Chng 8

Di truyn hc VirusMc tiu ca chngGii thiu s di truyn ca thc khun th, nghin cu s sp xp cc gen, cc c tnh ca virus v, trn c s bn cht ca genome ca virus xc nh kiu sao chp.

S tit: 3 Ni dung I. Di truyn hc th thc khun (Bacteriophage hay phage)1. S hnh thnh vt tan v cc th t bin phage Phage c pht hin d dng v trong chu trnh tan, mt t bo b nhim phage v ra v gii phng cc ht phage vo mi trng (Hnh 8.1). S to thnh cc m c quan st. Mt s ln t bo vi khun (khong 108 t bo) c tri ln trn mi trng c. Sau mt thi gian sinh trng, to mt lp t bo vi khun mu trng c. Nu phage c mt thi im vi khun c tri ln mi trng, n s nhim vo t bo vi khun. Sau t bo nhim phage b lm tan v gii phng nhiu phage mi. Th h sau ny ca phage li nhim vo vi khun gn , v tham gia vo chu trnh tan khc, cc vi khun ny b v gii phng ra nhiu phage, chng c th nhim vo cc vi khun khc vng ln cn. Chu trnh xm nhim ca phage c tip tc v sau nhiu gi, phage ph hu tt c cc t bo vi khun ca mt vng, to m (plage) trong sut khc vi lp t bo vi khun mu trng c.

142

T bo khng b xm nhim

Phn hy t bo ch

Phage hp ph ln t bo chPhage t do

Phage lp ghp bn trong t bo ch

Chu trnh sinh tanNucleic acid ca phage Phage protein

Vt cht di trun t bo ch b ph hy

Nucleic acid ca phage xm nhp vo t bo

Protein ca phage c tng hp, acid nucleic c nhn ln, vt cht di truyn t bo ch b ph hy

Hnh 8.1. Chu trnh sinh tan ca bacteriophage

Hnh 8.2. S xm nhp ca phage vo t bo vt ch theo c 2 dng b m ng thi. r+: m nh, r-: m ln, h+: m m, h-: m trong

143

Phage ch c th c nhn ln ch khi sinh trng trong t bo vi khun, v vy lm cn ngun dinh dng trong mi trng sinh trng, lm hn ch s nhn ln ca phage v kch thc ca m. V mi m l kt qu ca s nhim mt ht phage ban u, c th m c s lng cc m ring bit c trn mi trng (Hnh 8.2). Kiu gene ca cc th t bin phage c th c xc nh nh nghin cu cc m. Trong mt s trng hp, s xut hin ca cc m l y . Chng hn, t bin phage lm gim s lng phage th h sau t nhng t bo b nhim thng to m nh hn. Cc m ln c th c to ra bi cc t bin gy ra s tan sm cc t bo b nhim, nn mi m tip tc nhim nhanh hn. Kiu t bin khc ca phage c th c xc nh bi phage c kh nng hoc khng c kh nng to m trn nhng chng vi khun c bit. 2. Ti t hp di truyn trong mt chu k sinh tan (Lytic cycle) 2.1. Chu trnh tan (Lytic cycle)

Hinh 8.3 S kt hp u va ui tao phage hoan chinh

144

Cc bacteriophage lm cht t bo ch gi l c, chng sinh sn theo chu trnh tan. Chu trnh bt u khi si ui ca phage gn vo im nhn b b ngoi ca E.coli. ng ui co li to l thng xuyn vch t bo v bm DNA ca n vo. Capside ca phage cn li bn ngoi t bo. Sau khi b nhim cc t bo E.coli c qu trnh phin m v dch m cc gen ca virus. Phage T4 c khong 100 gen v phn ln c bit r. Mt trong nhng enzyme c to ra u tin ct DNA ca t bo ch. DNA ca phage c phin m u tin nh DNA polymerase ca t bo ch to ra mARN sm. Cc mARN mun hn c th c tng hp bi ARN polymerase ca phage hoc ARN polymerase ca vi khun b bin i phin m cc gen ca phage. Cc mARN mun c dch m to cc loi protein enzyme iu ha v cu trc. Cc protein iu ha ca phage kim sot s phin m ni tip ca cc gen. Khi DNA ca t bo ch b phn hy, b gen ca phage kim sot ton b hot ng ca t bo to ra cc cu phn ca n. DNA ca phage c sao chp ra hng trm bn sao. Cc protein ca capsid c tng hp thnh 3 phn ring: u, ng ui v cc si ui. Chng t rp vi nhau thnh cc virion con. Phage hon tt chu trnh khi enzyme lysozyme c to ra tiu ha vch t bo vi khun. T bo vi khun b v, 100-200 virion thot ra v chng c th lp li chu trnh mi. Ton b chu trnh t lc phage tip xc n tan din ra trong khong 20-30 pht 370C. Trong thi gian s lng phage T4 tng hn c 100 ln, trong khi s lng t bo E.coli mc nhanh nht cng ch tng gp i. Phn ln cc phage c theo chu trnh va nu trn. Tuy nhin c mt s ngoi l nh phage si M13 ca E.coli hu nh khng bao gi lm cht hoc lm tan t bo. Cc t bo vi khun v cc phage k sinh c s ng tin ha. Cc t bo vi khun c cc c ch bo v nh bin i mng t bo phage khng bm vo c hoc cc enzyme ct hn ch ct DNA ca phage. Phage cng bin i xm nhp c vo t bo vi khun. Cc phage tuy c kch thc nh b phi nhn di knh hin vi in t mi thy c. Nhng cc tnh trng ca phage c quan st da theo cc vt tan hoc bin ch. Cho hai dng phage T4 c kiu gene khc nhau nhim vo mt t bo vi khun E.coli, mt vi phage th h sau s thc hin 145

