt cho 4 loi nucleotid triphosphat ging nhau, khng phn bit
loi ny, loi kia. Khc vi s sao chp ADN, mi khng cn thit cho s phin m.
Khc vi ADN polymerase, ARN polymerase khng c mt hot tnh sa sai no, v nhng
nucleotid no kt hp khng chnh xc thng c thay th ngay bng nhng nucleotid ph hp. Hn
na, nu c li him hoi no xy ra th cng khng di truyn c v ARN khng phi l ni lu tr
thng tin di truyn.
- mARN thng cha thng tin nhiu gen ni tip nhau (polycistron mARN).
- Qu trnh tng hp mARN c tin hnh khi ARN polymerase bm vo vng khi ng
(promoter). Khi gn vo, polymerase khng tng hp mARN ngay. S tng hp ch bt u t du xut
pht, thng l TAC nm cch 7 - 8 base pha sau ch bm v pha u 5 ca mch khun.
- phn ln t bo nhn nguyn thu, qu trnh tng hp tip tc n khi c qua du kt thc. Kt
thc phin m, ARN polymerase v mARN tch ri khi ADN.
Hu ht s hiu bit v qu trnh phin m t bo nhn nguyn thu xut pht t s nghin cu
trn E. coli v thc khun ca n. C ch ca s phin m E. coli tng t hu ht t bo nhn
nguyn thu, ngoi tr Archaebacteria (c intron trong ADN).
Nhim sc th n bi ca E. coli l mt vng khp kn cha 4.106 bp (cp base) cng vi protein v
ARN. ADN ny c cu hnh tho xon cc i (negatievely supercoiled conformation). In vivo, nhim
sc th l mt cu trc lng lo gi l nucleoide v c gn vo mng t bo cht. B gen ca E. coli
cha khong 3000 gen cu trc khc nhau, hu ht c mt trong mt bn sao duy nht, sp xp thnh
100 n v phin m.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 77 of 203
Tiu n v (sigma) c th tch ra thun nghch t holoenzym v c phn tch bng sc k trn
ct phosphocellulose:
4.3.2. Promoter
Phn ln promoter E. coli v c bn u ging nhau. Nu k hiu base u tin phin m thnh
mARN (thng l Adenin) l + 1, th cc base theo hng ngc li vi chiu phin m k hiu " - ".
Promoter gm hai trnh t. l trnh t TATAAT, tn cng lun lun l base T, cch im xut
pht 6 - 8 base v c base trung tm l 10. Trnh t ny cn c gi l hp TATA hay hp Pribnow;
trnh t th 2 TTGACA c base trung tm - 35 (hnh 4.2). Hai trnh t ny chu trch nhim cho php
ARN polymerase gn vo vng khi ng phin m. Vng 35 tham gia vo vic gn khi u ARN
polymerase (bng 4.1). Khng phi promoter no cng c cu to ging ht cc trnh t ny, m c mt
t sai lch nh, do ngi ta gi trnh t ny l trnh t chung (consensus).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 78 of 203
Chc nng quan trng ca hp -10 v hp -35 c pht hin t hiu ng t bin (mt base c
thay th bi mt base khc) trong qu trnh phin m t promoter (hnh 4.3). Nhiu t bin nh vy c
ch tc khc s phin m ca gen. Tht ra, vi vng khi ng (promoters) E. coli c trnh t chung
rt ging vi trnh t chung l tng. Do , phn bit c chng c th da vo cng
(strength) hoc tn sut khi ng phin m. V d, t bo E. coli cha khong 12 phn t ca lac
repressor (th c ch lac - operon) v gen ca n c phin m ch mt ln mi 30 pht. Mt khc,
ARN ribosom, do i hi s lng ln, v khong 10000 ribosom phi c hnh thnh sau mi th h
t bo (khong 20 pht). Nn mi gen - rARN c phin m khong 2 giy mt ln. S khc bit ny
do cng vng khi ng.
Mi cho ti gn y, ngi ta vn cho rng ARN polymerase E. coli c mt loi holoenzym duy
nht c kh nng phin m tt c cc gen khc nhau. Tuy nhin, nhng nghin cu gn y trn s p
ng shock nhit cho thy rng kh nng phin m cc gen khc nhau c th t c do sinh ra cc
tiu n v khc nhau. Ngi ta chng minh s p ng shock nhit c lin quan n vic tng hp
nhng protein mi khi c s gin on nhit mi trng nui cy vi khun hay t bo. S p ng ny
bin thin rt rng t bo nhn tht v t bo nhn nguyn thu. Nhng protein mi ny, c gi l
protein shock nhit, c xem l bo v t bo chng li s bin i nhit.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 79 of 203
Vi khun Bacillus subtilis cng s dng yu t bin t dng sinh dng sang dng bo t.
Nhiu ADN thc khun th m ho ARN polymerase c trng cho ring chng. Mt s promoter
nh vy c s dng trong vic to dng plasmid nhm sn xut mt s lng ln mARN t mt gen
quan tm. Nhng bn m sao nh vy c th c s dng nh nhng on mi hoc c th c tng
hp in vitro thnh mt sn phm protein cn thit.
Trnh t chung ca promoter sc nhit c sp xp cho 6 gen sc nhit khc nhau. Nhng ch ci
dng trn ch s tng ng mnh, hng di tng ng yu, n l nucleotide bt k. V tr bt u
phin m k hiu +1.
Ngi ta thng dng h thng promoter v ARN polymerase ly t thc khun th SP6. SP6 l mt
thc khun th ca Salmonella typhimurium, m ho c mt ARN - polymerase virus c hiu. Cc
promoter ca t bo ch v cc promoter c trong plasmid ly trch t E. coli u khng c SP6
polymerase nhn bit. Promoter SP6 c chn vo vector plasmid pha trc polylinker (polylinker l
on ADN nhn to cha nhiu v tr nhn bit duy nht ca cc enzym ct hn ch). on ADN cn
sao chp c chn vo polylinker v plasmid ti t hp c bin np vo E. coli. Plasmid cha ADN
l c "lm dui thng" bi mt enzym ct hn ch. Plasmid dui thng ny c vi SP6
polymerase v cc NTP. Cc plasmid - ADN c loi khi cc bn sao mARN bng cch vi DNase
(hnh 4.5).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 80 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 81 of 203
E. coli, hu ht mARN c i sng bn hu c tnh bng pht. Chng nhanh chng b thoi ho
bi h thng enzym endonuclease v exonuclease. Tht ra, phn ln mARN c trong t bo cht bt
k thi im no cng u l nhng chui mi sinh. Chng gn vo phc hp ADN khun - ARN
polymerase. Ribosom gn vo v tr gn ribosom gn u 5 ca mARN va mi ri phc hp ADN
khun - ARN polymerase. Do , s phin m v s dch m t bo nhn nguyn thu c thc hin
gn nh song song vi nhau. Chnh iu ny lm xut hin mt dng kim sot phin m c gi l
phin m suy gim.
Chui ARN ni di cho n khi gp phi mt trnh t chuyn bit trong chui ADN. Chui ny
dng kt thc qu trnh tng hp ARN. E. coli c 2 c ch kt thc: mt c ch ph thuc vo protein
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 82 of 203
Qu trnh ny c trng bi s hin din ca mt trnh t giu GC trn ADN. Trnh t ny i xng
2 bn (dyad symmetry), c thm 5 hoc 6 adenin km theo. ARN c phin m t trnh t ny c th
hnh thnh mt nt vng hay mt cu trc kp tc do cc lin kt hydro ni phn t gia cc base b
sung (hnh 4.7). C th s hnh thnh xon kp ARN trong cu trc nt ny, thch hp cho vic hnh
thnh ADN - ARN lai gia si ADN phin m v chui ARN mi sinh. Nh vy, phn dn 5 ca nt
ko theo na ui 3 t ADN, ch li trnh t oligo (U) tip tc ni di theo ADN. Cc dng lai
oligoA - oligoU cho thy s bt cp t bn vng nht v ARN c th tch khi ADN. Sau , si kp
ADN c hnh thnh trong "bong bng phin m" v phn li ca enzym polymerase, c i lc thp
i vi ADN kp, c phng thch ra.
Khi bm dnh vo ARN, yu t thu gii ATP v nng lng t do c gii phng, gip n di
chuyn dc theo chui ARN mi sinh, hng ti bong bng phin m (hnh 4.8). Sau , yu t tch
i ADN - ARN lai bng mt c ch cha r, phng thch mARN vo t bo cht.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 83 of 203
- ARN polymerase II chu trch nhim tng hp mARN, ARN polymerase I tng hp rARN, ARN
polymerase III phin m cho ra cc ARN c kch thc nh, tARN, ARN 5S
- Bn phin m u tin cn gi l tin mARN khng c s dng trc tip m phi qua bin i.
T nm 1977, ngi ta pht hin nhiu gen ca t bo nhn tht c tnh gin on. Trn gen, cc
on m ho cho protein c gi l exon xen k vi cc on khng m ho c gi l intron. Bn
phin m u tin tc l tin mARN cha c trnh t phin m ca exon v intron. Tip theo cc intron
tc cc on khng m ho protein c ct ri ra, cn cc exon m ho protein c ni lin li vi
nhau to ARN trng thnh. Qu trnh ny c gi l ct ni (hnh 4.9).
Sau splicing, mARN va mi trng thnh khng cn intron, qua l mng nhn ra t bo cht
dch m.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 84 of 203
Nhn ca t bo nhn tht c 3 loi ARN polymerase (I, II, III) (bng 4.2).
Polymerase I trong nhn, phin m cc gen ARN ribosom. Bn sao ch yu l pre - rARN.
Polymerase II tm thy trong dch nhn, rt nhy cm vi s c ch ca - amanitin (khong 50% c
ch nng 10-8 M). N phin m cc gen m ho protein v cc bn sao s cp khng ng nht
(hnARN), l tin cht ca mARN t bo cht. Polymerase III cng trong dch nhn, phin m cc gen
5S rARN v gen tARN. N nhy cm trung bnh vi s c ch ca - amanitin (khong 50% c ch
10-6 M). Phn ng c xc tc bi cc polymerase t bo nhn tht, v mt ho sinh cng ging vi
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 85 of 203
Cc gen ARN ribosom thng l cc gen c sao chp mnh nht trong t bo, phn nh nhu cu
cao v rARN. Si nhim sc (chromatin) cha cc gen ribosom, hon ton khng to nucleosom. Cc
trnh t sao chp cc gen rARN c bo lu mc cao trong cc chng sinh vt, chnh v th tht
khng l khi tm thy c cc trnh t chuyn bit cho tng loi pha trc cc im xut pht phin m.
l mt vng khng phin m nm gia cc trnh t c phin m ca cc gen rARN ca Xenopus
laevis (cc chu Phi) v cc trnh t cn thit cho s phin m bi polymerase I. Cc trnh t cn thit
ny c xc nh bng tc ng xo b (hnh 4.10).
Promoter thc gm khong 150 bp c tm thy gia cc v tr - 146 v + 6. Tuy nhin, cc bn sao
chp hon chnh t di ca promoter ny c lp li cc v tr - 1200 v - 2300 bp trong vng v
tinh (vng khng phin m) v ci rt nhiu bn sao 60 hoc 81 bp ca promoter thc. C l iu ny
lin quan ti mt yu t gii hn s lng. Cc bn sao lp li 60/81 bp kch thch rt nhiu s phin m
ca promoter thc. Cc promoter v tinh ny c kh nng khi ng qu trnh tng hp cc bn m sao
rARN. Cc phn t polymerase "xp hng" trong vng v tinh trc khi chuyn ng ti promoter thc.
ngi, cc rARN c phin m bi ARN polymerase I. Bn phin m u tin l tin rARN 45S
(pre - rARN), sau ct ni to ra 3 loi 28S, 18S v 5,8 S. Cc gen ny khng phn tn m xp thnh
cm, mi cm c th hn 200 bn sao. ngi, cc cm ny nm trn vai ngn ca cc nhim sc th
tm u 13, 14, 15, 21 v 22. Cc nhm ny xp quanh yu t t chc hch nhn hnh thnh eo th cp
v gian k to nn hch nhn.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 86 of 203
Ngi ta cho rng cc trnh t chung ny c chc nng lin kt cc yu t phin m chuyn bit hn
l gn trc tip vi ARN polymerase II. V d, cc t bo ca ng vt c v cha 1 protein gi l SP1
c Mr 100 000, cn cho s phin m ca cc gen c hp GC trong promoter. Cc trnh t khi ng
khc nhau nm rt gn vi im bt u phin m l cn thit, nhng thng vn khng biu hin
cc gen ARN polymerase phin m. C cc phn t c a thm vo, gi l cc trnh t tng cng.
Chng khng c kh nng khi ng chnh mnh, nhng li c kh nng kch thch phin m mnh v c
th tc ng cc khong cch ng k (n vi kb ADN). Trnh t tng cng c hiu qu nh nhau
i vi cc gen cu trc do chng iu ho v c th nm rt gn u 3, 5 hoc nm ngay trong mt
cistron ca gen cu trc.
Trnh t tng cng lun th hin tnh chuyn bit, do khng lin quan ti vic iu ho biu
hin gen trong qu trnh pht trin ca c th. V d, trnh t tng cng min dch globulin ch hot
ng trong - lymphocyte.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 87 of 203
Cc bn m sao ca ARN polymerase II u l nhng ARN nhn khng ng nht (hnARN). Chng
c phn t lng rt ln (n 10 kb), v cha cc intron trong ADN. a phn cc bn m sao u cha
trnh t AAUAAA gm 20 nucleotid t u 3. Trnh t ny rt quan trng v: n c nhn bit bi
mt endonuclease c hiu ct ARN mi sinh khi polymerase II v n l du hiu c nhn bit
bi mt enzym poly(A) polymerase nhm gn ui 3 ARN.
ARN polymerase III chu trch nhim phin m ra rARN 5S, tARN v mt s ARN nh ca nhn v
ca t bo cht. Cc gen do ARN polymerase III phin m thng cha di 300 nucleotid. Gen 5S
rARN khng cha intron, trong khi , nhiu gen tARN cha cc intron nh.
Tham gia iu ho tng hp cc rARN 5S ca non bo X. laevis, ngoi ARN polymerase III, cn c
cc yu t phin m khc l TFIIIA, TFIIIB v TFIIIC. Hai yu t TFIIIB v C cng cn cho s phin
m cc gen khc bi ARN polymerase III (chng hn cc gen tARN), trong khi TFIIIA (mt protein
c Mr 40000) th chuyn bit cho non bo v gn vi vng ni kim sot ca gen rARN 5S. Sau ,
hng ARN polymerase III tng hp khong 50 nucleotid (hnh 4.13).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 88 of 203
Retrovirus l cc virus m vt liu di truyn ca chng l ARN, v d virus lm suy gim min dch
(HIV) gy ra hi chng suy gim min dch mc phi ngi (AIDS). S tng hp ADN t khun mu
ARN c xc tc bi cc enzym phin m ngc. Enzym ny bt u qu trnh phin m bng cc
dNTP nh l cht nn (hnh 4.14). Khc vi ARN polymerase, enzym phin m ngc phi dng ti
mi. on mi l mt tARN ca t bo ch, gn kt vo u 3ca retrovirus. Chui kp u tin l
chui lai ARN - ADN. ARN virus trong chui lai b hu bi RNaseH. Sau , mt mch ADN va c
tng hp to ra chui kp ADN. Chui kp li tch hp vo ADN k ch.
