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4-63).
1975 r.

Hpa I

5' ~ ~3'

3' ~ ~5'

 

PacLllenlleH111e

 

Eco RI

5'

3'

3'

~5'

 

PacLllenlleH111e

 

Hind Ill

5'

3'

 

 

 

3'

~5'

 

PacwenlleH111e

8

0

0

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MHor11e Myrau1111, BhI3hma10m11e B03Hl1KHOBeHtte reHHblX 6oJie3Hetl:, He nona,II,aIOT B Y'JaCTKl1, orneTcrneHHhie 3a Y3HaBaH11e ¢epMeHTOB pecTp11KUMH. B 3TOM cJiy'lae, ecJI11 nocJienonaTeJibHOCTh OCHOBaHHH B 06JiaCTH MYTaU1111 113BeCTHa, TO ee o6Hap)')KHBaIOTc noMOillhlO aJIJieJih-Cneu11¢tt'-IeC- Kl1X OJIHroHyKJJeOTH,!I,OB. L(nH 3TOro CHHTe3HPYIOT KOpOTKHe OJIHfOHyKJieOTH,!I,HbJe 30H,!I,bl' conep)l(a- ll{He 06bI'lHOOKOJ10 19 HYKJieOTH,!I,OB, KOMnJieMeHTapHbie yqacTKY HOPMaJihHoro tt MyraHTHoro aJIJieJIH B )],HK. 06JiaCTb reHOMa, CO,II,ep)Ka11.1,y10 HCCJie,II,yeMhlH reH, aMTIJIHWHUMPYIOT c nOMOUiblO OUP, 11 o6pa3UhI IlOJI)"feHHOM )],HK nepeHOCHT Ha HHTpoueJIJ1IOJI03Hbie cPHJibTpbl (JlOTHJIH CJIOT6JIOTnrnr). Ilpo6h1 Bh1Jlep:11rnna10T c 32 P-30H,II,aM11 MH BhrnBJlemrn HOpMaJibHOH HJIH MyraHTHOH nocJie)lOBaTeJlbHOCTH. y fOM03HroT no HCCJ1e,II,yeMOH MyraUHH )],HK 6y,II,eT m6pH,UH30BaTbC51 TOJibKO c 30H,IIOM, KOMrIJieMeHTapHblM MYTaHTHOH nocJienoBaTeJihHOCTl1. )],HK HOpMaJihHOfO fOM03HfOTHOro HH,!I,11BH,llyyl\1a CBH)l(eTCH c 30H,!I,OM, COOTBeTCTBYIOll{HM Hel13MeHeHHotl: Hy1<.i1eoTttnHoti noCJie,II,osaTeJihHOCTH, Torna KaK c )],HK reTep03HfOT 6y,II,yr m6pH,II,H30BaThCH o6a 30H,lla. Ha pttcyHKe 4-69 npe,IICTaBJTeHbl pe3yJibTaThl reHHOro 30H,!I,11pOBaHH51

7 nau11eHTOB Ha HOCHTeJibCTBO HaH60Jiee 'laCTO BCTpe'-Iaiowetlc51,II,eJieumi: 3 HyKJieorn,II,os (LiF508)

B re He, OTReTCTBeHHOM 3a pa3Bl1Ttte MYJ<OBHCU11 - no3a.

0JIHroHyKJieOTH,II,bI, aJIJieJih-cneu11¢11'-IHh1e no onpeneJieHHhIM MYTaUHHM, MO)l(HO HCnOJih30BaTh B Ka'leCTBenpai1Mepon B IlUP npH KJil1Hl1'-IeCKOM TeCTHpOBaHMH HaceJieHH51 Ha HaJiwme naTOJIOfH- '-IeCKOfO retta. EcJIH )],HK, noJTyqettHa51 OT nau11eHTa, aMJJJIH<l>ttu11pyeT c MyTaHTHbJM OJTHroHyKJieOTH,!I,OM, TO CJie,II,OBaTeJihHO nau11eHT HBJIHeTCH HOCHTeJieM Myrau1111. ECJTH HYKJieOTH,llHa51 nocJie- ,!I,OBaTeJThHOCTb B HCCJie)l)'eMOMrette He l13MeHeHa, TO OJIHroHyKJieOTl1Jl, CO,II,ep)l(all{HH MyraUHIO, He CB51)KeTCH C L(HK-MaTp11ueti, l1 IlUP He nOMJJ:eT.