ti t hp di truyn. Allele r- tan nhanh, kt qu to ra m ln, allele hnhim vo cc t bo ch, kt qu to m trong. Php lai nh sau: r-h+ r+hKt qu thu c bn kiu m. Hai kiu m c, ln v m trong, nh tng ng vi kiu hnh ca phage b m. Hai kiu hnh khc, m trong ln, m m nh l dng ti t hp tng ng kiu gene r-h- v r+h+. Khi nhiu vi khun b nhim s dng ti t hp thun nghch thng c tm thy trong s cc phage th h sau. Trong th nghim, mi kiu gene trong s bn kiu gene trn sinh ra kiu hnh khc nhau v dng m (Hnh 8.2). S lng kiu gene c th xc nh c bng kim tra cc m to thnh. Tn s ti t hp, c biu din di dng phn trm, c xc nh nh sau: Tn s ti t hp = S phagetait hp 100 Tng s phage

3. S sp xp ca cc gene trong nhim sc th phage Tn s ti t hp c th c s dng xc nh khong cch ca bn Eukaryote. Cc th nghim lp bn cho thy t bin T4 c lp bn thnh 3 cm ring bit. C ba cm ny c lin kt vi mt cm khc. George Streisinger v cng s (1964) chng minh bn di truyn ca phage T4 c dng vng trn. Trong mi php lai, lp ba n bn marker di truyn ln lt vi mi nhm v tin hnh qua ton b genome ca T4. Nhiu gene khc c xc nh v lp bn y trn phn t vng trn (Hnh 8.3). Nhng vng vng trn bn trong l 3 cm ca marker T4 c xc nh v lp bn di truyn. Vng ngoi c mt ca nhiu b marker ln to thnh ton b vng trn ca bn di truyn. Bn di truyn phage T4 cho thy gene ca phage T4 to cm m rng theo chc nng ca chng. Chng hn c cm ln cc gene dng cho sao chp DNA v tr phn t bn trn pha phi v c cm gene tng hp cc cu phn to nn u ca phage pha di ca vng trn. Phn t DNA ca phage T4 l phn t si n dng thng, mi u tn cng ca DNA phage T4 c nhn ln hoc lp on u cui (terminal redundant). Do vy, mi phn t DNA c kch thc tng thm 2%. Khi DNA c sao chp trong t bo, s ti t hp gia cc phn u 146

tn cng ca b gen T4 vi nhng trnh t tng ng ca b gen T4 khc, kt qu to ra sn phm DNA c kch thc ln hn kh nng cha ca phn u. Nhng phn t cha lp on c to thnh v s ti t hp trong b gen ca phage T4 xy ra thng xuyn, trung bnh c khong 20% s kin ti t hp xy ra trn mt nhim sc th. Khi phn t DNA c gi vo phn u, n c ct bng enzyme ch cn cha khong 102% ca chiu di b gen phage T4, v c cha on lp li ca phn u.

Mng Si ui Trao i nucleotide Tng hp DNA, ti bn v thay i Nhm gen lin kt xc nh ngun gc a gc ca ui

u, c, np gp c a gc ca ui u

Hnh 8.4. Bn di truyn ca T4 vi cc marker

4. Lp bn cu trc tinh vi vng rII ca phage T4 Cc nghin cu chi tit v cc t bin rII ca phage T4 lm sng t hn v cu trc gene. Phage T4 dng hoang di r+ c kh nng nhim ng thi hai ni E.coli B v K. Cc t bin rII ch nhim ni B nhng khng nhim ni K. Seymour Benzer (1955) nhn c 2400 t bin rII c ngun gc c lp vi nhau. ng cho lai cc t bin vi nhau v cn c vo s xut hin cc dng ti t hp hoang di r+ m lp bn cc im t bin. Mi t bin c th ti t hp vi cc t bin khc. t bin mt on ngn cn s ti t hp vi hai hoc nhiu t bin im cc v tr khc nhau ca gene. Mi mt on lm mt mt phn b gene ca phage 147

bao gm c vng rII. S dng t bin mt on l phng php n gin lp bn ca hng ngn t bin. Bn mt on (Deletion mapping) da trn s c hoc khng c dng ti t hp. Trong bt k php lai no gia mt t bin im cha bit v mt t bin mt on, s xut hin ca dng hoang di cho thy t bin im nm ngoi vng mt on. Ngc li, nu t bin im xut hin trong vng mt on, khng xut hin dng ti t hp kiu hoang di th h sau.rII A cistron rII B cistron

Khong cch t A1 n B c xc nh bng mt on cc khong 1272 qua 638

Khong cch t 1 n 47 c xc nh bng mt on khong 1364 qua 1519

Hnh 8.5 t bin mt on c s dng chia locus rII ca bacteriophage T4 thnh 7 vng v 47 tiu vng nh