Enzym phin m ngc ca HIV l mt protein gm 2 tiu n v. Mi tiu n v cha 560 acid
amin. Enzym phin m ngc c mt vai tr quan trng trong vic tng hp ADN b sung (cADN)
trong qu trnh iu khin gen. S tch hp ca virus vo b gen t bo ch thng c n nh (cc
provirus khng bao gi b ct ri nh prophage). Hin nay, nhiu ngi cho rng cc virus c th tc
ng n s biu hin ca cc gen ung th.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 89 of 203
4.6. M DI TRUYN
Ta bit, cc acid amin trong phn t protein c m ho bng nhm cc nucleotid trn phn t
ADN. C tt c 4 loi base, nu cc base c nhm i, tc 2 nucleotid m ho cho mt loi acid amin th
tt c ch c 16 t hp, khng cho 20 loi acid amin. Nh vy, n v m ho hay cn gi l codon
phi gm 3 hay nhiu nucleotid hn.
Nm 1961, F. Crick lm th nghim chng minh rng nhm nucleotid m ho c 3 hay ni cch
khc codon gm 3 nucleotid k tip nhau. Tt c s c 43 = 64 t hp.
M. W Nirenberg v H. Matthaei (M) dng enzym theo phng php ca Ochoa tng hp ARN
nhn to. Khi ch dng mt loi nucleotid l Uracil s nhn c ARN l polyuracil, nu ch Adenin s
nhn c polyadenin.
Nm 1961, khi dng polyuracil thay cho mARN tng hp protein trong h thng v bo (c acid
amin, enzym, nhng khng c ADN...), sn phm nhn c l mch polypeptid polyphenylalanin. iu
chng t codon UUU m ho cho phenylalanin. y l codon u tin c xc nh. Nirenberg v
Matthaei cng chng minh c AAA m cho lysin, GGG cho Glycin, CCC cho prolin.
Bng m di truyn (bng 4.3) cho thy trong 64 codon c 3 codon UAA, UAG, UGA khng m ho
cho acid amin c gi l codon v ngha (non - sense), ng thi l codon kt thc (stop) tc l "du
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 90 of 203
M di truyn c tnh "suy thoi" tc mt acid amin c nhiu codon m ho, ch tr methionin v
tryptophan ch c mt codon.
Cc codon ng ngha tc l m ho cho cng mt acid amin thng c 2 base u tin ging nhau,
nhng khc nhau nucleotid th 3. V d: CCU, CCC, CCA v CCG u m ho cho prolin. Trn thc
t U v C lun lun tng ng nhau v tr th 3, cn A v G tng ng nhau trong 14 ca 16 trng
hp.
Mt codon ch m cho mt loi acid amin, trng hp ngoi l l AUG va m ho cho Met ni,
va m ho cho acid amin m u N - formyl Methionin trong t bo t bo nhn nguyn thu hoc
methionin trong t bo nhn tht.
V tr th II
V tr th I U C A G V tr th III
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 91 of 203
Tm tt
Qu trnh phin m trong t bo nhn nguyn thu v t bo nhn tht c mt vi chi tit khc nhau
ng k. T bo nhn nguyn thu ch cha mt loi enzym nhn ARN polymerase, m khi tng tc
vi mt tiu n v iu ho (sigma) s to ra holoenzym quy nh tnh c hiu ca promoter. Promoter
gm 2 vng cha cc trnh t nm trung tm - 10 v - 35 k t v tr bt u phin m. Cc trnh t ny
tng tc vi holoenzym ARN polymerase lm cho s phin m c hiu qu v chnh xc.
Nhn t bo nhn tht cha 3 loi ARN polymerase khc nhau. Polymerase I phin m cc rARN;
polymerase II phin m cc gen m ho protein; polymerase III phin m rARN 5S, tARN v cc ARN
nh khc. Cc promoter ca nhng nhm gen trn c khc bit ng k. Cc gen c phin m bi
polymerase II c trnh t nhn bit ngay trong cc promoter ca chng, nhng cng c nhiu s khc
bit. Cc promoter thuc ARN polymerase III khc thng l nm ngay trong trnh t ca gen c
phin m.
M di truyn gm b 3 cc base (codon) trong mARN. Chng chuyn bit cho vic kt hp acid
amin vo mch polypeptid. M c tnh theo chiu 5 3 ca mARN, lin tc, ph qut v suy thoi,
trong nhiu loi acid amin c quy nh bi 2 hoc nhiu hn 1 codon.
CU HI
1. Phin m i xng c th xy ra :
a) Vi khun b) Lp th
e) Ti th t bo th
2. Tiu n v tch ra khi phc hp phin m khi ARN mi sinh t chiu di:
a) 4 base b) 6 base
c) 8 base d) 10 base
e) 12 base
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 92 of 203
e) a, b, c
a) b) '
c) ' d) ''
e)
e) Tt c
a) Mt base
c) Thm base
d) o v tr mt cp base
e) a, c
a) Cn mi
b) on mi l tARN ca t bo ch
d) on mi gn vo u 3 ca Retrovirus
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 93 of 203
c) Bn phin m u tin (pre - mARN) c s dng ngay cho vic tng hp protein
a) Leucin b) Methionin
c) Tryptophan d) Alanin
e) b v c
a) C ngoi l
c) Ph bin mi sinh vt l m b 3
Bi 5
SINH TNG HP PROTEIN
MC TIU
- M t c c ch dch m di truyn.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 94 of 203
5.1. M U
Giai on cui ca s biu hin thng tin di truyn xy ra khi thng tin c m ho bng trnh t
nucleotid trn mARN c dch m thnh trnh t acid amin tng ng trong chui polypeptid. Qu
trnh ny c gi l qu trnh tng hp protein. Mc d, v c bn tin trnh dch m xy ra nh nhau
mi sinh vt, nhng vn c nhng chi tit khc nhau gia t bo vi khun v t bo nhn tht. Nhn
chung, qu trnh ny t bo nhn tht phc tp hn. Phn ln qu trnh tng hp protein xy ra trong t
bo cht. Mt s bo quan ca t bo nhn tht cng c b my di truyn ring (lc lp, ty th) v cng
c kh nng tng hp c protein m ho bi chng. B my dch m ca cc bo quan ny ging t
bo nhn nguyn thu hn l t bo nhn tht.
5.2.1. Ribosom
Ribosom l xng tng hp protein ca t bo. Tuy c s khc nhau trong cu to v kch thc
gia cc gii sinh vt, nhng u gm 2 tiu n v. V ch c cc ribosom hon chnh, gm mt tiu
n v ln v mt tiu n v nh mi c kh nng dch m mARN.
Khi khng thc hin tng hp protein, mi tiu n v tn ti tch ri trong t bo cht (hnh 5.1).
Tiu n v ln ca t bo nhn nguyn thu gm 2 phn t rARN v 35 phn t protein, tiu n v
nh c 1 phn t rARN v khong 20 phn t protein. rARN c cu trc khng gian phc tp do nhiu
on bt cp vi nhau nh c trnh t nucleotid b sung.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 95 of 203
V t bo nhn nguyn thu khng c mng nhn, qu trnh phin m din ra trong t bo cht, cc
ribosom c kh nng gn vo mARN ang c phin m v dch m to ra protein trong khi ARN
polymerase vn tip tc phin m t ADN.
c t bo nhn nguyn thu ln t bo nhn tht, khi ribosom u tin dch m mARN c mt
on, th cc ribosom khc c th gn vo pha trc dch m. Do , cc ribosom xp thnh chui
trn mARN to nn cu trc polyribosom hay cn gi l polysome (hnh 5.2). Khong 15 - 20 ribosom
c th gn cng lc trn mARN. Nh vy, tc tng hp protein tng nhanh ng k.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 96 of 203
Cu trc ca mARN in hnh c minh ha hnh 5.3. Thng tin di truyn l mt trnh t thng
ca cc base nucleotid. Trnh t trong vng m ho ca mARN cha cc codon quy nh trnh t acid
amin ca protein. Trnh t ny c c theo hng 5 n 3 trn mARN c m u bng mt codon
m u (thng l AUG). Kt thc l mt trong cc codon kt thc (UAA, UGA hoc UAG).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 97 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 98 of 203
Cc ARN vn chuyn hot ng nh mt cht kt ni gia thng tin base - nucleotid ca mARN vi
trnh t acid amin ca phn t protein tng ng. Mi tARN c hiu cho mt loi acid amin ring bit,
mc d n c th gii m cho hn mt codon, s lng cc tARN bin ng theo loi: t bo nhn
nguyn thu 30 - 40, t bo nhn tht 50. Do , cc tARN phi c nhn bit chuyn bit ng thi
bi mARN v bi enzym gn acid amin vo tARN tng ng (enzym tARN - aminoacyl synthetase).
iu ny gip gii quyt tr ngi khng gian trong qu trnh dch m. Kch thc ca codon ln hn
nhiu so vi acid amin, nn nu mt acid amin nhn bit v gn trc tip ln mt codon trn mARN th
n s cch xa vi acid amin tng ng vi codon k cn hnh thnh lin kt peptid. V c ti 20 loi
acid amin khc nhau cu trc nn bn dch, do , cn phi c ti 20 loi aminoacyl - tARN synthetase
khc nhau. l Alanyl - tARN synthetase, Arginyl - tARN synthetase, Asparaginyl - tARN
synthetase,... Mc d, c th c ti mt vi loi tARN khc nhau cho mi mt loi acid amin ( gii m
cc codon khc nhau tng ng vi acid amin ), nhng ch c mt aminoacyl - tARN synthetase cho
mi loi acid amin. Vn mt aminoacyl - tARN synthetase nhn din c vi tARN khc nhau nh
th no hin vn cha r rng. Dng nh, s nhn din chnh xc tARN ca synthetase thng qua vic
nhn din anticodon. Enzym aminoacyl - tARN synthetase phi ph hp v chn ng tARN: tARN ch
ng cho mt synthetase th c gi l tARN duy nht (cognate tARN). Cc loi tARN lin quan n
cng mt acid amin th c gi l tARN ng nhn (isoaccepting tARN).
5.2.4. Aminoacyl ho
Acid amin phn ng vi ATP c enzym xc tc to thnh aminoacyl adenylate hot ho.
Pyrophosphat to ra b thu gii thnh 2 phn t phosphat v c. Phn ng ny to ra mt nng lng
thu gii t do tng i cao, y phn ng theo chiu to aminoacyl - AMP trung gian:
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 99 of 203
C hn mt codon trong m di truyn cho tng loi acid amin, ngoi tr methionin v tryptophan.
Nhng codon ng ngha (synonym) ny thng lin quan n cc tARN khc nhau. Cc tARN li c
trnh t i m (anticodon) khc nhau. Th d, trong trng hp ca serin, mc d c ti 6 codon (UCU,
UCC, UCG, UCA, AGU, AGC) u c ngha nh nhau, nhng khng phi tt c u c s dng
thng xuyn nh nhau. Tht vy, c mt vi codon cho mt acid amin c s dng v c mt vi
phn t tARN cho mi codon. Hin tng ny c gi l s d bit codon v y l im khc nhau
quan trng nht gia loi ny v loi khc.
(1) tARNmMet kt hp vi codon AUG nm gia phn t mARN, c nhim v gn Met vo chui
polypeptid ang hnh thnh.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 100 of 203
t bo nhn tht, cc tARN chuyn bit cho cc codon AUG khi u v AUG bn trong mARN,
nhng tARNMet m u ch vn chuyn methionin khi u c gi l tARNiMet v tARNmMet cho
cc codon AUG bn trong (bng 5.1).
Qu trnh dch m mARN c m t trong hnh 5.6, bao gm cc giai on: khi u, ni di v
kt thc.
5.3.1. Khi u
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 101 of 203
c trng ca s khi u chui peptid vi khun l s tng tc trc tip gia mARN v rARN
ca tiu n v nh 30S (hnh 5.7). S tng tc ny dn n vic gn mARN vo ribosom v quan
trng hn l nhn din c codon khi u ca trnh t mARN. Cc mARN vi khun c cha mt trnh
t dn u 5 kh ngn. N bao gm trnh t polypurin ngn b sung vi mt trnh t giu pyrimidin
tn cng 3 ca rARN 16S (hnh 5.8). Shine v Dalgarno cho rng vic ghp i base gia cc trnh t
ny c th l c ch lm cho ribosom ca vi khun hng ti ng codon khi u ca mARN. Do ,
ngi ta gi trnh t ngn ny l trnh t Shine - Dalgarno.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 102 of 203
Vai tr IF - 2 l lm trung gian trong vic ghp tARN khi u (fMet - tARNfMet) vo tiu n v
30S. iu ny ph thuc vo GTP. Bi v, IF - 2 to ra phc 3 yu t vi GTP v vi tARN khi u.
l phc [IF - 2 - GTP - fMet - tARNfMet] m tARN gn vi ribosom. Cc phn t tARN lun
lun gn vi ribosom nh nhng phc c cha GTP v yu t protein. Chc nng chnh xc ca IF - 1
cha c xc nh, nhng trong mt s trng hp cho thy n to iu kin cho tc ng ca IF - 2.
C th mARN hoc phc hp tARN gn trc tin vo tiu n v 30S. Ngi ta mi ch bit c
rng, c 2 bc ny u xy ra trc khi tiu n v 50S gn vo to ra phc hp 70S cho vic ni
di. Vai tr ch yu ca IF - 3 l gi s gn kt mARN vi ribosom.
S khi u chui peptid vi khun bao gm s nhn din trnh t Shine - Dalgarno, nm gn im
xut pht ca trnh t m ho mARN. Do , s khi u dch m khng nht thit nm gn vi im
khi u 5 ca mARN. ng vy, cc mARN t bo nhn nguyn thu c cha ti vi vng m ho,
ng hn l vi khun thng c polycistron mARN. c bit, r nht l mARN cha operon m ho
cho 3 loi protein khc nhau lac operon.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 103 of 203
Qu trnh khi u chui peptid t bo nhn tht phc tp hn v c nhiu im khc bit vi t
bo nhn nguyn thu. Trong khi, E. coli ch c 3 yu t khi u th t bo nhn tht c ti 10.
iu ny c phn nh trong cc cch thc gn kt mARN v xc nh codon khi u.
Trnh t dn mARN t bo nhn tht khng c xem nh trnh t Shine - Dalgarno t bo nhn
nguyn thu. u tn cng 3 ca rARN 18S (tng ng vi rARN 16S vi khun) khng c trnh t
CCUCC. V mARN t bo nhn tht l monocistron, chng ch cha mt trnh t m ho v khi u,
thng xy ra codon khi u AUG trong mARN. Nhng nhn xt trn gip cho Kozak xy dng m
hnh khi u sinh tng hp protein t bo nhn tht (hnh 5.9).
(a) Tiu n v nh 40S gn vo mARN chp 5 v qua chp (Cap) hnh thnh nn phc hp chp
- cc yu t khi u.
(b) Tiu n v 40S qut dc theo mARN, tm AUG khi u. Qu trnh ny cn ATP v c th tho
xon nt kp tc.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 104 of 203
Tiu n v 40S s b qua codon AUG bnh thng cho n khi nh v ti codon AUG khi u
nm trong ng cnh tt.
Yu t t bo
Yu t vi khun Chc nng
nhn tht
eIF - 4E
eIF - 4F
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 105 of 203
eIF - 4A c kh nng tho xon cc cu trc bc 2 (cn thu phn ATP). Vai tr ca eIF - 4B cha
r, nhng c th lm gia tng tc ng ca eIF - 4A (hnh 5.11).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 106 of 203
5.3.2. Ni di
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 107 of 203
Bng 5.3. Cc yu t ni di
t bo nhn
t bo nhn tht Chc nng
nguyn thu
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 108 of 203
Theo sau s gn kt phc hp vo v tr A, l qu trnh thu phn GTP gii phng GDP v Pi (hnh
5.13). GDP c phng thch khi ribosom s gn kt vi yu t ni di to phc hp [EF - Tu - GDP]
hoc [EF - 1 - GDP]. yu t ni di li c th tham gia vo qu trnh gn kt aminoacyl - tARN tip
theo vo ribosom th GDP phi c thay th bi GTP. vi khun, yu t EF - Ts ng vai tr trung
gian cho vic chuyn i GDP/GTP (hnh 5.14), tng t cc yu t ca protein - G lin quan ti s ti
np tn hiu hormon. Gia acid amin sau cng ca chui mi sinh vi acid amin trn tARN v tr A,
hnh thnh lin kt peptid. Phn ng ny c xc tc bi enzym peptidyl - transferase. Kt qu l
tARN v tr P lc by gi khng cn mang acid amin hoc l tch khi chui polypeptid.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 109 of 203
- Ribosom chuyn dch theo tng codon trn mARN, do , codon tip theo c a vo ging
hng vi v tr A.