221

 

 

Y"laCTKl.1

 

 

 

 

pecTp1.1K1..11.11.1

 

 

 

 

1

 

 

 

1

 

HopManbHb1i;t

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

reH 13-rno61.1Ha

 

 

 

.....GAG ......

 

 

 

 

 

 

 

 

 

 

 

 

<l>parMeHT pecTp1.1K1..11.11.1 1,1 K1.1no6a3bl

 

 

 

 

 

 

 

 

 

 

 

1

 

 

 

l

 

 

 

 

 

 

 

 

My,-aHTHb1i;I reH

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

j3-rno61.1Ha

 

 

 

.....GTG......

 

 

 

 

 

 

 

<l>parMeHT pecTpi.1Kl..ll-11.1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1,3 K1.1no6a3bl

 

 

 

AA SS

AS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1,3 K1.1no6a3bl

 

 

 

CJ

CJ

1, 1 Kl.1no6a3bl

 

 

 

 

 

 

 

 

 

PaA1.1oaerorpaMMa nocne rn6p1.1,Q1.13a1..11.1111 c 30HAOM K l3-rno61.1Hy

PMc. 4-68. PecTpMKl.IMOHHblM aHanMs JlHK reuorno6MHa <1enoeeKa, 6onbHOro cepnOBMAHO-KneTO'IHOMaHeMMeM (HbS). M111c- ceHc-My,-a4111S1, OTBeTCTBeHHa51 aa B03HlllKHOBeH111e cepnOBlllAHO-KfleTOLJHOM aHeMllllll, CB513aHa c 3aMeHOM B reHe ~-4en111 rno6111Ha TplllnneTa GAG Ha GTG. np111 3TOM y,-paLJ111eaeTC5I yLJaCTOK peCTp111K4111111 ¢epMeHTa Mstll, yaHat0ll.1ero nocne,:ioeaTenbHOCTb CCTNAGG, ri:ie N

MO>KeT 6b1Tb nt06biM OCHOBaH111eM. np111 Hanl/ILJllllll My,-a4111111 reHHblM 30HA r1116p111,:i111ayeTC5I c 6onee KpynHblM C!JparMeHTOM !lHK pa3MepoM

1,3 Klllno6a3bl, 111Mel0ll.1111M np111 sneKTpo¢opeae MeHbWYIO nOABlll>KHOCTb, LjeM npo,:iyKT pecTplllK4111111 HOpManbHOro reHa, j:lnlllHa KOTOporo paeHa 1,1 K111no6aab1.

HopManbHas:i npo6a

Myra1..11.1s:i i1F508

1

2

3

4

5

 

 

 

2

3

4

5

••o••

 

 

oeeoo

6

7

8

9

10

 

 

6

7

8

9

10

•••••

 

 

oeeoe

 

 

 

 

 

 

PMC. 4-69. reHHOe 30HAMPOB8HM8 HS HOCMT8nbCTBO MyKOBMCl.IMAOSa. c nOMOlJ.1blO nL-lP aMnn111¢1114111pyt0T reHOMHYIO