Nhiu php lai c thc hin lp bn t bin chi tit gene rII. Khong cch t A1 n A6 v B c trnh by hnh 8.4. Mt t bin c bit c kim tra nh v vng A4. t bin ny khng ti t hp to dng kiu di trong php lai vi cc t bin mt on ln nh r1272, r1241, rJ3 v rPT1 nhng n c th ti t hp to dng kiu di trong php lai vi rPB242, rA105 v r638. Cc t bin c to ra bi cng mt khun, kt qu lai vi cc t bin mt on ln s c xp vo vng A4. Bn di truyn trong vng A4 c th c to ra bi mt b cc t bin 148

mt on c trnh by phn di ca hnh 8.5. Xc nh 7 tiu vng trong A4 (t a qua g).

rII A cistron

rII B cistron

Vng xc nh t bin mt on t A1 n A6 v B trong gen rII

t bin trong vng b s to ra dng ti t hp hoang di vi tt c cc mt on m trong vng b ca dng hoang di c mt

Mt on vng xc nh a n g ca vng A4

Hnh 8.6 Xc nh vng rII lin quan vi cc marker di truyn dng thng ca bn di truyn phage T4

V d, mt t bin trong vng A4 kt qu ti t hp to dng kiu di vi t bin mt on r1368, nhng li khng th thc hin c vi t bin r221 s c sp vo tiu vng c. mc chi tit hn, cc t bin trong mt tiu vng c sp xp nh lai gia chng vi nhau. phage T4, cc im t bin rt gn nhau, c tch nhau nh ti t hp. 1% ti t hp tng ng vi khong cch khong 100 bp. V vy, bt k hai t bin khng th ti t hp c vi nhau c th c xp vo cng v tr trong gene. Bn di truyn cho s ln cc t bin rII c ngun gc c lp c m t hnh 8.6. Nghin cu t bin vng rII v lp bn di truyn c vai tr quan trng, qua c th rt ra cc kt lun sau: + S trao i di truyn c th xy ra trong gene v c th gia cc nucleotide gn nhau. 149

+ Cc t bin khng c to ra cng tn s vi tt c cc im trong gene, chng phn b khng u nhau. Chng hn, 2400 t bin rII c xc nh ch 304 im. Mt trong nhng im ny c th c n 474 t bin (Hnh 8.6). Nhng im c tn s t bin cao nh th c gi l cc im nng (hotspot mutation). nhng im khc, t bin c phc hi mt ln hoc vi ln.Mi hp th hin s xut hin ngu nhin ca cc t bin ti v tr

"im nng" t bin

Nhiu t bin xut hin mt im to thnh mt "im nng"

Hnh 8.6 Bn di truyn locus rII ca phage T4

Kt qu phn tch vng rII rt quan trng, gip cho chng ta phn bit c 3 khi nim v gene. Ph bin nht, gene lin quan vi mt n v chc nng. iu ny tng ng vi mt on DNA m ha cho mt phn t protein. Benzer a ra thut ng cistron ch chc nng ny, thut ng cistron thnh thong vn c s dng. n v chc nng c xc nh qua th nghim b sung (complementation test), xc nh c 2 t bin c allele vi nhau khng. Trc th nghim ca ng rII c coi l mt locus. Th nghim cho thy cc t bin xp thnh hai nhm rIIA v rIIB. Lai cc t bin rIIA 150

rIIB s c r+, nhng lai rIIA rIIA v rIIB rIIB th thu c kiu hnh t bin r. Ngoi ngha l n v chc nng, gene cn l n v ti t hp (recon) v n v t bin (muton). C hai n v ny, u tng ng vi nhng nucleotide ring l trong gene. 5. Tnh tim tan (Lysogeny) v phage Chu trnh tim tan bt u khi phn t DNA ca phage gn vo nhim sc th ca vi khun v tin hnh sao chp nh mt phn nhim sc th vi khun. Cc ht phage khng c to thnh. Phn t DNA ca phage c gn vo b gen ca vi khun c gi l prophage, t bo vi khun sng st c gi l t bo tim tan (lysogen). Mt chng tim tan cho phage c k hiu theo tn ca phage. V d chng E. coli K12() l chng K12 tr thnh t bo tim tan ca phage .Phage

Phage DNA

Phage tn cng t bo ch v bm DNA vo

NST vi khun

Mt s prophage tn ti trn NST vi khun, khi u cho chu trnh sinh tan Nhiu t bo phn chia to ra khun lc vi khun c cha prophage

T bo b phn gii, gii phng phage

Chu trnh sinh tan

Ti to vng DNA phage

DNA v protein ca phage c tng hp v lp ghp to thnh phage mi

T bo vi khun phn chia bnh thng, sao Prophage chp prophage v truyn cho th h sau DNA ca phage tch hp vo NST vi khun to thnh dng prophage