EF - Tu c gii phng khi ribosom sau mi chu k ni di thnh phc hp vi GDP (bt hot),
ngha l phc hp ny khng th gn kt aminoacyl - tARN. cho EF - Tu tham gia vo vng ni di
tip theo th GDP cn phi c bin i thnh GTP to phc hp c hot tnh [EF - Tu - GTP]. S bin
i nucleotid Guanin cn c mt yu t protein khc, l EF - Ts.
5.3.3. Kt thc
Chu trnh dch m thc hin thm khong 15 acid amin mt giy vo mch polypeptid. Chu trnh
c chm dt khi tri qua codon kt thc l UAA, UAG v UGA.
vi khun c 3 yu t phng thch: RF - 1 chuyn bit cho cc codon UAA v UAG; RF - 2 lin
quan ti UAA hoc UGA v RF - 3 kch thch c 2 yu t kia. y cn cn yu t ni di EF - G v
yu t phng thch ribosom (RRF) chuyn bit.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 110 of 203
RF - 3 (Kch thch RF - 2)
S kt thc lin quan n vic phn gii lin kt gia acid amin cui cng ca chui peptid mi sinh
v tARN cui cng. Phn ng ny c xc tc bi enzym peptidyl transferase ca ribosom v i hi
GTP. Nh , polypeptid hon chnh c phng thch. Sau , 2 tiu n v ca ribosom c phng
thch cng vi mARN. Tt c cc thnh phn tham gia chu trnh u c s dng li.
- Cung cp nng lng thc hin cc phn ng c bit, nh hot ho acid amin bng cch gn
chng vo tARN.
- Cung cp nng lng cho mt qu trnh no nh tho xon cu trc bc 2 ca mARN hoc cho
s chuyn dch ribosom trong cc bc chuyn v trong qu trnh ni di.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 111 of 203
- Cung cp nng lng cho s bin dng ca ribosom, nh sau khi gn kt aminoacyl - tARN vo v
tr A.
C th tnh nng lng chuyn ho cn dng cho s tng hp mt chui polypeptid in hnh vi
khun gm 300 acid amin nh sau:
Tm li, tnh ra chnh xc c 4 lin kt phosphoanhydrid c s dng cho mi acid amin trong qu
trnh sinh tng hp protein.
Mt tARN c th nhn thm mt acid amin khng ng hoc enzym aminoacyl - tARN synthetase
dng tARN c anticodon khng chuyn bit cho cc acid amin c gn vo n. C mt s acid amin c
cu trc tng t vi acid amin cn c a vo, cho nn cc enzym aminoacyl - tARN synthetase
cng phi c cch thc chn ng.V d, enzym isoleucyl - tARN synthetase, mt mt phi phn bit
gia isoleucin v cc acid amin nh hn nh valin, mt khc phi phn bit gia isoleucin vi cc acid
amin k nc, cng knh hn nh leucin, phenylalanin. C ch iu chnh xy ra nh sau:
Phc hp sai gia codon v anticodon s dn n vic gn vo acid amin khng ng. Cc nghin
cu t bin vi khun cho thy cc protein chuyn bit trong cc tiu n v ca ribosom ng vai tr
quan trng trong vic gn ng cc codon v anticodon trong sut qu trnh dch m.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 112 of 203
c) Ch c isoleucin gn vo v tr A to isoleucyl-tARN
nhng khng th gn vo v tr H v do khng b thu phn.
Kt qu: Acyl ho chnh xc isoleucin
Ngi ta c tnh rng, sai st tng ln t l 1/3000 gc acid amin c kt hp. Ngha l, nu mt
phn t protein c 300 gc acid amin th ch 1 phn t protein trong 10 phn t c cha 1 li v thng
nhng li ny s c nh v m khng quan trng i vi cu trc hoc chc nng ca protein.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 113 of 203
(bng 5.6). Trong s ny c nhng cht c ch chn lc s tng hp protein vi khun m khng nh
hng ti sinh tng hp protein t bo nhn tht. V chng c trin vng s dng nh nhng khng sinh
loi tr s nhim khun m khng nh hng n qu trnh chuyn ho protein ca t bo ch (bng
5.7).
Hp cht p dng v ch dn
Acid fusidic Ph khng khun hp, dng cho cc Staphylococci khng penicillin
Spectinomycin iu tr lao
Nhm tetracyclin Khng sinh c ph khng khun rng, nhng nhiu vi khun khng
vi nhm ny.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 114 of 203
chnh s dch m.
Tm tt
- ARN thng tin: mang thng tin di truyn m ho di dng codon, mi codon l mt t hp 3
nucleotid m ho cho mt acid amin.
- ARN ribosom: l thnh phn ca ribosom, ni hnh thnh chui polypeptid ang tng hp.
- ARN vn chuyn: yu t mang cc acid amin tng ng ti cc codon trn khun ARN n gn
vo chui polypeptid ang hnh thnh ti ribosom
- Khi u: trc tin l hnh thnh mt phc hp gm 3 thnh phn: (1) tiu n v nh ribosom,
(2) tARN mang methionin (Met - tARNiMet), (3) mARN. Mt yu t khi u s pht hin codon khi
u AUG gip phc hp v tiu n v ln ca ribosom gn vo, v s dch m bt u.
- Kt thc: khi cc yu t kt thc nhn ra du hiu kt thc trn mARN, s dch m dng li.
S khc bit gia cc thnh phn v c ch chi tit ca s dch m vi khun v t bo nhn tht l
nn tng cho vic s dng rng ri cc cht khng sinh trong vic c ch chn lc qu trnh sinh tng
hp protein vi khun.
CU HI
e) Gn kt mARN vi ribosome
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 115 of 203
c) Methionin d) AUG
e) GUG
d) Ti hi EF - Tu
e) Ti hi EF - 1
a) mARN b) Tiu n v nh
c) Tiu n v ln d) V tr P
e) V tr A
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 116 of 203
e) a, b v d
a) RF - 1 b) RF - 2
c) RRF d) EF - G
e) EF - Ts
a) c ch chuyn peptid ho
b) c ch peptidyl transferase
a) c ch chuyn peptid ho
b) c ch peptidyl transferase
e) Gy kt thc sm qu
Bi 6
IU HO HOT NG GEN
MC TIU
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 117 of 203
- Gii thch c cch thc tng tc gia cc protein v cc trnh t ADN c bit v cc
phn t khc trong qu trnh iu ho hot ng gen.
6.1. M U
vi khun c ba loi enzym permease vn chuyn qua mng: permease dipeptid, permease tripeptid
v oligopeptid permease. Oligopeptid permease c oppA, B, C, D, F ln lt chuyn vt cht ln nhau
v qua mng, trong oppA c tnh c hiu.
Mc iu ho qu trnh sinh tng hp protein gm nhiu giai on: cc thi k chun b sao chp
b gen v phin m ngha l sinh tng hp cc ARN, thi k kt thc - dch m to ra nhng phn t
protein, mt xch di truyn cui cng ca t bo.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 118 of 203
t bo nhn nguyn thu c h thng a enzym: ADN - polymerase I, II, III, ADN - ligase. Ngoi
ra, li xon v tho xon c helicase v topoisomerase I, phc hi dng siu xon c topoisomerase II
(ADN - gyrase) ph thuc ATP.
Ngoi sao chp b gen, cn c s sa cha v ti t hp, ng thi c s sao chp nhng yu t di
truyn ngoi nhn - plasmid.
Ngi ta xc nh c khong 15 locus di truyn (Dna) kim sot s sao chp ADN. Mi mt gen
nh vy m ho cho mt polypeptid cn thit cho qu trnh sao chp. V d, DnaA cn cho s tng hp
primer (ARNo) v ng vai tr iu ho dng trong qu trnh ny. Song vic to ra sn phm ny li
tn hi n qu trnh t iu chnh. Mt s protein c tnh a chc nng. V d, protein do DnaC tng
hp li cn thit cho vic gn primer vo chui ADN v sau protein ny tr thnh thnh phn ca
phc h sao chp v tham gia vo qu trnh ko di.
Ni mt cch tng qut, kim sot dng l s tch ly cht hot ho cho n ngng khi
ng mt chu trnh sao chp mi, cn thit cho s cn bng vi vic nhn i ca sinh khi t bo.
Ngc li s kim sot m, khi cht kim hm cn c tng hp mt cch gii hn tip ngay khi
bt u chu trnh sao chp trc (cht km hm nh vy c th l sn phm ca gen nm gn v tr bt
u sao chp, m s phin m ca n ch c thc hin trong giai on sao chp ca vng ADN ny).
Trong mi t bo, khng phi gen no cng hot ng nh gen no. Trong khi sn phm ca gen ny
cn c tng hp lin tc, th sn phm sinh tng hp ca gen khc li ch c tng hp trong nhng
giai on cn thit tu thuc mi trng. Thm ch, khi gen "m" th hm lng protein cho chng
cng b kim sot. Do hot ng ca hu ht cc gen u c iu ho trong mt hay mt vi con
ng lm cho hiu ng s dng ca t bo v mt nng lng t c cao nht. C ch iu ho ny
i vi s biu hin gen c th xy ra cc mc khc nhau. S iu ho gen c th xy ra ngay ti
thi im phin m hay trong qu trnh dch m. Sau qu trnh dch m, mt s protein c th b bin i
cu hnh thnh protein chc nng.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 119 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 120 of 203
lm tng s lng ARN polymerase khi ADN phin m. Enhancer c th nm cch gen cu trc m
n tng cng.
Hiu qu tng cng c thc hin qua mt loi protein gn vo mt trnh t ADN c hiu. Khi
protein gn c hiu ny gn vo v tr tng cng, cc nuleotid xen gia ni nt ra a v tr tng cng
ln tip xc vi promoter ca operon cn tng cng. Cu trc m nt ny gip cho ARN polymerase
gn c vo gen phin m.
Khi protein hot ho gn vo v tr khi ng hay v tr tng cng s kch thch s phin m ca
gen cu trc trong operon. Ngi ta gi c ch ny s kim sot dng. S kch thch do cc gen iu
ho p ng c th xy ra t nhng phn t nh (ng, acid amin...) n nhng cht ln hn nh cc
phc hp steroid ca hormon (t bo nhn tht) v cc protein th th ca n.
Cht "m" gen, lm cho gen phin m c gi l cht cm ng. Cht "ng" s phin m c gi
l cht c ch. Cc gen c th cm ng thng tham gia trong cc phn ng d ho, v d nh khi phn
gii polysaccharid thnh ng n.
- Kim sot cm ng m.
- Kim sot c ch m.
Kiu khi nguyn ca s kim sot m c thc hin bi operon c th cm ng, l "h thng
lactose" ca E. coli.
- galatosidase l mt enzym c hai chc nng. Chc nng ch yu ca n l phn gii lactose
thnh glucose v galactose. Chc nng th hai ca n l bin i lin kt 1 - 4 glycosid ca glucose v
galactose trong ng lactose thnh lin kt 1 - 6 trong ng allolactose. Bnh thng, khi lactose
thiu ht trong mi trng th enzym ny khng nhiu lm. Ngay sau khi thm lactose vo mi trng,
m trong khng c glucose, enzym ny lp tc c sn sinh. Protein vn chuyn c tn l
galactoside permease cn thit cho s vn chuyn c hiu qu lactose qua mng t bo. Protein ny cng
t ngt tng ln sau khi cho lactose vo mi trng. "H lactose" hoang di gm gen iu ho (I+) v
operon cha trnh t promoter (p+), operator locus (O+) v 3 gen cu trc ca - galatosidase (Z+),
permease (y+) v transacetylase (a+). t bin ca tng locus tng ng c tm thy trong t
nhin.
Mt s k hiu:
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 121 of 203
Cc allen ca promoter
- p+: promoter kiu hoang di, hot ng bnh thng vi ARN polymerase
- ps: tng cng s nhn bit ca ARN polymerase, nng cao mc phin m ca operon.
- picr: nh hng ti v tr gn ca CRP - cAMP lm gim mc biu hin ca operon lac di 10%
kiu hoang di (icr = insensitive to catabolite repression).
Cc allen ca operator
- O+: allen iu khin ny cho php cc cistron cnh n (tc l v tr cis) biu hin, ngha l c
phin m. Allen ny mn cm vi cht c ch, cht ny ngn chn s phin m phn cn li ca operon.
Cc allen - galactosidase
- Z: t bin c ngha, sinh ra mt sn phm bin tnh khng hot tnh enzym gi l Cz protein.
Cc allen permease
Cc allen regulator
- i-: gen iu ho b hng, khng c kh nng sinh cht c ch hot tnh do t bin v ngha hay
lch ngha.
- is: to cht siu c ch khng nhy cm vi lactose v khng hot ho vi bt k operon O+ no,
ngha l khng b bt hot bi lactose.
i khi c s chng cho gia promoter v cc v tr operator ca "h lac"; trong mt vi operon
ngi ta thy cc locus operator ny ph hon ton ln promoter. Operon iu ho thng xuyn sinh
ra cc protein c ch mc thp, bi v n c mt promoter km hiu sut. Promoter ny c k
hiu bng pi (inefficient promoter). Vic tng hp ra cht c ch khng thay i theo mc lactose trong
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 122 of 203
t bo. Ngc li, promoter bnh thng (P+) ca operon lactose (lac - p) li gn vi ARN
polymerase rt hiu qu. Khi thiu lactose (iu kin khng cm ng) th protein c ch c hot tnh
(sinh ra bi i+) s gn vo operator O+ (lac O). ARN polymerase lc by gi c th hoc l khng gn
c vo promoter hoc l khng c c operator, bi v protein c ch bao vy c vng promoter.
T lc by gi, s phin m ca 3 gen cu trc ca lac operon b ngn cn.
Khi lactose xut hin (iu kin cm ng c) song li khng c chuyn qua mng t bo vo t
bo cht mt cch hiu qu bi v lc by gi ch mi c vi phn t permease xut hin. Bn trong t
bo, mt vi phn t lactose b bin i thnh allolactose nh - galatosidase. Allolactose l cht cm
ng ca lac operon. N gn vo protein c ch, lm thay i cu hnh ca protein c ch v lm thay i
v tr gn ca protein c ch vo operator. S thay i cu hnh trong phn t protein do mt phn t
khc gn vo, c gi l s bin hnh d lp th. Phc hp allolactose - protein c ch khng th gn
lu vo operator c v n b ri khi ADN ngay (hnh 6.2). Lc ny, ARN polymerase c th c
operator phin m cc gen cu trc trn operon. Khi lng permease tng ln, lactose c chuyn
qua mng nhiu ln v ng lactose li b phn hu bi - galatosidase. Khi lactose trong mi trng
tr nn cn kit, protein c ch va c tng hp s c gii phng khi allolactose, do chng
(cht c ch) c th gn cht vo operator, lm ngng phin m ca cc gen cu trc trong operon lac.
Cht c ch ni chung l cc protein c cu trc oligomer, trong mi mt tiu n v ca n u c
hai tm c th. Trong hai tm ni trn, tm no c i lc cao vi trnh t ca ton b promoter th c
coi l tm gn cht c ch, cn tm cn li s l tm gn cht cm ng. S gn cht cm ng vo cht c
ch tm bin cu th hai s lm thay i cu hnh ca cht c ch, chnh v vy m lm gim i lc
ca tm gn cht c ch i vi operator v gip operator t gii thot khi cht c ch.
Allolactose lun c xu hng tch khi protein c ch, ngay sau khi mc lactose trong t bo xung
thp. Thm ch khi "h lac" b c ch, i khi protein c ch tch khi operator mt cch nhanh chng.
ARN polymerase c th ln vo operator m operon v tng hp nn phn t mARN a cistron. iu
ny gii thch c mc rt thp ca permease v - galatosidase lun lun thng trc trong t bo
vi khun.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 123 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 124 of 203
Gen iu ho cho h thng ny lun lun sn sinh ra mt loi protein khng chc nng, c gi l
cht c ch gc. Khi tryptophan sn xut d tha, th s qu ti ca tryptophan s tc ng nh mt cht
ng c ch. Cht ng c ch gn vo cht c ch gc hnh thnh nn mt phc hp c ch chc nng.
Phc hp c ch chc nng ny gn vo operator trp, v c ch s phin m ca 5 gen cu trc trn
operon. Cc vng promoter v operator ca operon ny nhiu khi ph ln nhau v gn c phc hp co
- aporepressor, do ARN polymerase kh chen vo c. Khi nng tryptophan gim xung,
tryptophan tch khi cht c ch gc v cht c ch gc ri khi operator, th ARN polymerase li tng
hp c mARN a cistron cho 5 enzym tng hp tryptophan.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 125 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 126 of 203
Trnh t dn cha mt phn ng vai tr b suy gim, chnh b suy gim ny m ho phn t peptid
dn cha 14 acid amin bo hiu cho s phin m dng li.
Trnh t dn cha 4 vng, trong vng A cha hai codon m ho cho 2 codon m sao cho
tryptophan (UGGUGG) c k hiu XX. Phn D trc khi ni vi Tryptophan operon c mt trnh t
m ho cho 7 - uracyl trn mARN.
ARN polymerase chuyn ng dc si c ngha ADN theo chiu 3 5 v phn t mARN tng
ng s c tng hp theo chiu 5 3. Gn u 5 ca mARN l v tr gn ribosom (hnh 6.7).
Ribosom gn nh chuyn dch lin tip theo sau ARN polymerase trong qu trnh phin m v gii
m. Ribosom gn v trt trn mARN. Cc vng A v B c th bt cp vi nhau v c cha nhng trnh
t b sung v vng C, D cng vy. Nhng nu B cng bt cp c v tr vi C th s c th hnh thnh
nn 3 nt kp tc A/B, B/C v C/D.
S phin m suy gim ca tryptophan operon cng nhy cm vi cc nng ca mt s acid amin
khc ngoi tryptophan nh histidin v leucin.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 127 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 128 of 203
Sn phm protein ca araC (AraC) l cht c ch ca nhm BAD khi mi trng khng c
arabinose. N lun lun gn vo araO lm ADN c dng loop nn ARN polymerase khng gn vo
c phin m. Tuy nhin, sau khi gn vi arabinose th repressor ny b bin i cu hnh thnh
phc hp hot ho ri khi operator (araO) v gn vo initiator (araI), to iu kin cho ARN
polymerase gn c vo promoter ca BAD, nh cm ng c s phin m ca operon. Bn thn
AraC t iu ho chnh n bng cch gn vo araC.
Adenosine monophosphat vng (cAMP), protein hot ho gen d ho (CAP) v protein nhn AMP
vng (cyclic AMP receptor protein = CRP) tham gia vo s iu ho ca h thng arabinose. AMP vng
c kh nng hot ho cho nhng promoter c i lc yu nhng bn thn n khng gn c vo
promoter. Mun lm iu ny n phi lin kt vi CAP hoc vi CRP. Khi AMP gn c vo promoter
n s hot ho ARN polymerase v ARN polymerase s gn c vo promoter.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 129 of 203
V d, sn phm cui cng isoleucin trong E. coli khi xut hin nng cao, lin kt vi enzym
u tin (e1) ca qu trnh sinh tng hp v do vy km hm ton b con ng ny cho n khi
isoleucin tr li mc bnh thng thng qua mc tiu th ca t bo.
Cht khi u ca chui gm 5 phn ng ny l threonin. Phn ng u tin threonin bin thnh
acid - ketobutyric do enzym threonin - desaminase, qua mt lot qu trnh chuyn ho to ra sn phm
cui l isoleucin. Vo mt thi im no , isoleucine khng c t bo s dng n s lin kt tr li
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 130 of 203
Tm tt
Vi khun sng trong mt mi trng cnh tranh gay gt, chng cn phi thay i nhanh chng trong
c ch chuyn ho sng st. Nhiu vi khun to ra cc phng thc chuyn ho thch ng vi s
thay i ny, cc enzym trong qu trnh chuyn ho ch c tng hp khi cn thit. S kim sot nh
vy c thc hin mc gen.
phng thc ng ho, cc enzym cn cho s sn xut mt acid amin c bit nh tryptophan ch
c tng hp khi tryptophan khng c sn vi khun. trong trng hp ny, cc cht c ch l sn
phm cui cng ca qu trnh.
Cc phng thc khc ca s kim sot biu hin gen l bng cc protein hot ho, y l s kim
sot dng. C s tham gia ca cAMP vo vic iu chnh s biu hin gen vi khun.
CU HI
b) Thuc dng iu ho m
d) Gn vo v tr tng cng
e) a, b
e) a, c
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 131 of 203
a) Gen iu ho (Is)
e) a, b, d
e) a, b ng
5. Cht c ch gc l:
b) Tryptophan
d) Trnh t dn
e) b, c v d
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 132 of 203
8. Operon gm:
b) Cc gen cu trc
c) V tr iu ho
d) Chp GMP
e) a, b v c
9. Operator l:
a) on mARN gn c protein iu ho
c) a vo co - repressor
d) Loi b co - repressor
e) a v d
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 133 of 203
Bi 7
B GEN T BO NHN THT
MC TIU
7.1. M U
Cc b gen t bo nhn tht phc tp hn t bo nhn nguyn thu. S gen m ho cho protein cng
nhiu hn gp trm ln. S d ADN c th hin nhiu loi trnh t khc nhau. ADN ca t bo nhn
tht dng thng, cn t bo nhn nguyn thu th dng vng.
Nm 6 000 1,3.107
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 134 of 203
ssADN virus
Parovirus 5 5 000
dsARN virus
Coronavirus 7 20 000
Infuluenza 12 13 000
TMV 4 6 400
Viroid
Nghch l gi tr C l mt khi nim phn nh s khng tng xng gia kch thc b gen (gi tr
C) vi s gen ca mt loi. V d, ngi c b gen ln hn giun n 30 ln nhng ch gp i v s gen.
Nh vy phn ln trnh t tn ti trong b gen khng lin quan n gen.
Khi tch ADN ca t bo nhn tht bng siu li tm trn thang nng Cesium chlorid, c 3 vt
c ghi nhn (hnh 7.1).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 135 of 203
S khng ng nht ca ADN t bo nhn tht th hin r hn khi thc hin phn ng ti hp
(reassociation). ADN c ct nh v cho bin tnh ri sau cho hi tnh. Khi hi tnh, cc on c
trnh t b sung d ti hp vi nhau, nh vy nhn bit cc trnh t lp li.
ng hc ti hp thnh cp ca ADN t bo nhn tht khc vi t bo nhn nguyn thu (hnh 7.2).
ng cong ti hi ca t bo nhn nguyn thu c dng hnh sigma in hnh, chng t s ng nht
ca cc trnh t trong ti hp. t bo nhn tht ng cong phc tp hn, ko di mt khong rng cc
gi tr C0t (n v ca n l s mol nucleotid/lit/giy) v c cha 3 thnh phn lp li mc khc
nhau:
- ADN lp li trung bnh (ti hp nhanh va), chim khong 25 - 40% b gen.
Phc hp ca mt vi b gen bit kch thc c th c tnh ton bng cch so snh ng cong
C0t ca n vi ng cong C0t ca mt ADN mt phc hp c bit trc (thng l ADN E. coli
vi kch thc b gen c tng chiu di ca cc trnh t l 4,2.106 bp). Cch so snh nh vy s dng
ca mt dn s ADN, l gi tr C0t thi im 50% ADN ti t hp.
C0t 1/2 ca ADN t b gen th nghim = phc hp hin din ca b gen th nghim.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 136 of 203
Cc trnh t ny cng c bit nh l ADN v tinh (satellite DNA), bi v khi ADN ca b gen
c ct ra v ly tm trong cc nng gradient t trng cesium chlorid. Cc chui lp li cao thng
hnh thnh cc di bng v tinh cch xa nh chnh (kh rng) so vi cc chui lp li trung bnh hay
duy nht. iu ny cho thy rng ADN lp li cao thng c mt thnh phn base khc vi thnh phn
ca b gen (v v vy t trng cc phn ni khc nhau).
- Loi th ba: c nhiu trnh t lp li mc cao, phn tn pha ngoi cht d nhim sc nh trnh
t CEN v trnh t TEL.
- Cc trnh t TEL:
Cc trnh t TEL thuc cc nhm telomere (u mt ca nhim sc th) vi nhiu vai tr khc nhau
nh bo v u mt nhim sc th khi b ct, gi chiu di ca nhim sc th khi sao chp, gn vi
mng nhn v km hm s biu hin ca cc gen u mt.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 137 of 203
Cc trnh t TEL c tnh bo tn trong tin ho. Chng c s ln lp li cao, giu C v A: CC(C)
ACACA(CA) nm men, CCCTAA ngi. u mt ca nhim sc th giu G gp li hnh kp tc c
cu trc 4 mch. Cu trc ny bo v u mt nhim sc th khi b ct bi nuclease, ng thi khi sao
chp gi u mt khi b mt cc trnh t m ho.
Loi trnh t ny chim 25 40% b gen ngi. Chng cng gm cc trnh t lp li nhng di hn
(100 1000 bp) v a dng hn nhiu so vi loi lp li cao (v tinh). Cc trnh t ny phn tn trong b
gen v nu ct b gen thnh on 20 40 kb th c 90 n 100% s on c trnh t lp li trung bnh.
Trong s cc trnh t lp li trung bnh, c cc h trnh t c hiu l SINE v trnh t LINE.
Mt t bo nhn tht c th c hng trm nhm ADN lp li trung bnh, vi mi nhm c khong t
50 - 105 on lp li.
C mt s lng ln cc nhm khc ADN lp li trung bnh trong b gen ngi, nh chui 6400 cp
base c lp li t 3000 - 4600 ln c tnh cho khong 1% trong tng s ADN ngi.
Mt trong nhng nhm ln nht ca ADN lp li trung bnh ngi l phc hp ca cc chui c
gi l h Alu. S d c tn ny v n c cha v tr nhn din cho enzym ct gii hn AluI. Chui Alu
xut hin khong 3x105 ln trong b gen n bi ca ngi, v vy chim khong 3% ADN ngi.
S chuyn v nghch to bn sao ca cc phn t v tr mi, trong khi phn t cho ban u vn gi
nguyn cu trc khng bin i. Do vy, chuyn v nghch to nn mt t on t v s ti cu trc ca
b gen t bo ch. Nhng bin i ny s lm ngng hay hot ho cc gen, m mt s bin i ny c
th gy ra ung th.
Hai n v lp li ca trnh t Alu ging nhau v trnh t 7SL ARN, thnh phn cu to ca du hiu
nhn din, vn chuyn protein qua mng li ni sinh cht. Do iu ny ngi ta tin rng trnh t Alu
tng ln l nh s chuyn v 7SL ARN, do vy to thnh gen gi ct 7SL ARN. Qu trnh chuyn v
trnh t Alu xy ra nh enzym phin m ngc m ho bi LINE - 1 (Kpn) v c th l nguyn nhn gy
cc vn lm sng. C th s lng ln cc bn sao gen gi ct lin quan n s hin din trnh t
promoter trong trnh t 7SL ARN (gen 7SL ARN, ging gen tARN c m ho bi ARN polymerase
II t mt promoter ni). Ngc li, gen gi ct t bn sao ARN polymerase II thiu trnh t promoter v
chng ch c th biu hin nu chng gn trnh t promoter c chc nng.
Hin nay chc nng ca trnh t Alu cha uc bit. Mc d tn s thng gp l 1 bn sao trn 4
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 138 of 203
- Cc trnh t LINE
Trong h LINE 1, nhiu on c kh nng chuyn v. Chiu di ton b l 6.1 kb v c 2 ORF, tuy
nhin chng khng hin din trong hu ht trnh t ring r. ORF 1 gn cui (u tn cng 5) v m
ho protein cha r chc nng p40 (trng lng phn t khong 40 kDa). ORF2 c nhng vng ging
vi trnh t nucleotid m ho cc enzym phin m ngc khc nhau v cc protein virus khc. Cu trc
hon chnh ca LINE 1 cha mt promoter trong vng ADN khng dch m trc ORF1 (gi l 5 -
UTR) trong khi ti u 3 c trnh t (A)n/(T)n, thng c ui poly A. Trong trng hp cc cu trc
khc c th chuyn v v cc cu trc LINE - 1 b kp bi cc trnh t i ngn. Cu trc LINE - 1 hon
chnh kh him (ch khong 3500 bn sao) v hu ht cc on lp li b ct u 5 do vy chng c
chiu di khc nhau v thng c ui polyA. Cc cu trc LINE - 1 thng vng cht nhim sc
nhng khc vi trnh t lp li Alu do thch nhnh G ti (Giemsa dng) ca nhim sc th k gia.
Ging Alu, chng thiu cc trnh t m ho nhng c th tm thy cc trnh t khng m ho ni sinh.
Do vy, chng hin din trong bn sao ARN u tin ca cc gen ln nhng khng c trong mARN.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 139 of 203
Mt lot cc gen gi vai tr quan trng trong sinh tng hp protein nh cc gen ribosome, tARN,
ARN 5S v ARN 7SL c s lp li hng nghn ln.
Cc gen rARN c phin m bi ARN polymerase I. Bn phin m u tin l pre - rARN, sau khi
ct ni to ra 3 loi rARN l rARN 28S, rARN 18S v rARN 5,8S. Cc gen ny khng phn tn m xp
thnh cm (cluster), mi cm c th hn 200 bn sao. ngi, cc cm c tm thy trn vai ngn
ca cc nhim sc th tm u (acrocentric) 13, 14, 15, 21 v 22, chim khong 0,4% b gen. Cc nhm
ny xp quanh yu t t chc hch nhn hnh thnh kiu cu trc c bit trn nhim sc th v gian
k to nn cc hch nhn (nucleolus).
Cc gen 28S, 5,8S v 18S rARN bt thng do cc gen nhn chim s lng ln, c phin m
ring r, u tin chng c biu hin nh l cc bn sao a gen, theo cch ca gen ty th. on sao
chp ca 13 kb biu hin tin 45S rARN sau tri qua nhiu phn ng khc nhau to thnh 28S,
5,8S v 18S rARN trng thnh. Ging nh cc sn phm ca gen ty th, cc sn phm rARN ring r
ca nhm rADN l cc thnh phn c lin quan vi mt chc nng ring bit. Do vy, vic s dng bt
thng cc bn sao ARN khng khc so vi cc sn phm dch m u tin t t c tch ra thnh 2
hay nhiu chui polypeptid c chc nng thng thng. V d, phn t insulin c 2 chui polypeptid
c to thnh t sn phm dch m duy nht. Cch di truyn ny c lin quan n cc sn phm khng
trng lp t tin phn t duy nht him khi xy ra: cc thnh phn ring r ca a s protein a tiu n
v ngi c m ho bi cc gen khc nhau, thng trn cc nhim sc th khc nhau.
ngi c hn 200 gen m ho cho ARN 5S. Cc gen ny tp trung thnh cm mt s im nht
nh ca cht nhim sc th. mt s loi nh Xenopus s bn sao ca ARN 5S c th hn 20 000.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 140 of 203
- Cc gen gi.