Chu trnh tim tan

Hnh 8.7 Chu trnh tan v tim tan phage

Phn t DNA ca phage c u cc u cui cha 12 nucleotide khng kt cp, m dng si n to u dnh (cohesive end) b sung. Khi vo t bo, u cui b sung gn li to phn t vng trn. S to vng trn xy ra sm c chu trnh tan v chu trnh tim tan (Hnh 8.7). C khong 75% t bo vi khun b nhim phage, phn t DNA vng trn sao chp v 151

chu trnh tan xy ra tip theo. Cn khong 25% t bo b nhim, phn t DNA vng trn ca phage v phn t DNA vng trn ca E. coli tng tc v xy ra ti t hp im chuyn bit (site-specific recombination) v DNA ca phage gn vo nhim sc th ca vi khun. V tr ca ti t hp im chuyn bit DNA ca vi khun v phage c gi l im gn vo ca vi khun v phage (bacterial and phage attachment sites). Mi im gn c cha 3 on: on trung tm c cng trnh t nucleotide c 2 v tr gn v l vng m s ti t hp thc s xy ra. im gn vo ca phage c k hiu bi POP (P: phage) v im gn vo vi khun c biu din bng BOB (B: bacteria). So snh bn di truyn ca phage v prophage POP nm gn vng trung tm ca phn t DNA dng thng. Mt protein ca phage, integrase, xc tc cho ti t hp im chuyn bit. Enzyme integrase nhn ra im gn vo ca phage v vi khun, gy ra s trao i vt l, kt qu l phn t DNA ca phage gn vo phn t DNA ca vi khun. Kt qu ca s ti t hp lm bn di truyn ca prophage khc vi bn di truyn ca phage. Bn di truyn prophage l s chuyn i vng trn bn di truyn phage t do. Prophage c chn vo nhim sc th ca E. coli gia gene gal v gene bio. S chn vo ca phage lm tng khong cch gia gene gal v gene bio. Khong cch gia gene gal v gene bio t bo tim tan vi phage l khong hai pht so vi mt pht t bo khng tim tan.

Hnh 8.8 M hnh gn ca phage vo NST ca E.coli

152

Khi t bo tim tan, cc gene ca phage tr thnh mt phn nhim sc th ca vi khun v vy c th lm kiu hnh ca vi khun b thay i. Nhng hu ht cc gene ca phage prophage u c gi trng thi bt hot nh protein repressor - sn phm ca gene phage. Protein repressor c bt u tng hp nh s nhim vo ca phage v n c tip tc tng hp nh prophage. Gene m ha cho repressor thng ch l gene ca prophage c biu hin chu trnh tim tan. Nu t bo tim tan b nhim bi phage ging vi prophage, s c mt ca repressor trong prophage ngn cn s biu hin cc gene ca phage nhim vo. Tnh khng vi nhng phage ging vi prophage c gi l tnh min nhim (immunity). y l tiu chun xc nh t bo vi khun cha phage c bit. Chng hn phage khng to m trn vi khun cha prophage . Trong t bo tim tan, s sao chp khng gii phng cc phage mi. Tuy nhin, cc prophage i khi tr nn c hot tnh, tri qua chu trnh tan, to ra s lng ln phage th h sau. Hin tng ny c gi l s cm ng prophage (prophage induction), n c bt u bng s h hi DNA ca vi khun. i khi prophage c th tch ra khi DNA ca vi khun mt cch ngu nhin nhng thng n c gy ra do cc tc nhn ca mi trng nh ha cht hoc chiu x. Kh nng b cm ng l mt thun li cho phage bi v DNA ca phage c th thot khi t bo b h hi. C ch sinh ha ca s cm ng l phc tp nhng s thot ra ca phage xy ra d dng. S ct ra ca phage l s ti t hp im chuyn bit khc, ngc vi qu trnh gn vo. S ct ny yu cu enzyme ca phage, integrase thm protein ca phage l excisionase. Nghin cu di truyn ca s gn vt l cho thy escisionase gn vi integrase v sau nhn ra im gn vo ca prophage BOP v POB, gn vi cc im ny. Integrase ct trnh t O v to ra li BOB v POP. Qu trnh tch din ra ngc li vi s gn vo.

II. c tnh ca cc virus1. Tnh a dng v cu trc v thnh phn di truyn Virus c b gene rt a dng. B my di truyn ca virus c th l DNA mch kp (double strand - dsDNA), DNA mch n (single strand ssDNA), RNA mch kp (dsRNA) hay RNA mch n (ssRNA). B gene RNA ca virus l mt phn t hoc mt on, si n phn cc mch (+) hoc mch (-), c th dng vng trn hay dng thng. Virus nh nht c nht c khong 4 gene, virus ln c khong vi trm gene. B gene ca 153

virus cu trc a dng nhng u m bo yu cu chung l phi sao chp c trong t bo ch to ra c genome cho lp rp virion th h sau v cc mRNA phi tng hp protein ca virus. 2. Tnh c th v vt ch (Host specificity) Mi kiu virus c th nhim v k sinh ch mt bin gii hn ca t bo c gi l bin ch (host range). Cc virus nhn bit t bo ch theo nguyn tc ng kha v cha kha cc protein bn ngoi ca virion lp va cc im nhn trn b mt t bo. Mt s virus c bin ch rng xm nhp vo vi loi. Chng hn, cc virus bnh di c th nhim nhiu loi c v gm gm nhm, ch v ngi. Bin c th rt hp nh nhiu phage ch nhim vi khun E. coli.