Cc gen m ho cho protein thuc nhm II v n c phin m do ARN polymerase II. Tr cc gen
m ho cho protein histone, cc gen nhm II c trnh t c nht hoc vi s t bn sao. Cc gen nhm II
ny hu nh ch m ho cho mt loi protein.
- H cc gen Globine.
- H cc gen Actine.
- H cc gen Myosine.
Cc gen Globine tp hp thnh cm: phc hp ca cm Globine nm trn vai ngn ca nhim sc
th 16 v phc hp Globine nm trn vai ngn ca nhim sc th 11. Trnh t sp xp ca gen tng
ng vi trnh t biu hin trong qu trnh pht trin c th.
- tng t (xi)
- tng t (epsilon) G , A ,
Gen gi c xc nh bi vic cha cc trnh t c lin quan mt thit vi cc gen cu trc, nhng
khng dch m thnh protein chc nng c.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 141 of 203
Phn tch trnh t 2 cho thy rng mc d gen 2 c quan h mt thit vi cc trnh t khc trong
cm, nhng n li tch t, mt s t bin bao gm lch khung v chui kt thc, ngn chn 2 m ho
protein bt k phng din no. Do , 2 ca th l mt v d ca gen gi. Gen gi c biu th theo
ch Hylp l , v vy gen ny c k hiu l 2. Loi gen gi ny c xem l kt qu tin ho ca
mt gen c khi biu hin nhng by gi li l gen t bin ln. C mt vi v d khc ca cc gen gi
loi ny, nh cc gen tng t - tubulin ngi, cc gen rARN 5S Xenopus.
Gen gi 2 ca th cng cha intron v exon lin quan mt thit vi gen cu trc 1. Nhng vic
mt mt cp base codon th 20 ca n gy nn hin tng t bin lch khung lm chm dt ngay
s dch m sau . Gen gi cng c th c to ra khi 2 intron c xen gia l mt exon v khng c
intron no c ct ra khi exon khi gen phin m.
DNaseI ct ADN b gen mt s vng lm tho xon cho cc gen biu hin (1) ca hnh 7.6.
Hai vng c lu : nhy cm v tng nhy cm. Cc vng nhy cm c lin quan n cc gen c
hot tnh cao v nhng gen trc y biu hin (gen hot ng phi).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 142 of 203
7.3.2. Mc phin m
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 143 of 203
- S tc ng ca trnh t cis (cng pha) nm trn cng mch ADN nh enhancer (on tng
cng) (4).
7.3.4. Mc dch m
Sau khi mch polypeptid c tng hp, cc protein nhiu khi chu cc bin i th cp trc khi
biu hin hot tnh. V d, trypsin l enzym phn gii protein trong d dy ch c c hot tnh sau khi
tin cht ca n (proenzym) khng c hot tnh, b ct mt mt on polypeptide.
- Glycosyl ho, phosphoryl ho... (11): gn thm cc nhm cht nh ng, phosphat protein c
hot tnh.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 144 of 203
- Trnh t iu ho 5' pha trc cc promoter t bo nhn tht thng rt di, c khi hng chc
kb.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 145 of 203
bo nhn nguyn thu phn ln p li tn hiu ngoi sinh. Ngc li, phn ln s iu ho t bo
nhn tht l p li cc tn hiu ni sinh.
7.4.1. Cc promoter
Tng t nh t bo nhn nguyn thu, cc promoter ca t bo nhn tht cng nm pha trc
im xut pht ca mARN v cng c nhng trnh t chung trong tin ho. Hp TATA nh hng cho
ARN polymerase bt u phin m, pha ngc chiu vi chiu phin m khong 30 bp ADN ng
vt c v v 60 n 120 bp ADN nm men. Hp TATA hot ng c hiu qu cng vi hai trnh t
tng ng pha trc khong 40 bp l CCAAT v 110 bp l trnh t giu GC (hnh 7.8).
Hp TATA v cc trnh t pha trc phi c nhn bit bi cc protein iu ho. Chnh cc
protein ny gn vi cc im nht nh trn hp TATA v hot ho s phin m.
Trnh t th hai c bit n l hp GC hay hp Sp1, cng hin din ngc chiu mt s cc gen
t bo nhn tht, thng l trong cc bn sao nhiu ln. V d, trnh t promoter ca gen Thymidine
kinase c 2 hp GC ngc chiu vi hp TATA (hnh 7.9).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 146 of 203
Trnh t tng cng u tin c m t virus SV40, c nh v khong 200 bp ngc chiu vi
v tr bt u phin m ca gen sm. Nu loi b trnh t ny s lm gim ng k kh nng sao m t
trnh t promoter ca gen sm. Tuy nhin, s ti chn ca trnh t tng cng vo bt k ni no ca b
gen SV40 s gi li mc biu hin bnh thng. Hn th na, nu trnh t tng cng SV40 c ni
vi gen - globine th s t ngay s biu hin cch xa 200 bp pha trc hay pha sau promoter -
globine v ngay c khi trnh t khuch i SV40 nm cch v tr khi u phin m ti vi nghn cp
base. Gn y, ngi ta tm thy cc trnh t tng cng c cc gen chui nng globine min dch
ng vt c v v gen albumine ca chut. Khong cch gia mt trnh t tng cng v mt trnh t
khi ng ln nht gp gen albumine ca chut l 10 kb.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 147 of 203
Cc trnh t ti cis c c im chung l chng thng c cu trc gm hai phn i xng nhau. V
d, trnh t p ng vi hormon tuyn gip (TRE - Thyroid hormon Respone Element) di y:
- Vng tc ng ln s phin m.
Tin hnh ghp hai trnh t ADN c ngun gc khc nhau: vng tc ng phin m ly t mt yu
t trans ng vt c v v vng gn vo ADN ly t yu t trans nm men. Kt qu to ra mt yu
t "lai" c kh nng hot ho cc gen ch tng ng nm men. Gen ch l gen mang trnh t tip
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 148 of 203
nhn yu t trans. Ngoi hai vng k trn, nhiu yu t trans cn mang mt s vng khc nh: vng
gn hormon, cc ion... (hnh 7.11).
Hnh 7.11 cho thy cu trc ca mt th th tuyn gip (THR - Thyroid Hormon Receptor) bao gm
nhiu vng cu trc v chc nng, trong hai vng c bit r nht l:
- C: vng gn ln ADN.
Nh vy, cc gen t bo nhn tht c hot ho bi hai trnh t ADN c tc ng cis l promoter
v enhancer. Chng c nhn bit bi cc yu t protein c tc ng trans. Cc yu t ny cho php
ARN polymerase khi s phin m v t tc phin m ti a (hnh 7.12).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 149 of 203
7.4.4. Hormon
- Vng gn vo hormon.
- Hot ho protein hiu ng, to thnh phc hp hot ho, kch thch ARN polymesare gn vo
phin m.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 150 of 203
Mt s nhm phn t hin din nhiu trong t bo nhn tht nh histone, cc thnh phn ca b my
dch m, cc thnh phn ca mng, t bo,... duy tr s lng ln chng trong t bo phi c cc
chng trnh:
- Lp li cc gen.
7.5.1. S di do ARN
Nhiu 50 1
t 35 12.500
Nhiu 22 9
t 37 11.500
Mt t bo cha vi trm mARN d trung bnh, chim khong 33% tng s mARN t bo, nhng
mi loi mARN d ch chim 0,1 - 0,5% tng s mARN. Phn mARN cn li ca t bo gm cc bn
m sao d t. C khong 10000 bn m sao nh vy trong mt t bo in hnh, mi bn sao i din
cho t hn 0,01% tng s mARN.
Cc loi ARN khc cng c hin tng d. Xenopus c vng t chc hch nhn cha 450 bn sao
ca ADN m ho cho rARN 18S v 28S. Ngc li, trong mi nhn c t 20000 bn m sao ca cc gen
m ho cho rARN 5S v cc gen ny khng nm trong vng t chc hch nhn.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 151 of 203
Trong nhn ca t bo trng Xenopus c ti hng trm nhn con ngoi nhim sc th vi kch thc
khc nhau. Mi nhn con cha cc vng rARN c kch thc khc nhau, vai tr cha r. Cc ADN vng
ny sn sinh nhiu rARN lp rp thnh nhiu ribosome.
Tm li, s biu hin ca cc gen t bo nhn tht rt phc tp. Tuy vy, hng nghn promoter
c iu ho to ra cc mARN mc hp l.
Tm tt
Kch thc v cu trc b gen t bo nhn tht phc tp hn nhiu so vi b gen ca t bo nhn
nguyn thu. ADN ca b gen t bo nhn tht c mc lp li cao, trung bnh v n c.
S iu ho biu hin gen c thc hin t bo nhn tht nhiu mc khc nhau:
CU HI
1. Gi tr C l:
2. Nghch l gi tr C
a) L gi tr nghch o ca gi tr C
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 152 of 203
a) SINE b) LINE
c) CEN d) TEL
e) Tt c
4. Trnh t TEL
e) a v b
5. Trnh t SINE
e) a v d
6. Gen gi l:
e) c v d
b) Tc ng nh mt cht cm ng
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 153 of 203
e) Tt c
e) Tt c
9. Enhancer
a) C tc ng cis
e) a v c
d) Ch yu p ng li tn hiu ni sinh
e) a v d
Bi 8
T BIN GEN
MC TIU
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 154 of 203
- Trnh by c tc ng ca cc yu t gy t bin.
8.1. M U
t bin theo ngha rng ch cc bin i di truyn xy ra t ngt. Mt t bin gen l mt s thay
i trong trnh t nucleotid trn gen, dn n kt qu l to ra mt protein t bin vi trnh t acid amin
thay i hay tc ng n qu trnh iu khin s tng hp ca cc sn phm gen. Nu protein t bin
c chc nng khc vi dng t nhin th s to ra mt thay i tng ng nhng c tnh c th quan
st c v to ra th t bin. S khc bit c th do hot tnh hay tnh n nh ca enzym. Mt s t
bin c th tn ti trong qun th bn cnh cc dng t nhin lm thnh mt th a hnh. Trong th a
hnh, khng th phn bit gia dng t bin v dng t nhin.
ngi, cc gen c t l t bin cao (khong 10-4) l bnh u x thn kinh (NF) v bnh lon dng
c Duchenne (DMD), c t l t bin thp (khong 10-6) l gen bnh Huntington. S cch bit hng
trm ln cho thy t l t bin gen c th khc nhau tht s.
- Kch thc ch: gen NF v DMD c vng m ho protein rt ln, c nhiu base c th b thay i
hoc lm mt chc nng ca gen.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 155 of 203
Sinh vt t bin Tn s Cn c nh gi
Tn s t bin c th hiu mt cch o ngc. V d, rui dm thn xm bnh thng (e+) t bin
thnh thn c mu en h phch e (ebony) vi tn s 2.10-5, ngha l trong 105 giao t c hai t bin t
e+ e. Hoc v d E. coli t bin nhy cm vi streptomycin thnh khng streptomycin vi tn s
4.1010 t bin trong mt t bo mt th h. d hiu ta c th tnh ngc li, tc l trong 10 t t
bo ca mt th h c 4 t bin StrR xut hin ngu nhin.
Tuy tn s t bin ca tng gen l rt thp, nhng tng cc t bin ca nhiu gen l mt s ng
k, c ngha quan trng trong tin ho. Cc kt qu nghin cu in vivo v in vitro trn t bo ngi cho
thy t l t bin ngi khong 106/gen/th h. Cc vi sinh vt t bo nhn nguyn thu v t bo
nhn tht cng c t l t bin tng t. C th c tnh cc thay i xy ra trong mi th h da vo t
l t bin ngi nh sau: 106 t bin/gen x 5.104 gen/b gen n bi bng 5.102 t bin trn giao t
(5/100 hoc 1/20). Mi hp t c (1/20).2 giao t trn hp t bng 1/10 c hi mang t bin mi u
trong b gen. Con s ny c v rt cao nhng cn nh rng hu ht cc t bin l ln v nh vy s
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 156 of 203
K thut ti t hp ADN cho php chn mt t bin c hiu vo mt gen quy nh trc (t bin
im nh hng) c th to ra cc t bin tt v hu ch.
8.2.1. t bin im
Ph bin nht l chuyn v, pyrimidin c thay th bi pyrimidin khc hay purin bi purin khc (A
G, C T). S chuyn v, bt cp sai, c th gy bi acid nitr hoc cc ng ng base nh 5 -
bromo - 2 - deoxyuridin (BrdU). Cch t bin im t ph bin hn l o chuyn, pyrimidin c thay
th bi purin hay purin bi pyrimidin (C/T A/G). t bin im c khuynh hng c trng l t
bin li (hi bin), ngha l chuyn li dng nguyn thu. iu ny xy ra v ln t bin tip theo ti
cng mt im c mt phn ba c hi quay li chui ban u. C ba loi t bin im tu theo codon
mang t bin m ho:
t bin ln hay cn gi l t bin cng ngha: m ho cho cng acid amin, khng c nh hng
n protein cui. V d, codon GCA hoc GCG trong mARN u ngha l arginin (loi ny thng ng
vi t bin chuyn v v tr th ba - base "linh hot" ca codon).
t bin lch ngha: m ho cho acid amin khc, c th lm protein khng c chc nng. V d,
trong chui protein - globin gy bnh thiu mu hng cu hnh lim, GAG m ho cho glutamat; nu
thay bng GUG s m ho cho valin. t bin lch ngha c th gy hu qu rt nghim trng nh thiu
mu t bo li lim, hoc nh nh hemoglobin C (acid amin v tr 6 ca - globin b thay th bi acid
amin khc) hoc khng c kiu hnh.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 157 of 203
t bin thay th base c th xy ra trong promoter hoc cc vng iu ho 5 ca gen hoc trong
intron v c th nh hng n s phin m, dch m hoc mc xon bn ca ADN. Nhiu -
thalassemia l hu qu ca cc t bin khng cu trc ny nh hng mc biu hin cc gen globin.
Mc nh hng tu thuc nucleotid b thay th v v tr ca n trong ADN.
8.2.2. t bin a im
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 158 of 203
C hai loi t bin l t bin t nhin (xy ra mt cch t pht) v t bin cm ng, gy ra bi
cc tc nhn t bin.
- Kh amin: Cc phn ng kh amin thng thng l phn ng thu phn cytosin thnh uracil, qu
trnh ny phng thch ammonia v kh amin 5 - methylcytosin thnh thymin v ammonia (hnh 8.2b).
Trong ADN, s kh amin cytosin t pht c chnh li bng cch loi b uracil (v uracil khng c
trong ADN) v thay th bng cytosin, cn s kh amin 5 - methylcytosin khng c chnh v c ch
sa cha khng nhn din Thymin l li. Trong ADN b gen cytosin ti cc trnh t CG (CpG) thng
b methyl ho, iu ny ngha l u CpG xy ra trong gen, n l "im nng" cho t bin. Mi y
ngi ta tm thy trong gen lin quan n lon sn sn c nhng im nng nh vy.
- t bin lch khung (chn hoc mt trn mt si), thng l li ca polymerase khi sao chp cc
on lp li ca mt nucleotid. Mi polymerase c chnh xc khc nhau. Yu t chnh nh hng n
chnh xc ca polymerase l c exonuclease c sa 3 - 5, lm loi b cc base bt cp khng ng
chn vo bi polymerase.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 159 of 203
8.3.2. t bin cm ng
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 160 of 203
Base ng ng:
- Bromouracil (BU) l hp cht nhn to dng rng ri trong nghin cu. Ging vi thymin (c
nguyn t Br thay cho nhm methyl) v s gn vo ADN v bt cp vi Adenin ging nh Thymin. C
nhiu kh nng h bin thnh dng enol (BU*) v c kh nng bt cp vi Guanin. Nh vy, BU c th
xm nhp v bt cp b sung vi Adenin thay cho Thymin to A - BU v vng sao chp tip theo BU
li h bin thnh BU* v bt cp vi Guanin to BU* - G, do A - T c thay th thnh G - C (hnh
8.3a). Trong khi BU* c th xm nhp sai, khi gn thay ch cho Cytosine to G - BU*, sau BU*
li h bin thnh BU v tip theo bt cp vi Adenin to A - BU v vng sao chp tip theo Adenin
bt cp vi Thymin thnh A - T (hnh 8.3b).
- Cc cht kh amin. V d in hnh: acid nitr to bi s phn gii cc nitrit (cht bo qun)
trong thc phm lm Cytosin thnh Uracil, Methylcytosin thnh Thymin, v Adenin thnh Hypoxanthin
kh amin, Hypoxanthin trong ADN bt cp vi C gy chuyn v. Methylcytosin c mt trong cc gen
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 161 of 203
Cc cht alkyl ho. V d in hnh: Nitrosoguanidin (NTG), Methyl Methane Sulfonat (MMS),
Ethyl Methane Sulfonat (EMS) l cc tc nhn ho hc gy t bin phn ng vi cc base v thm cc
nhm Methyl hoc Ethyl nn cn gi l cc tc nhn alkyl ho (bng 8.2). Tu yu t b tc ng, chng
c th gy t bin t nht bng 3 cch:
Thm nhm Methyl (- CH3) hay ethyl (- C2H5) vo Guanin to base ng ng ca Adenin dn
n s bt cp b sung sai (hnh 8.4).
Mt purin do Guanin b alkyl ho to l hng trn ADN, khi sao chp c th lm t mch
(hnh 8.4).
Lin kt cho gia cc mch ca mt hoc cc phn t ADN khc nhau lm mt nucleotid.
Guanin l base c th d b tn cng nht, vng pyrimidin, c bit l cc nhn pyrin d dng b
alkyl ho. Cc v tr ny l cc v tr lin quan trc tip n vic ghp cp nh 6 - keto oxygen ca
guanin hay 4 - keto oxygen ca Thymin v cc v tr cch xa v tr ghp cp nh N - 7 ca Guanin.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 162 of 203
Ethylen imin EI
Nitrosoguanidin NG
Nhm ny gm cc cht Proflavin, cam Acridin, Ethidium bromide dng trong phng th nghim
lm cht nhum. Tt c u l cc phn t a vng phng tng tc vi cc base ca ADN v chn vo
gia chng. S chn ny lm "dn" si i ADN v ADN polymerase b "nh la" chn base vo i
din nhiu hn bnh thng, lm lch khung ADN to thnh.
Cc tc nhn ny c th l:
- Cc phn t ln (knh cng) gn vo base trong ADN v lm chng tr thnh khng m ho. V
d, N - acetoxy - 2 - acetylaminofluorene (NAAAF)
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 163 of 203
- S lng t bin t l thun vi liu lng phng x, khng ph thuc vo cng v thi gian
chiu x.
Cc bc x in t:
Cc sng di nht (radio AM) c nng lng t nht, trong khi cc sng ngn hn l FM, TV, viba,
hng ngoi, kh kin, tia t ngoi (UV), tia X v tia gamma ln lt c nng lng gia tng. Tia t
ngoi (cn gi tia cc tm) c nng lng phng x cao nn c ngha sinh hc.
Bc x ion ho:
n v dng cho tt c cc loi bc x ion ho hin nay l rem (roentgen equivalent man = 0,01J/kg
ngi b chiu x), do 1 rem ca bt c bc x ion ho no cng c tc dng sinh hc nh nhau. n
v c dng trc y l rad (radiation absorbed dose). Tuy nhin rad ca cc bc x khc nhau s
khc nhau: 1 rad ca cc ht c tc dng ph hu ln hn 1 rad ca cc tia gamma, ngha l cc ht
c RBE (relative biological effectiveness) ln hn 1 rad ca cc tia gamma. Mi tng quan gia cc
n v ny l: rad RBE = rem.
Ngoi ra, cn xt n loi nng lng v liu bc x ton phn, t l liu: cng s rem, nu chiu tp
trung (t l liu cao) gy bng v ph hu da, trong khi chiu nh trong thi gian di (t l liu thp) s
ch lm tng nguy c t bin v ung th.
Ngun bc x:
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 164 of 203
Ngoi ra, con ngi to ra ngun bc x nhn to, gp phn tng mc bc x. Trong c cc th
nghim y t (v d chn on bng tia X), th ht nhn v cc nh my nng lng ht nhn, cc sn
phm khc (tivi, my d khi, kim tra bng tia X sn bay).
Tc dng sinh hc ca bc x:
nh hng di truyn ca bc x:
- Ph hu cc base (t bin).
Tia cc tm (UV):
UV c phn loi theo bc sng: UV - C (180 - 290 nm) c tnh st trng mnh nht v gy cht,
khng c trong nh sng mt tri v n c hp ph bi tng ozon; UV - B (290 - 320 nm) l phn
on gy cht/t bin chnh ca nh sng mt tri; UV - A (320 nm - kh kin) l "cn UV", cng c
tc dng xo mt (v n to cc gc oxy) nhng n sn xut rt t dimer pyrimidin. Cc tn thng gy
cht chnh l cc dimer pyrimidin trong ADN (to bi UV - B v UV - C), cc dimer ny l lin kt
ng ho tr gia cc pyrimidin k nhau trong mt si. Cc dimer ny lm ngn chn s phin m v sao
chp ADN v gy cht nu khng sa cha. Chng c th kch thch t bin cng nh sp xp li
nhim sc th.
Trong t bo, cc cht hu c c mch vng ch yu nh purin v pyrimidin hp thu trc tip UV.
Tia t ngoi c kh nng xuyn thu thp nn ch tc ng ln cc sinh vt n bo v giao t. ADN hp
thu tia t ngoi mnh nht bc sng 257 nm ( 260 nm), y chnh l bc sng lm tng tn s t
bin ht phn cy bp (ng). Di tc ng ca tia t ngoi, cytosin gn thm phn t nc vo lin
kt C=C ca mch vng (Hnh 8.5) v thymin b t lin kt C=C mch vng, ni 2 phn t thnh
thymin dimer.
Stone v cc cng s nhn thy tn s t bin tng ln Staphylococcus aureus khi mi trng
nui chng c chiu tia UV trong thi gian ngn trc khi cy vo. y l tc ng gin tip ca tia
t ngoi.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 165 of 203
Hin tng quang phc hi l mt c trng trong tc ng ca tia UV. Sau khi chiu tia t ngoi
ln t bo, nu ngoi nh sng, th cc sai hng phn ln c phc hi. nh sng c tc ng hot
ho enzym sa sai, ct t cc thymin dimer.
- o nghch sai hng: c ch ny n gin nht, enzym hot ng phc hi cu trc bnh thng
khng c gy khung.
- Loi b sai hng: ct b v thay th base hoc phn nucleotid sai hng hoc khng thch hp.
- Dung np sai hng: khng sa cha tht s, chp sai hng v tip tc sng.
Enzym photolyase xc tc phn ng ny, n c nhiu trong vi khun, t bo nhn tht bc thp, cn
trng v thc vt. Khng c trong t bo ng vt. Gen (phr) c trong ng vt c v nhng m ho cho
protein c chc nng h tr loi sa cha khc.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 166 of 203
E. coli gen m ho cho enzym ny (39 kDa) l ada. V tr Cys69 trn enzym nhn gc metyl t
phosphodiester v thay i cu hnh cho php n hot ho promoter ca cc gen ada, alkA
(methyladenin DNA glycosylase), alkB, v aidB, nh tng kh nng khng vi cc tc nhn alkyl
ho. Trong khi v tr Cys321 nhn gc metyl t O6 - MeGuanin v O4 - MeThymin, khi phc base
bnh thng.
8.4.1.3. Ni cc ch t ca si n
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 167 of 203
trong c bc x ion. Ngoi ra, hi chng Bloom cng lin quan vi khim khuyt ADN ligase
(mc d protein ca hi chng Bloom l ADN helicase); t bo nui cy ca bnh nhn ny c mc bin
dng nhim sc th v t bin t pht cao.
Loi b ch sai nh mt hoc vi enzym N - glycosylase. N - glycolase nhn bit base bin i v
thu gii lin kt N - glycosilic ni base vi ng pentose. iu ny to ra mt v tr AP (khng c
purin hoc pirimidin), ngha l c mch ng - phosphat nhng b thiu base nit. V tr AP sau
c ct b bi AP endonuclease v ADN polymerase chp y li da vo khun l mch b sung i
din v c ligase ni lin li.
Qu trnh ny xy ra sau sao chp ADN nh l "nh vn kim tra" cui cng s chnh xc ca sao
chp. E. coli, c ch ny lm tng chnh xc ca sao chp ln thm 100 1000 ln. Sa cha ny
c tin hnh bi mt nhm cc protein c th qut ADN v tm cc base bt cp khng chnh xc
(hoc cc base khng bt cp) lm sai lch hai chiu trong xon kp. Nucleotid khng chnh xc phn
di s c loi b v ADN polymerase s hot ng ln th hai c trnh t ng.
H thng ny lm vic trn "khi" ADN sai hng, lm ngng sao chp ADN v phin m. Qu trnh
gm ct si ADN cha sai hng bi endonuclease pha sai hng, tip theo loi b on ngn cha
vng sai hng bi exonuclease, khong trng to thnh c lp vo bi ADN polymerase (hnh 8.8).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 168 of 203
Nhiu sinh vt b t bin khim khuyt NER, d b dit v t bin bi UV v cc ho cht hot
ng ging UV. Ngi c bnh di truyn xeroderma pigmentosum nhy cm vi nh sng mt tri, h
c nguy c cao b ung th da nhng vng c th phi di Mt Tri v c cc khim khuyt trong cc
gen tng ng vi h thng NER t bo nhn tht n gin. Cc t bin NER sinh vt bc thp
nhy vi UV v tng mc t bin v ti t hp cm ng bi UV (v chng khng th dng phng
php NER chnh xc loi b cc dimer pyridin v phi dng cc h thng gy t bin hoc gy ti t
hp).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 169 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 170 of 203
Khi c nhiu sai hng cp cu, lexA b kch thch, thay i cu hnh, t ct v mt hot tnh km
hm. Lc , cc gen SOS c m ra. Nu sa sai khng kp, t bo phi chp nhn hoc b t bin
hoc b cht.
Mt sinh vt b t bin n gin l mt sinh vt biu hin nhng tnh trng bt thng di truyn
c. Hu ht nhng tnh trng ny c th thy c nh nhng t bin sc t gy nn bnh bch tng,
thay i mu sc bn ngoi ng vt v mu sc hoa bt thng.
Trong y khoa, tm thy cc loi t bin khc nhau ngi. ng dng cc nh lut di truyn
Mendel, ngi ta chng minh rng mt vi bnh ngi c di truyn nh l cc bnh do mt gen
ring l quy nh.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 171 of 203
Hnh 8.11. Chui phn ng sinh ho ca bnh Alkapton niu v Phenylketon niu,
hai s ri lon di truyn ca s thoi qu acid amin thm
Bnh t bin u tin do Garrod nghin cu l bnh Alkapton niu. Nc tiu ca bnh nhn tr
nn en khi ngoi khng kh. Bnh xy ra do thiu enzym homogentisate oxydase trong gan nn
khng phn hu c acid homogentisic (tn c l alcapton) thnh mt sn phm khng mu l acid
acetoacetic. Lc by gi acid homogentisic tch t li trong nc tiu v c mu en khi gp khng kh.
Lin quan ti ri lon chuyn ho bm sinh, cn c bnh thiu ht enzym chuyn ho phenylalanin
v tyrosin. Mt trong cc v d quen thuc v d tt bm sinh c di truyn do yu t gen ln Mendel
n trn nhim sc th thng vi tn s cao (khong 1/10.000) l Phenyl ceton niu. Bnh ny l do
thiu enzym phenylalanine hydroxylase trong gan c th oxy ho phenylalanine thnh tyrosin (hnh
8.11). Hu qu trc tip l tch t phenylalanine trong mu v bi tit thi qu tng phn qua nc tiu
di dng cc cht chuyn ho bt thng nh acid phenyl pyruvic.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 172 of 203
khng cn pht trin trn mi trng gin n na tr khi thm vo mt cht khc, thng l acid
amin, vitamin nucleotid hoc mt phn t vi lng no . Nhng c th mang t bin ny c gi l
th khuyt dng.
Bng 8.3. Mt s bnh di truyn ngi c lin quan n protein chuyn bit
hay khim khuyt enzym
Tn s tnh
Triu chng Hot tnh sinh ho b nh
Loi ri lon Tn trn 1000
in hnh hng hay khim khuyt
ngi
Dystrophin, mt protein
Teo c
C Thoi ho c lin quan n mng t 0,1 (n ng)
Duchenne
bo c
Mc
phenylalanine,
Chuyn ho acid Phenylketon mc Phenylalanine
0,1
amin niu phenylacetic hydroxylase trong gan
acid trong huyt
tng v nc
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 173 of 203
tiu cao; tr tr
chm pht trin
Tn thng
Hypoxanthine guanin
Hi chng nron thn kinh,
Chuyn ho purin phosphoribosyl 0,1 (n ng)
Lesh - Nyhan thng c hnh
transferase
vi t hu hoi
Da nhy cm
vi nh sng,
gan b tn Enzym Ferro - chelatase
Chuyn ho Hem Protoporphyria thng, ca qu trnh sinh tng Him
protoporphyrin hp Hem
trong huyt
tng
Cc thc th lipoprotein
X va ng
t trng thp (LDL) trong
Vn chuyn Cao cholesterol mch
mng t bo LDL v 1
lipoprotein mu gia nh (Atherosclero -
cholesterol khng c
sis)
hp thu
Chm tr tu, tn
Thoi ho Bnh Acetylglucosamindase
thng gan v 0,01
Mucopolysaccharid Sanfilippo B trong lysosom
khp xng
B thoi ho
thn kinh, tch 0,3
Thoi ho BnhTay - Hexoseaminidase A
ly gangliosid (Ashkenazi
Gluosephingolipid Sachs trong lysosom
trong t bo Jews)
thn kinh
Rt nhiu nguyn nhn gy ung th khc nhau, nhng c ch phn t u lin quan n cc bin
i di truyn trn ADN lm tn thng tin trnh tng sinh bnh thng.
- Th nht, cc bin i lm tng hot gen kch thch, kiu t bin ny c hiu qu tri. Alen bin
i c gi l oncogen (gen ung th), cn alen bnh thng l proto - oncogen (gen tin ung th).
- Th hai, cc bin i lm bt hot gen km hm, kiu t bin ny thng c hiu qu ln. Gen
km hm c gi l gen c ch khi u.
Cc retrovirus bng con ung sao chp ngc tch hp c ADN ca mnh vo nhim sc th
t bo ch. Chng c th ngu nhin mang cc gen ung th lm bin i t bo ch bng cch xen on
ADN ca mnh cnh gen tin ung th ca t bo ch. Hng lot cc gen ung th c xc nh t
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 174 of 203
H gen Ngun gc
Chc nng ca gen tin ung th U do virus gy ra
ung th virus
abl = Bch huyt Abelson; erb = Erythroblastose (tng nguyn hng cu);
Sis = Simian sarcoma virus; Src = Sarcoma.
Mt s gen ung th khc c pht hin khng do virus. Hin nay, pht hin c hn 60 gen
tin ung th.
- Khuch i gen.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 175 of 203
Hnh 8.12. Ba cch bin i gen tin ung th thnh gen ung th
- Gen p53 to protein p53 c ch nhiu dng ung th. Cc nghin cu gn y cho thy protein p53
tham gia h thng cp cu, sa cha nhiu tn thng ca t bo ang phn chia.
Cc h thng chn lc t bin gip pht hin c hiu qu cc t bin him hoi trong ngun t
bin rng ln. C nhiu h thng chn lc t bin v p dng tu thuc vo cc t bin (bng 8.5).