III. Ti bn ca cc virusBn cht genome ca virus xc nh kiu sao chp. Virus c phn loi da trn cc c im: - Phn loi theo bnh: chia ra virus gy bnh ngi, ng vt v cy trng Vn ch yu i vi h thng phn loi ny l nhiu loi virus khc nhau li gy ra cng mt triu chng. Chng hn, s nhim trng h hp vi st c th c gy ra do nhiu virus khc nhau. - Phn loi theo hnh thi: phn loi virus c bn da trn cu trc ca ht virus. Kiu phn loi ny c hn ch trong phn bit gia cc virus c hnh thi tng t nhng gy ra triu chng bnh khc nhau. - Phn loi theo chc nng: trong nhng nm gn y, nhiu nghin cu c tin hnh da trn phng thc sao chp ca virus. Cn xc nh thnh phn v cu trc genome ca virus v t xc nh cch sao chp. Kiu t bo b nhim bi virus c nh hng quan trng i vi qu trnh sao chp. i vi virus ca prokaryote, s sao chp phn nh mi quan h m rng n gin ca cc t bo ch. i vi virus ca t bo eukaryote, vn phc tp hn. Kh nng m ho ca genome buc virus chn mt phng thc sao chp. Phng thc sao chp ca virus ph thuc vo bn cht vt liu di truyn ca chng. V phng din ny, virus c chia thnh 7 nhm: Nhm I: Virus cha DNA si i. Nhm ny c chia nh thnh hai loi: 154

+ Sao chp l ch ca nhn. S sao chp ca cc virus ny ph thuc tng i vo cc yu t ca t bo. + Sao chp xy ra trong t bo cht. Nhng virus ny c lin quan vi cc yu t cn thit cho phin m v sao chp genome ca chng v v vy ph thuc nhiu vo b my t bo. Nhm II: virus cha DNA si n. S sao chp xy ra trong nhn lin quan s to thnh qua trung gian si kp c xem nh l khun cho tng hp li DNA si n th h sau. Nhm III: virus cha RNA si kp. Nhng virus ny c b gene c chia on. Nhng on ny c phin m ring to ra cc monocistronic mRNA. Nhm IV: Virus cha RNA si n mch (+), c th chia nh thnh 2 nhm: + Virus vi polycistronic mRNA. RNA genome to ra mRNA, phn t ny dch m to sn phm l mt polyprotein, thng c phn ct to cc protein trng thnh. + Virus phin m phc tp. Cch dch m (nh Togavirus) hoc cc RNA ca subgenome (Tobamovirus) cn thit to RNA ca b gene. Nhm V: Virus cha RNA si n mch (-), genome ca virus ny c chia thnh 2 nhm: - Genome khng chia on (Mononegvirales). Bc u tin trong sao chp l phin m RNA si (-) ca genome nh RNA polymerase ph thuc RNA ca ht virus to ra monocistronic mRNA, c xem l khun cho sao chp genome. - Genome c chia on (Orthomyxoviridae). Sao chp xy ra trong nhn vi monocistronic mRNA cho mi gene ca virus c to ra nh enzyme transcriptase t genome y ca virus. Nhm VI: Virus cha mRNA si n mch (+) qua trung gian DNA. B gene ca retrovirus l mRNA mch (+) nhng dng lng bi. Chng khng trc tip to ra mRNA m phin m ngc to DNA. Nhm VII: DNA si i qua trung gian RNA. Virus nhm ny da vo enzyme phin m ngc, nhng khc vi retrovirus, qu trnh ny xy ra bn trong ht virus trong sut qu trnh trng thnh.

155

Si RNA (-) virus b phn ct5' C Tng hp mRNA 3' Ti bn 5' 3' 3' 5' 5' 3' 5' 3' C

Phn t duy nhtC C Tng hp mRNA 5' si RNA (-) genome Ti bn 3' si (+) c chiudi y

si RNA (-) 5' genome

Si RNA (+) virus Flavi v picornaviruses5' C Ti bn 3' 5' C 5' C 5' 3' 5' C

Alphaviruses

Ti bn

si RNA (+) genome (mRNA)

5' si (-) c chiudi y

5' C

Tng hp mRNA si RNA (+) genome (mRNA)

Ambisence RNA virus5'C RNA genome

RNA virus si isi (+) 5' si (-) 3' Tng hp mRNA

C 5'

Tng hp mRNA

C 5' mRNA 3' 5' C 5' Anti Tng hp -genome RNA mRNA mRNA

3'

C 5'

3'

C 5'

Si (+) c chiu di y (mRNA) Dch m Ti bn si (+) 5' si (-) 3'

Protein

RNA genome

C 5'

Hnh 8.9 Sao chp RNA ca virus

156

1. Cc virus ca vi khun C 3 pha bt u cho xm nhim ca virus. - Bt u nhim - Sao chp v biu hin genome ca virus - Gii phng cc virion trng thnh t t bo b nhim Bacteriophage c thm vo nui cy vi khun ang sinh trng mnh v sau mt vi pht nui cy b gim, ngn cn tng tc gia cc ht phage v t bo. Ngay sau khi lm gim nui cy, c giai on khong 1015 pht khng pht hin thy cc ht phage, y l giai on che khut (eclipse period). iu ny xy ra mt thi gian sau khi nhim vo t bo, lin quan vi genome ca chng nh l iu kin trc tin cho sao chp. giai on ny khng c s nhim na v vy khng th pht hin nh vt m. Giai on mun l thi gian trc khi ht virus mi u tin xut hin v khong 20-25 pht cho hu ht cc bacteriophage. Khong 40 pht sau khi t bo b nhim, ng cong v s lng ht virus tng s v virus ngoi bo hp nhau thnh mt v lc ny, t bo b nhim lm tan v gii phng cc ht phage ngoi bo. Cc bacteriophage lm cht t bo ch gi l c (virulent) v chng sinh sn theo chu trnh tan Cc virus n ho (temperate virus) c th sinh sn m khng l cht t bo ch. Chng c hai kh nng sinh sn: chu trnh tan v chu trnh tim tan khng lm cht t bo ch. Chu trnh sng bt u khi phage gn vo b mt t bo E. coli v bm DNA vo trong gy nhim. DNA ca phage sau khi vo t bo to DNA vng trn v s tham gia vo mt trong hai chu trnh. DNA ca phage c th hoc tham gia vo chu trnh tim tan ca phageT4 hoc gn vo nhim sc th ca vi khun nh ti t hp im chuyn bit bc vo chu trnh tim tan. 2. Cc virus thc vt Hu ht virus thc vt c genome RNA, tuy nhin 2 nhm virus thc vt c nghin cu nhiu nht c cha genome DNA: Cauliflower mosaic virus (CaMV) v gemini virus. - Cc virus RNA Phn ln virus thc vt c b gene RNA si n mch (+) v nhiu dng c capsid hnh que, cc protein capsomer hnh xon. 157

+ Tobacco mosaic virus (TMV) Genome TMV l RNA si n. Genome ca chng m ho t nht 4 chui polypeptid. Protein 130 v 180 kDal c dch m trc tip t cng mt codon bt u trn RNA b gene. Hai protein khc, 30 kDal v protein v c dch m t on RNA. Protein 130 v 180 kDal lin quan vi sao chp virus, trong khi protein 30 kDal cn cho s di chuyn ca virus t t bo ny n t bo khc. V vy 3 loi protein ny cn cho s nhn ln ca virus trong ton b cy. - Cc virus DNA Virus thc vt c b gene DNA rt him, ch gm 2 nhm: + Cauliflower mosaic virus: CaMV c nghin cu nhiu nht trong nhm Caulimovirus. y l nhm virus dng cu, cha genome DNA vng trn, mch kp, kch thc khong 8 kb. Caulimovirus gy ra mt s bnh lm thit hi kinh t cy trng. Chng c ph vt ch hn ch, ch nhim cy 2 l mm. DNA ca CaMV c cu trc khng bnh thng, c 3 im gin on trn si kp, hai im trn mt si v mt im trn si cn li vi nhng vng trnh t overlap. Ngoi ra DNA CaMV c ribonucleotide gn vi u cui 5 ca im gin on. T cc nghin cu CaMV, Hull v Covey (1983), Pfeiffer v Hohn (1983) cho rng sao chp CaMV lin quan vi phin m ngc qua trung gian RNA ca b gene. Chu trnh sao chp ging vi retrovirus v hepatitis B virus. + Gemini virus: Gemini virus l nhm virus c ph xm nhim rng, c cy mt l mm v 2 l mm.Virus sc vn l ng (maiz streak virus MSV) l mt gemini virus ly nhim qua l, c truyn do cn trng. B gene ca geminivirus cha phn t DNA vng trn si n. Sao chp DNA virus c ngh l xy ra nh trung gian DNA v genome ca virus sao chp cho nhiu bn sao trong nhn ca nhng t bo tng sinh nhanh. Virus gy ra s c ch sinh trng v l c sc vng ca nhng cy ng b nhim. 3. Cc virus ng vt Chu trnh sao chp ca virus ng vt c nhiu im tng t vi cc virus khc vi nhiu bin dng ng k. Virus ng vt thng c promoter mnh, c th p dng cho biu hin gene. Trong nhiu trng hp, chng c kh nng sao chp genome ca chng vi s lng ln bn sao trong t bo. 158