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 176 of 203
H thng chn lc t bin c nh gi bng nng lc phn gii, l kh nng pht hin cc t
bin rt him so vi khng t bin. Nng lc phn gii cng ln th pht hin c cc t bin cng
him. V d, pht hin cc t bin khng c phn gii cao v khi cy s lng rt ln t bo ln
mi trng chn lc c cha cc tc nhn th phn ln t bo cht, ch c rt t t bo c t bin
khng mc c thnh khun lc.
Dng tc nhn l thuc hoc phage chn lc vi khun. Cc t bin d dng c pht hin cc
khun lc mc trn mi trng thch c thuc hay phage.
Dng pht hin c cc t bin khuyt dng. Dung dch vi khun pha long c cy trn b
mt mi trng thch ti thiu mc thnh cc khun lc ri. Ph mt lp mng mi trng tng t
ln mt mi trng thch va ri, cc khun lc bnh thng mc ln. Sau li ph thm mt lp
mi trng dinh dng c b sung v tip cho cht b sung khuch tn. Cc t bin khuyt dng s
mc sau khi c cht b sung, nn khun lc nh hn do mc chm.
Penicillin c tc ng dit khun dng phn chia. Vi khun c cho vo mi trng ti thiu c
penicillin. Cc vi khun ang tng trng b dit, ch cc t bo t bin khng tng trng cn sng
st. Sau , hn hp c cy ln mi trng khng c penicllin th cc t bin khuyt dng mc ln
vi t l tng i cao hn.
Dng chn la cc t bin khuyt dng nm si. Dung dch bo t c nui trong mi
trng dinh dng thiu cht b sung. Cc t bin thiu cht b sung khng mc c, cc dng bnh
thng mc ra thnh nhiu si. Khi lc qua mng lc si thu tinh, cc dng bnh thng nhiu si b
gi li, cc dng t bin i qua mng lc. Dung dch c nhiu dng t bin c cy trn mi trng
c cht b sung v kim tra tm cc dng t bin.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 177 of 203
Vi khun c cy mc ri tng khun lc trn mi trng dinh dng. Dng ming nhung (c
nhiu lng mn bc trn khc g) in ng v tr khun lc trn mi trng ti thiu. Cc khun lc t
bin khng mc ln c. Cn c v tr khun lc khng mc bn sao, tch cc t bin khuyt dng
(hnh 8.13).
Tm tt
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 178 of 203
Cc h thng chn lc t bin vi sinh vt hon thin cc phng php chn lc t bin trong
phng th nghim.
CU HI
a) 105 b) 106
c) 107 d) 108
e) 109
a) Base ng ng b) Alkyl ho
Chn vo ADN
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 179 of 203
e) b v c
e) Ti t hp
d) a v b
e) a, b v c
a) Mt nhim sc th b) Mt t bo
e) b, c, d
a) H bin ca base
b) Kh amin AP site
c) Kh amin to base ng ng
e) B cm ng bi ho cht
9. C ch chng li t bin
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 180 of 203
e) Tt c
e) a v b
Bi 9
CC PHNG PHP PHN TCH ADN
MC TIU
ADN cha thng tin di truyn ca sinh vt, do mun nghin cu mc sinh hc phn t cn
chit tch ADN. ADN gm cc loi: ADN b gen, ADN ty th; ADN plasmid, ADN chloroplast v
ADN virus. Tu kch thc ADN, loi t bo m chn phng php chit khc nhau m bo chit
c ADN tinh khit v c hiu sut cao.
ADN l phn t c kch thc ln, khi chit tch cn lu trong thao tc trnh mi tc nhn c hc
hay ho hc qu mnh c th lm t gy phn t ny.
- Bc 2: Loi b cc thnh phn khng mong mun trong mu (protein, lipid, polysaccharid...).
Thng dng hn hp phenol - chloroform - iso amyl alchl (25 : 24 : 1) lm bin tnh protein, ng
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 181 of 203
thi khng ho tan acid nucleic; sau ly tm: protein bin tnh s ta thnh mt lp nm gia pha
nc cha acid nucleic v pha phenol - chloroform, thu nhn li pha nc cha acid nucleic.
9.1.2. Tinh ch
- Siu ly tm trn gradient lin tc cesium chlorid (CsCl). K thut ny c th t phn gii 1%
n v t trng, cho php phn tch hai phn t ging nhau trong mt phn t c nh du bng
mt ng v nng.
- Siu ly tm trn m cesium chlorid (gradient khng lin tc). K thut ny thng c p dng
tinh ch mt lng ln acid nucleic.
- Siu ly tm trn gradient saccharose. K thut ny dng phn tch hn hp cc acid nucleic c
kch thc chnh lch nhau nhiu kb.
- Sc k i lc: s dng pha tnh l U - Sepharose hay oligodT - cellulose tinh ch mARN.
- Sc k trao i ion trn vi ct, p dng thu hi nhng lng ADN rt nh.
- Sc k lng hiu nng cao: dng tinh ch cc oligonucletid tng hp ( phn gii l 1
nucleotid), plasmid, phn tch cc on ADN.
9.2.1. Quang ph k
Phng php ny cho bit kt qu tng i nng acid nucleic trong mu.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 182 of 203
Tuy nhin cch tnh ny ch ng vi cc dung dch acid nucleic tinh khit. kim tra tinh khit
ca dung dch, ngi ta o thm gi tr OD 280 nm (OD280nm). 280 nm l bc sng cc protein
c mc hp th cao nht, nhng cc protein cng hp th nh sng bc sng 260 nm nh cc acid
nucleic nn lm sai lch gi tr tht ca nng acid nucleic. Mt dung dch acid nucleic c xem l
tinh khit (khng tp nhim protein) khi t s OD260nm/OD280nm nm trong khong 1,8 - 2.
Phng php ny c p dng c trong phn tch nh tnh ln trong vic thu nhn mu acid
nucleic.
Tc in di ca ADN qua gel tu thuc vo bn thng s chnh: (1) Kch thc phn t ADN - l
thng s quyt nh nht. Phn t ADN si i dui thng di chuyn vi tc t l nghch vi logarit
ca trng lng phn t ca chng; (2) Cu dng ca ADN, 3 dng tn ti ca mt ADN l dng siu
xon, dng vng v dng thng tuy c cng trng lng phn t v thnh phn, nhng di chuyn qua gel
vi tc khc nhau; (3) Nng gel xc nh tc di chuyn v hiu qu tch cc phn t ADN, s
di chuyn ca phn t ADN t l nghch vi nng ca gel; v (4) in th s dng - in th thp,
tc di chuyn t l vi in th s dng.
in di trong trng xung c p dng phn tch cc phn t ADN rt ln (>50 kb). Nguyn
tc phng php da vo s i hng in trng trong qu trnh in di. Mi ln hng in trng
thay i th phn t ADN phi t nh hng li. Thi gian cn cho vic t nh hng li tu thuc
kch thc ca phn t, phn t cng di th thi gian nh hng li cng ln khin cho n di chuyn
chm hn phn t kch thc nh.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 183 of 203
Da vo kh nng nhn bit v ct mt trnh t xc nh trn phn t ADN, ngi ta chia RE thnh
ba loi: (1) Loi I - enzym ct phn t ADN ti im cch trnh t nhn din khong 1000 - 5000
nucleotid v gii phng vi chc nucleotid; (2) Loi II enzym ct ngay v tr nhn bit trnh t; (3)
Loi III - enzym ct phn t ADN ti im cch trnh t nhn din khong 20 nucleotid. Trong k thut
di truyn, cc RE loi II c s dng rt nhiu thao tc trn ADN nn sau y ch trnh by cc c
tnh ca loi ny.
9.3.1.2. c im ca RE loi II
Trnh t c trng RE nhn bit thng bao gm 4 - 8 nucleotid. Mi enzym ch nhn nhn mt
trt t chuyn bit c cu trc palindrome, ngha l hai mch ca trnh t hon ton ging nhau khi
chng c c theo chiu 5 3, v d trnh t GAATTC ca enzym EcoRI.
T nm 1962, khi Werner Arber ln u tin pht hin ra RE trong t bo vi khun, ti nay ngi ta
pht hin hn 500 loi RE v hn 100 loi ang c thng mi ho.
Cc RE loi II c hai kiu ct: (1) ct to u t do RE ct hai mch ADN ti cng mt im v (2)
ct to u so le hay u dnh. Cc enzym ct to u dnh c s dng rt nhiu trong ti t hp di
truyn in vitro, hai phn t ADN c ngun gc khc nhau nhng cng c ct bi mt RE s c kh
nng kt hp thnh mt thng qua cc u dnh.
9.3.1.3. ng dng
Vic s dng cc RE c ngha quan trng trong nghin cu di truyn phn t. Chng cho php ct
nh b gen khng l ca cc sinh vt t bo nhn tht, lp bn gii hn, so snh b gen cc loi
khc nhau thng qua k thut RFLP.
Cc enzym ligase xc tc s hnh thnh lin kt ni hai on ADN (ADN ligase) hay ARN (ARN
ligase).
Cc enzym ny c th c ly trch t E. coli (E. coli ADN ligase) hoc t phage T4 xm nhim E.
coli (T4 ADN ligase v T4 ARN ligase).
E. coli ADN ligase c kh nng xc tc phn ng ni hai trnh t ADN c u dnh; T4 ADN ligase
cng chc nng vi E. coli ADN ligase nhng c bit cn c kh nng ni hai trnh t ADN c u
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 184 of 203
bng; v T4 ARN ligase xc tc ni hai trnh t ARN bng lin kt phosphodiester thng dng
nh du phng x u 3 ca cc phn t ARN, sn xut on d.
Khi nhit c nng ln ti nhit "bin tnh" (Tm), hai mch phn t ADN tch ri nhau, s
bt cp tr li s khng xy ra nu nhit phn ng h xung t ngt v phn t ADN s tn ti trong
mi trng dng mch n di mt cu hnh khng gian v trt t. Ngc li, nu sau khi hai mch
tch ri, nhit c lm gim t t cng vi iu kin thch hp, hai mch s bt cp tr li. Hin
tng ny c gi l s lai phn t, n c tnh c hiu tuyt i.
- Nhit .
- di ca cc trnh t.
- Lc ion.
Cc phng php lai phn t rt a dng, trong bi ny ch cp kiu lai trn pha rn v lai ti ch.
Nguyn tc ny l mt trong hai trnh t b sung (cc mch n) nm trong mi trng lng l mt
dung dch m v trnh t kia (thng l trnh t ch, tc trnh t cn tm) c c nh trn mt gi th
rn.
Cc phng php lai trn pha rn c ng dng nhiu nht l Southern blot, Northern blot v dot
blot.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 185 of 203
Phng php dot blot cho php xc nh tng i mt ARN c trng trong mt hn hp ARN m
khng cn phi phn tch chng ra. Phng php ny c th s dng cho ADN.
9.3.3.3. Lai ti ch
Nguyn tc lai ti ch l mt kiu lai phn t trong trnh t acid nucleic cn tm (trnh t ch)
nm ngay trong t bo hay trong m. Lai ti ch cho php nghin cu acid nucleic m khng cn phi
tch chit chng ra khi m, t bo.
Cc ng dng ca kiu lai ny rt a dng i t k thut nh v gen trn nhim sc th, pht hin
dng vi khun ti t hp trong phng php to dng n vic nghin cu mt mARN chuyn bit trong
t bo v m.
9.3.3.4. Cc loi mu d
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 186 of 203
- Phn tch trn gel polyacrylamid, v tr ca cc oligonucleotid trn gel tng ng vi kch thc
ca chng v c pht hin nh u c nh du phng x.
- Tng hp ADN trn khun ADN mch n nh enzym ADN polymerase, vi s hin din ca bn
loi nucleotid, trong c mt loi c nh du ng v phng x (35S).
Phn ng tng hp c tin hnh trong bn phn on ring, mi phn on c cho mt trong
bn loi dideoxyribonucleosid (l nhng deoxyribonucleosid c nhm 3OH c thay bng H) vi hm
lng rt nh. Do hm lng thp nn thnh thong mi c mt dideoxynucleotid c s dng vo
phn ng tng hp mt oligonucleotid, v lp tc s tng hp oligonucleotid dng li.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 187 of 203
l phng php xc nh trnh t bng my t ng v phng php PCR dng trong xc nh trnh
t acid nucleic.
9.5. PCR
9.5.1. M u
PCR l phn ng tng hp ADN nh enzym polymerase do Karl Mullis v cng s pht minh nm
1985. PCR a li mt cuc cch mng trong di truyn phn t: khuch i in vitro acid nucleic c
chn lc bng to dng in vitro m khng cn s hin din ca t bo, khc phc c nhc im thao
tc phc tp v cn thi gian di ca k thut to dng in vivo. Nh PCR, mt on ADN mt vng
bt k trong b gen c khuch i ln rt nhiu ln khi trnh t nucleotid hai u on bit.
9.5.2. Nguyn tc c bn
PCR s dng cc c im ca qu trnh sao chp ADN. ADN polymerase dng cc on ADN
mch n lm khun tng hp nn si mi b sung vi si ny. Cc si ADN khun mch n c
th to ra theo cch n gin l un nng dung dch ADN mch kp n gn nhit si.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 188 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 189 of 203
li c dng tip cho chu trnh tip theo, bao gm cc bc gn on mi, tng hp ADN v
tch ri cc on. Kt qu cui cng ca phn ng PCR l sau n chu k phn ng, tnh theo l thuyt ta
s c 2n-2 bn sao cc phn t ADN mch kp nm gia hai on mi (bng 9.1).
1 0 17 32768
2 0 18 65536
3 2 19 131072
4 4 20 262144
5 8 21 524288
6 16 22 1048567
7 32 23 2097152
8 64 24 4194304
9 128 25 8388608
10 256 26 16777216
11 512 27 33544432
12 1,024 28 67108864
13 2,048 29 134217728
14 4,096 30 268435456
15 8,192 31 536870912
16 19,384 32 1073741824
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 190 of 203
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 191 of 203
Bc 1: Trong mt dung dch phn ng bao gm cc thnh phn cn thit cho s sao chp, phn t
ADN c bin tnh nhit cao hn nhit "chy" Tm ca phn t, thng l 94oC 95oC trong
vng 30 giy 1 pht. Ti nhit ny, cc phn t ADN mch kp tch nhau ra hon ton, to nn cc
si n dng lm khun cho cc on mi v ADN polymerase. y l giai on bin tnh.
Bc 3: Nhit c tng ln n 72oC gip cho ADN polymerase chu nhit hot ng tt nht.
Thi gian tu thuc di ca trnh t ADN cn khuch i, thng ko di khong 30 giy hay hn
na. on ADN nm gia hai mi c tng hp. y l giai on tng hp hay ko di.
Tm li, mt chu k gm ba bc: bin tnh (un nng) tch cc si ADN kp thnh si n
lm khun, lai ho (gn kt) cc on mi vo si khun v ko di (tng hp) bng ADN polymerase,
s c lp i lp li nhiu ln v mi ln li lm tng gp i lng mu ca ln trc. y l s
khuch i theo cp s nhn (hnh 9.6).
Vt liu gm trc ht l ADN c cha on cn nhn ln. Khng cn thit phi tch ring on cn
nhn ln v vic c cc on mi dng trong phn ng xc nh. Hm lng ADN cn cho phn
ng rt nh. Trong cc th nghim bnh thng phng th nghim ch cn mt microgram ADN b gen
l . Trong nhiu trng hp, phn ng PCR c th nhn cc on ADN ch t mt phn t ring l.
Hai on mi xc nh im bt u tng hp ADN, ADN polymerase, v hn hp ca 4
deoxyribonucleotid ( dng dNTP) v dung dch m c ion Mg2+ cn c a vo ng nghim c
cha ADN khun. Dung tch tng s thng l 50 l hoc 20 l. Hn hp phn ng c cho vo ng
nha nh chu nhit, c np y.