Mt s virus ng vt nh retrovirus gn DNA ca chng vo nhim sc th t bo ch nh l mt phn chu trnh sao chp ca chng. - Cc virus RNA nh retrovirus, paramyxo virus Retrovirus c ph vt ch rng gm chim, ng vt c v v nhng ng vt khc. S nhim ca retrovirus khng dn n lm cht t bo. Biu hin gene ca virus mnh nh promoter mnh. Retrovirus cha genome RNA. Ht virus cha 2 bn sao RNA. Mi genome RNA c nhiu tnh cht tng t vi mRNA eukaryote: c trnh t poly(A) khong 200 n v u mt 3 v cu trc m u 5. Virus xm nhim vo t bo km theo enzyme reverse transcriptase v intergrase. Enzyme reverse transcritase tham gia phn ng tng hp cDNA, dn n s hnh thnh bn sao DNA mch kp ca RNA virus, c gi l DNA provirus. DNA provirus c ng vng trn nh protein intergrase v c xen vo genome t bo ch. Thng ch c mt bn sao DNA provirus c gn vo trong mt t bo ch. im gn vo genome l ngu nhin. Retrovirus l mt tc nhn gy ung th. Hu ht chng c cu trc genome cha trnh t oncogene. Oncogene virus thng c to thnh t gene ca t bo v thng l kt qu ca s dung hp gene t bo vi gene ca virus. Kt qu l nhng virus gy ung th nh th b mt chc nng gene ca virus. Chng c th sao chp trong ly nhim hn hp vi mt helper virus cung cp chc nng b mt. - Cc virus DNA: SV40, Bovine papilloma virus (BPV) Ht virus SV40 cha DNA mch kp, vng trn, khong 5,2 kb c gn vi 4 phn t histon: H4, H2a, H2b v H3. SV40 c th tham gia vo hai kiu chu trnh sng ph thuc vo t bo ch. Trong t bo cho php virus xm nhim (permissive cell) thng l cc dng t bo n nh c ngun gc t kh chu Phi, s sao chp virus xy ra nh cc trng hp nhim bnh thng. Trong t bo khng cho php (non-permissive cell), thng l nhng dng t bo chut, khng c s nhim lm tan t bo v virus khng th sao chp ton b DNA ca chng. nhng t bo kh b nhim v lm tan do SV40, c th phn ra 3 giai on. Trong sut 8 gi u tin, ht virus khng v v DNA ca chng di chuyn n nhn t bo ch. 4 gi tip theo l pha sm (early phase), c s tng hp mRNA sm v protein sm v c s kch thch tng hp DNA ca t bo ch. Trong pha mun xy ra 36 gi tip theo, trong giai on ny c s tng hp DNA ca virus, mRNA mun v protein mun, lp rp virus v lm tan t bo. 159

+ Bovine papilloma virus (BPV) BPV gy nn bnh bu (wart) tru b. BPV c genome DNA si kp, vng trn khong 7,9 kb. 4. Cc virus gy ung th, HIV/AIDS Khong 15% ung th ngi c c ch hnh thnh lin quan vi virus. Chng c cc nhm sau : + DNA virus: h Apovavirus (Papilloma virus), h HepDNAavirus (Hepative-B virus), h Herpesvirus (Eptein-Barr virus) + RNA virus: h Retrovirus (HIV-1, virus AIDS) Cc virus gy ung th c th tc ng do xen on DNA ca chng vo b gene ch. Ngoi ra c th thc hin : + Hot ho b my sao chp DNA ca t bo ch. + Km hm tc ng ca cc tumour suppressor gene ch yu hot ho sao chp DNA Nh vy bng nhiu cch khc nhau cc virus khi xm nhp t bo c th lm t bo mt s kim sot bnh thng. AIDS l hi chng do virus lm suy gim min dch ngi (HIV). Ht virus l mt khi cu, b ngoi g gh, gm v bn ngoi, trong l cht nn protein bao quanh li c mt ct dng nn. Trong li c genome gm 2 si RNA ging nhau gn vi enzyme DNA polymerase l reverse transcriptase. Trong qu trnh nhim, virus HIV bm v nhim li ca n vo t bo h thng min dch ca ngi. Tip theo chng s dng enzyme reverse transcriptase sao RNA genome ca chng thnh phn t DNA si kp trong t bo cht ca t bo ch. Phn t xon kp ny chuyn n nhn, gn vo nhim sc th t bo ch nh mt enzyme khc. Khi gn vo nhim sc th ca t bo ch, genome ca virus. Mt khi gn vo nhim sc th ca t bo ch, genome ca virus c th thc hin mt trong 2 qu trnh. N c th trng dng b my tng hp protein ca t bo ch c th to ra hng trm ht virus mi sinh sn bng ny chi, tch khi mng t bo v i khi lm cht t bo ch. N cng c th tim tng trong nhim sc th ca t bo ch, ri sao chp v truyn genome virus sang 2 t bo mi khi t bo phn chia.

160

Glycoprotein

Mng bao Capsid

HIV xm nhp vo t boT BO CH RNA virus Enzyme sao m ngc

Enzyme sao m ngc a. Cu trc ca HIV

Lai DNA-RNA RNA (2 si

phn bit)

DNA Protein virus

DNA NST

Provirus

RNA NHN

b. S sinh sn ca HIV

HIV mi

Hnh 8.10 Chu trnh sinh sn ca virus HIV

Ngoi ra sinh hc hin i cn pht hin ra hai dng sng c bit l cc viroid v prion - Viroid: l nhng phn t RNA rt nh (200-400 nucleotide), dng que c mc cu trc bc hai cao (Hnh 8.11). Chng khng c capsid v v bao (envelope) v ch cha mt phn t acid nucleic n. Viroid gn lin vi bnh thc vt. Viroid u tin c pht hin v nghin cu y nht l viroid ng thoi khoai ty (potato spindle tuber viroid-PSTVd). Viroid khng m ha cho bt k mt protein no v n c sao chp nh RNA polymerase ca t bo ch hoc c th nh mt sn phm ca mt gene RNA polymerase ph thuc RNA trong mt vi t bo eukaryote. Chi tit ca sao chp n nay ngi ta cha r, dng nh n xy ra nh c ch vng trn xoay c s ct (autocatalytic cleavage) v gn (self-ligation) to ra viroid trng thnh.