Mt phn ng PCR c th gm cc thnh phn hay cc vt liu ban u: mch khun cho s khuch
i, cc on mi oligonucleotide, dNTP, v ADN polymerase chu nhit vi dung dch m phn ng
thch hp v MgCl2. Nng ca mi thnh phn ny tu yu cu. Tr ADN khun v cc on mi,
cc thnh phn cho PCR u c sn t cc nh cung cp.
Enzym ADN polymerase c s dng u tin trong PCR l on Klenow ca ADN polymerase I.
c tnh ca on ny l xc tc s tng hp ADN theo chiu 5 - 3 v c hot tnh exonuclease (thu
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 192 of 203
gii lin kt gia cc nucleotide t u phn t ADN) theo chiu 3 - 5. Tuy nhin enzym ny
khng chu c nhit nn thao tc phc tp v hiu qu thp (phi thm enzym mi vo phn ng sau
mi ln bin tnh v enzym c b nhit phn hu, nhit lai thp khin s khuch i khng c hiu
vv...).
Hin nay thng dng ADN polymerase l Taq polymerase chu nhit, c tch chit t mt loi vi
khun sui nc nng c tn Thermus aquaticus. Enzym ny khng b ph hu nhit bin tnh v
xc tc s tng hp t u n cui qu trnh phn ng.
Taq polymerase c sn trn th trng t cc cng ty cng ngh sinh hc. Cc polymerase chu nhit
khc c trn th trng bao gm Vent polymerase c phn lp t Thermococcus litoralis, xc tc tng
hp nhiu bn sao chnh xc t mch khun hn l Taq polymerase. Ngoi ra cn c Tth polymerase
c phn lp t Thermus thermophilus. Cc polymerase chu nhit l thnh phn t nht ca PCR v
thng c s dng 1 n v cho mt phn ng. Hm lng ny xc tc cho s khuch i
khong 40 chu k bng my PCR.
Trc y, vic la chn polymerase chu nhit cho 1 th nghim th khng my kh khn v ch c
mt loi ADN polymerase chu nhit (Taq). Hin nay c nhiu loi polymerase c ngun gc khc nhau,
nhiu bn sao ca cc polymerase ny c bin i di truyn v chng c th u vit hn polymerase
gc trong phm vi mt vi ng dng (bng 9.2). S chn la mt polymerase chu nhit cho mt th
nghim PCR ph thuc vo yu cu ca tng th nghim.
Mc n nh Hot ng Cation cn 3 5
Enzym Ngun gc Ni 95oC (t1/2) ti u thit Exonuclease
Thermus Sui nc MgCl2
Taq 40 pht 50 - 75 -
aquaticus nng
AmpliTaq Tag ti t hp 40 pht 50 - 75 MgCl2 -
AmpliTaq AmpliTag ct
b 289 aa N - 80 pht 75 - 80 MgCl2 -
Stoffel tn
Dng chy
Thermotoga
Ultima nng +
maritima
bin su
Dng chy
Thermococcus >95% hot MgSO4
Vent nng 80 +
litoralis ng sau 1 h
bin su
Vent (Exo - Vent ti t MgSO4 -
) hp
Pyrococcus Dng chy
>95% hot MgSO4
Deep Vent sp. Strain GB nng 72 - 80 +
ng sau 1 h
-D bin su
Dng chy
Pyrococcus >95% hot MgCl2
Pfu nng 75 +
furiosus ng sau 1 h
bin su
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 193 of 203
Phn ng khuch i ti u xy ra trn ADN tht tinh khit nhng nhiu k thut chn on bng
PCR vn t kt qu tt vi ADN thu nhn trc tip t dch chit mu th (vt mu, vt liu lu tr,
ADN c v ngay c vi khun tit trng). Tuy nhin, trong nhiu trng hp khc, cc qu trnh chun
b mu phi c thc hin m bo cho vic khuch i, v cc cht c ch nh heparin, EDTA, hay
cc acid c th hin din trong mu.
Mch ADN khun mu cng c th nh hng n phn ng chui. Nu ADN ny c cha nhiu
GC th s kh khn cho vic tch ri cc chui ADN nhit cao. Trong cc trng hp ny, vic
thm formamide hay dimethyl sulfoxide (DMSO) vo hn hp phn ng c th gii quyt c vn .
ADN khun c th l b gen tch ra t t bo, ADN t cc ngn hng genome hoc cADN (ch cn
nng di ng), cc ADN bt k, ARN ton b hoc ARNm.
9.5.4.3. Cc on mi
- Cc mi chn phi c trng cho trnh t ADN cn khuch i, khng trng vi cc trnh t lp li
trn gen.
- Khong cch gia hai mi khng di qu 3 kb. Phn ng PCR s ti u trn nhng trnh t nh
hn 1 kb. Tuy nhin hin nay c mt s enzym polymerase chu nhit c th tng hp c on ADN
c kch thc khong 30kb.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 194 of 203
Tm = 2(A + T) + 4(G + C)
Nhit = Tm 5oC
Nng MgCl2 nh hng n s thnh cng ca phn ng, nu khng c mt Mg+2 th s khuch
i s khng xy ra. MgCl2 cn thit cho hot ng ca ADN polymerase khi ADN vi cc on mi
v n c mt mi tng quan vi nucleotid triphosphat. Nng MgCl2 ti u cho PCR phi c xc
nh cho tng phn ng bng thc nghim. C th dng phng php nh phn bng cch thit lp mt
lot phn ng vi cc nng MgCl2 khc nhau t 1 mM n 5 mM v xc nh nng MgCl2 ti u
cho mu mong mun.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 195 of 203
cc k thut cng ngh di truyn "c in" phi mt mt tun hay hn na.
- Nhy: Phn ng PCR cc nhy v c th ch cn dng mt phn t ADN lm khun cng thu c
sn phm.
Tuy nhin, cn phi nhn mnh rng cc ng dng rng ri ca k thut PCR i hi phi bit r
trnh t ADN khun mu. y l mt im gii hn ca k thut ny, ngha l n vn phi da vo nn
tng ca cc k thut cng ngh di truyn ch khng hn l thay th cc k thut ny. Do vy ngi ta
khng s dng k thut ny trong cc k thut to dng gen thit k. Trong mt vi trng hp phng
php PCR khng p dng c vi cc on ADN kch thc ln hn 3 kb (thng 1 kb l tt nht).
Ngoi ra kh nng ngoi nhim i vi phng php ny l rt ln.
t c nhy cao trong PCR, ngi ta c th thc hin 2 PCR lin tip. Trn mt in di
ca cc on thu c sau PCR th nht, ngi ta quan st thy ngoi mt vt chnh cn c cc vt nh
khc. iu ny l do cc mi c th bt cp vi vng khc ca ADN ch bi s lai ho khng c hiu.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 196 of 203
ADN ch c khuch i bng PCR, sau cho lai vi on d (l mt phn c hiu ca ADN
ny). Tn hiu c lai ho c hiu vi on d theo cng cch thc ca ADN ch c gn (cht hunh
quang, cht d quang hay cht mu), tn hiu s c to ra nh cht nh du hin din sau cng my
khuch i. PCR "nhnh" c c hiu cao.
K thut ADN "nhnh" c dng pht hin ADN ca virus gy bnh vim gan B. Trong k
thut ny s dng 5 on d. Mt on d thnh phn l mt trnh t cho php s lai ho cng mt lc
trn 2 on ch khc nhau.
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 197 of 203
Trong k thut ny, 2 hay nhiu ADN ch c khuch i ng thi. PCR a thnh phn c th
c s dng thng xuyn trong cc xt nghim chn on, s dng 2 b mi khc nhau: b th nht
xc nh s ng nht ca PCR v b th hai nhm vo cc trnh t ADN d tha. PCR a thnh
phn cng c th c s dng trong phng th nghim vi sinh lm sng vi cng on d cho mt mu
xt nghim cho vi sinh vt khc nhau trong mt ng PCR.
Khi tin hnh PCR a thnh phn cn phi xem xt mt s yu t trong thit k on mi. Cc mi
phi c nhit lai tng t nhau. S khc bit 10oC trong nhit lai gia cc b mi c th lm cho
cc sn phm khuch i rt khc nhau hay to s khuch i khng th pht hin c ca sn phm
ny hay sn phm khc. Cc on mi c s dng cng c thit k sao cho cc sn phm khc
nhau v kch thc mi sn phm c th c xc nh nhng khng qu khc nhau cho s
khuch i ca mt ADN ch c u tin hn so vi s khuch i ca ADN ch khc. PCR a thnh
phn c cc ADN ch khc nhau nhiu v kch thc thng u tin khuch i cc ADN ch ngn
trc cc ADN ch di, kt qu l c s khc nhau v s lng cc sn phm c khuch i. Tuy
nhin, s khc nhau ng k v nhit lai hay chiu di ADN ch l khng th trnh khi. Ngi ta
c th iu chnh quy trnh thc hin lm cn bng phn ng v t c s lng sn phm tng
ng nhau hoc iu chnh nng mi cn bng vi mt phn ng a thnh phn.
ARN ca retrovirus c ly trch v c chuyn i thnh cADN bi enzym sao m ngc enzym
phin m ngc, cADN mi c dng lm khun mu cho PCR. Tuy nhin, cc phn ng nh enzym
phin m ngc kh thc hin v vic s dng cc enzym khng chu c nhit trn 42oC l mt bt
li v xy ra s lai khng nghim ngt. V vy, s phin m ngc km hiu qu l mt tr ngi lm cho
s pht hin cc ARN ch km nhy v km c hiu.
Ngi ta tm ra ADN polymerase chu nhit c hot tnh enzym phin m ngc l ADN
polymerase ti t hp c trch t vi khun chu nhit Thermus thermophilus (Tth pol). ADN
polymerase chu nhit ny hot ng hu hiu khi c s hin din ca ion Mn2+. Hn hp phn ng ca
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 198 of 203
PCR ca ARN bao gm: Tth pol, 2 loi mi oligonucleotide (mt c s dng cho s tng hp
cADN, v c 2 c s dng cho PCR), ion mangan dng MnCl2, v tt c cc thnh phn khc cn
cho s phin m ngc v PCR. My chu k nhit c un nng trc n 70oC, v nhng thnh phn
phin m ngc c trong 15 pht. Sau phn ng nh enzym phin m ngc, hn hp phn ng
c nng nhit ln 95oC lm bin tnh phc hp ARN - ADN. PCR sau c bt u vi 2
chu k 95oC trong 15 pht v 60oC trong 20 giy, tip theo l 38 chu k 90oC trong 15 giy v 60oC
khong 20 giy. S phin m ngc c thc hin 70oC do vy c hiu v nhy ca vic pht
hin ARN ch cao.
Trong nghin cu khoa hc, PCR gip cho vic xc nh trnh t nucleotid ca cc on ADN c
nhn ln; c th s dng PCR tch dng nhng on ADN c hiu, mc d trong nhiu trng hp
tch dng khng cn n PCR; n gip pht hin t bin, nghin cu mARN hoc to cc t bin gen,
cho php phn tch lin kt gen t nhng t bo ring l; gip nghin cu qu trnh tin ho mc phn
t. Thm ch gip phc hi nhng gen trong cc c vt tn ti cch y hng chc triu nm.
Trong y hc, PCR c th chn on chnh xc cc bnh nhim trng t virut n vi khun v cc
bnh do nm, k c HIV - AIDS, chn on sm v theo di iu tr ung th...
Trong t vn di truyn y hc, PCR cho php chn on nhanh, chnh xc cc bnh di truyn k c
chn on trc sinh gii tnh v cc d tt bm sinh c th c t khi thai mi c 8 tun tui v iu
quan trng l khng cn chc i, ch cn ly mt git mu ngoi vi ca m lm mu th nghim.
Trong khoa hc hnh s, PCR l khng th thiu, n gip chn on nhanh, chnh xc th phm ch
t mt vt mu kh, mt si tc hoc mt sinh phm no m th phm li trn hin trng. Ngoi
ra k thut ny cn cho php xc nh chnh xc quan h huyt thng cha - con, ng - chu v.v... cng
ch trong vi gi.
Nhng ng dng thc tin a dng ca phn ng chui tng hp ADN tht v cng tn. Nu nh
nm 1985 mi c 3 cng trnh c cng b v PCR th 5 nm sau k thut ny c s dng trong
hng nghn phng th nghim trn khp th gii. Ngay nc ta, PCR cng v ang c dng trong
nhiu trng i hc, vin nghin cu v ngy cng tr nn ph bin. Phn ng chui tng hp ADN
tht s a li mt cuc cch mng trong lnh vc ng dng thc t ca sinh hc phn t. Ngy nay
c th ni mi lnh vc nghin cu sinh hc u s dng PCR.
Tm tt
Chit tch v tinh ch ADN ng vai tr quan trng trong cc nghin cu sinh hc phn t. Cc
bc chnh chit tch ADN l ph v t bo, loi b protein, v ta acid nucleic. Tinh ch ADN c
th p dng k thut siu ly tm hoc sc k. Nng ADN c th o bng quang k bc sng 260
nm, kch thc tng i ca ADN c th xc nh bng k thut in di trn gel i chiu vi thang
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 199 of 203
Cc thao tc ct, ni v lai ADN l cc thao tc c bn ca k thut di truyn phn t. Trnh t ADN
c th xc nh bng phng php ho hc (Maxam v Gilbert) hoc phng php enzym (Sanger v
cng s). Phng php Sanger c ci tin n gin hn v c th t ng ho.
Phng php PCR s dng enzym ADN polymerase v my lun nhit cho php khuch i in vitro
acid nucleic ln gp t ln sau 30 - 40 chu k ch trong vi gi. K thut ny c nhiu u im nh n
gin, nhanh v nhy nn c ng dng rng ri trong nghin cu khoa hc, chn on, khoa hc hnh
s, chn ging...
CU HI
a) Sc k i lc.
b) Sc k lc gel.
e) Sc k kh
2. Tc in di khng ph thuc:
b) Cu dng ca ADN
c) Nng ADN
d) Nng gel
e) in th s dng
a) I b) II
c) III d) I v III
e) II v III
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 200 of 203
c) di ca cc trnh t d) Lc ion
e) Tt c
c) Nucleotid c nh du.
e) C s dng dideoxynucleotid
a) Formamid b) MgCl2
c) DMSO d) EDTA
e) a v c
c) Di t 6 - 30 nucleotid
d) L oligonucleotid
e) Tt c
a) Bin tnh mi
b) Mi gn vo khun
e) Mi gn vo cc on khc nhau
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 201 of 203
e) c v d
10. PCR t:
e) Tt c ng
P N
Bi 1. Nhp mn sinh hc phn t
1. b 2. e 3. c 4. e 5. a
1. d 2. b 3. d 4. e 5. e
6. e 7. a 8. b 9. b 10. b
Bi 3. Cc loi ARN
1. e 2. a 3. a 4. d 5. b
6. b 7. e 8. c 9. d 10. e
Bi 4. S phin m v m di truyn
1. e 2. e 3. e 4. c 5. e
6. b 7. c 8. c 9. e 10. a
1. e 2. d 3. a 4. a 5. b
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 202 of 203
6. d 7. e 8. e 9. d 10. c
Bi 6. iu ho hot ng gen
1. e 2. e 3. d 4. b 5. a
6. e 7. d 8. e 9. b 10. b
1. b 2. d 3. e 4. e 5. e
6. e 7. e 8. e 9. e 10. e
Bi 8. t bin gen
1. c 2. e 3. e 4. d 5. d
6. e 7. e 8. d 9. c 10. a
1. e 2. c 3. b 4. e 5. a
6. e 7. e 8. b 9. e 10. e
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009
New Page 2 Page 203 of 203
Bin tp v sa bn in:
TRN NGC OANH
Trnh by ba:
BI QUANG TUN
Ch bn:
THI SN
SINH HC PHN T
M s: 7K721M7 DAI
file://C:\Windows\Temp\nlddyqkdsp\sinh_hoc_phan_tu.htm 30/09/2009