161

Vng kt thc bn tri

Vng gy bnh

Vng trung tm c tnh bo th

Vng bin d

Vng kt thc bn phi

Hnh 8.11 Cc vng chc nng ca phn t RNA viroid

Gia cc viroid khc nhau c cc trnh t khc nhau, n c s dng trong phn loi chia viroid thnh ging (genera) v loi (species). Tuy nhin, tt c viroid u c c im chung l vng trung tm c tnh cht bo th lin quan vi sao chp ca chng (Hnh 8.12). Mt nhm cc viroid c kh nng to thnh cu trc u ba (hammerhead) lm cho chng c tnh cht enzyme ca mt ribozyme. Hot tnh ny c s dng ct cu trc nhiu n phn to ra trong qu trnh sao chp. Cc viroid khc s dng cc enzyme cha c bit trong t bo ch thc hin iu ny. Mt vi viroid gy bnh trm trng v gy cht thc vt ch. Cc viroid khc gm cc loi t khng c biu hin bnh bn ngoi n c triu chng bnh nh.

Hnh 8.12 Cu trc RNA viroid

- Prion: Mt nhm bnh ly nhim h thn kinh kinh nin, pht trin nhanh chng v gy cht c bit nh l bnh xp no ly nhim (Transmissible spongiform encephalopathics - TSE). Tc nhn ly nhim lin quan n TSE khng phi l acid nucleic (Tikvah Alper, 1967). Cc bnh TSE ngi nh l bnh Kuru v Creutzfeldt-Jacob c th gy nn do cc phn t protein gy nhim v Standley Prusiner (1982) gi chng l prion (proteinacious infectious particle). Bn cht ca prion l cha c chng minh mt cch chc chn. Chng l mt hin tng mi vt ra ngoi hiu bit thng thng. Tt c cc bnh prion u c bnh l ging nhau c bn, mc d gia chng c s khc bit c ngha nhng iu 162

kin khc nhau. Cc bnh khc nhau c c trng bi s lng (deposition) ca cc protein bt thng trong cc m khc nhau nh thn, lch, gan hoc no ... S lng cc amyloid ny bao gm s tch lu cc protein khc nhau dng tm hoc dng cun ri to ra protein amyloid. Chng l kt qu t nhng sai hng ca b gene trong trao i cht do nhiu loi nhn t cha c bit.. Mc d c ch phn t lin quan vi s cht ca t bo l cha r rng, nhng tc ng gy ra xp no do to l m no c quan st thy di knh hin vi. Nhng l ny gy ra do s mt t bo thn kinh v gliosid. Php chn on xc nh TSE khng ch tin hnh trn lm sng m yu cu chng minh s lng protein prion (prp) bng nhum ho hc min nhim m no ngi sau khi cht.

Cu hi v Bi tp1. Hy trnh by qu trnh ti t hp genome ca bacteriophage. 2. Genome ca vi khun v virus tng tc vt l theo cch no? 3. ngha ca vic phn tch cu trc locus rII ca phage T4. 4. Hy nu c im v chu trnh sng ca phage. 5. Virus thc vt c nhng nhm no? 6. Hy trnh by phng thc sao chp ca virus cha RNA si n. 7. c im chu trnh sinh sn ca virus HIV. 8. Hy nu tn cc dng sng ch c acid nucleic hoc ch c protein. 9. C bao nhiu m tan ca phage c to thnh trn mi trng nui cy t mt phage ring l ban u? 10. Bacteriophage c trt t cc gene l ABC att DEF. Hi trt t ca cc gene ny prophage nh th no?

Ti liu Tham khoPhm Thnh H. 2000. Di truyn hc. NXB Gio Dc. L nh Lng, Phan C Nhn (1998). C s di truyn hc. NXB Gio dc. Hong Trng Phn. 1995. Di truyn hc phn t. Trung tm o to T xa, i hc Hu

163

Anthony J. F. Griffiths, Susan R. Wessler, Richard C. Lewontin, William M. Gelbart, David T. Suzuki, Jeffrey H. Miller. 2004. An introduction to genetics analysis. W.H. Freeman Publishers. Cann AJ. 2001. Priciple of molecular virolory. Academic Press. London, UK. Flint SJ, Enquist LW, Krug RM, Racaniello VR, Skalka AM. 2000. Principles of Virology. Molecular Biology, Pathogenesis, and Control. ASM Press, Washington DC. Printed in the United States of America. Harlt D.L., Jones E.W. 1998. Genetics - Principle and analysis. Jone and Bartlett Publshers, Toronto, Canada. Hartwell et al. 2003. Genetics: From genes to genomes, Second editor. The McGraw-Hill Companies. Old RW & Primrose SB. 1989. Principles of gene manipulation. Blackwell Scientific Publication. Stansfield W.D. 1991. Schaums outline of theory and problems of genetics. McGraw-Hill, Companies, Inc., United States of America. Watson D.J, Baker T.A., Bell S.P., Gann A., Levine M., Losick R. 2004. Molecular biology of the gene. Benjamine Cummings, San Francisco, United States of America.

164