!lHK, Ill npo,:iyKT nepeHOC51T Ha 2 Hei4nOHOBblX qllllnbTpa, OAlllH 1113 KOTOpbiX rn6p111,:i111ayt0T c 32 P-30HAOM Ha HOpManbHblM annenb, a BTOpoi4 c 32 P-30HAOM Ha MYTaHTHblM annenb t.F508. 06paaoBaH111e rn6p111AOB 06Hapy>K111Ba10T paj:11110aBTOrpaC!J111LJeCKlll. npo6bi 1-3 cny>K111n111 KOHTponS1M111: nepea51 co,:iep>Kana npo,:iyKTbl nL-lP !lHK OT rOM03111roT no HOpManbHOMY reHy; BTOpa51 - OT reTepoa111roTHoro HOC111Ten51 MyTa4111111 t.F508; TpeTb51 - OT 6onbHoro MYKOBlllC4111A030M, roM03111rDTHOro no MyTa4111111 t.F508. npo6b1 4-10 nonyLJeHbl np111 06cne,:ioeaH111111 7 na4111eHTOB Ha Hoc111TenbCTBo t.F508: 4, 5, 6 111 9 OKaaan111cb roMoa111roTaM111 no HOpManbHOMY reHy, a 7, 8 Ill 10 - reTep03111rOTHblMlll HOClllTen51Mlll MyTaHTHOro reHa.

E. llcnoJih30BAHME ,IJ;HK-TEXHOJior11fi ,U.Jlll

JTOJIYllEHMH JIEKAPCTBEHHbIX JTPEIIAPATOB

II JIEqEHMH PA3JiffqHblX liOJIE3HEH

Baiill,Hllb1 - O'IBI..UeHHb1e6eJI1<H, aHTIITeHHhie .uerepMllliaHTbI PMa B036y.Il,l1reJieii BttpycHhIX tt 6aicrepttaJibHhIX HHQ:>emttll:. B rrOCJie.IJ,Hee BpeMH MX nonyllaIOT, IIOJih3Y5!Cb TeXHHKOH peKoM6MHaHTHbIX .llHK. flepooii BaKUI.fHOll, CIDITe3HpOBaHHOH 3THM cnoco6oM, 6hma BaKUMHa npoTHB Bttpyca renaTIITa B.

EeJIKH, HMe1mn:ue TepaneBTH'ICCKoe3Ha'leuue, rronyqaIOT c HCIIOJib30Bamt:eM 3TOH TeXHOJIOfHH BO MHOfHX CTpaHax MHpa. TaK, O)J,HHM ll3 rrepBblX CHHTe3ttpOBaH HHCymrn qenoBeKa (pttc. 4- 70).

B KJieTKax E. coli, rpattcQ:>opMHpOBaHHbIX TVIa3MH-

.uaMtt, co.uep)l(aI.IJ:HMH ,llHK, KOTOpa5! KO.UHPOBana A- tt B-uerrtt HHCYJIHHa, ttapa6aTbIBaIOT 6e11KOBb1e rrpo.uyKTbl A- 11 B-uerretl:. flocne OllttCTKH HX no.u- BepraIOT <Pon.umrry tt oKHcnemno, KOTopoe o6ecnellHBaeT o6pa30BaHtte COOTBeTCTBYIOI.IJ:HX )J,HCyJibQ:>IDJ.- HbIX MOCTHKOB.

222

 

 

 

 

1. X111MepHb1e nna3MV1Abl,

 

 

 

1

co,lleplt<aw.111e

 

1

 

reHbl f1 B·L\enelil

 

 

f1HCYflV1Ha

 

 

 

 

 

("(.·~.~

;£))') 2. 0T6op KfleTOK,

 

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<::i-::7

"XJ} . /

co.a.ep>Kalll,111x x111MepHb1e

 

 

"'--~~,-.-~-.--,,.~------

BeKTop

b8KTepvianbH85'1

nna3M111Abl

 

 

xpOMOCOMa

 

l

l

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\j'\J\ . j

'v'\..F\_/

 

A·L\erlb

 

B·L\Onb

 

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A-L\eno-Nf-1;,

Yt COOH

AKH-1 BHblVt

 

B-L\enb-Nf-1;, _j---1\-- COOH

"1HCYf1"1H

 

 

 

A1t1cynbcp111.nHaSo1

 

 

 

 

CBH3b

 

 

Pi.tc. 4·70. nonr1eHi.te lt1HCyni.tHa 1.1enoseKa B 1<11eTK8X E.coli. 1 -

Tp8HCcj:JOpM8L\lllH KJ18TOK E. coli nna3MlllA8Mlll, KOTOpb18 COA8P)l(8T

reHbl, KOAlllPYIOLL\1118 CTPYKTYPY A- 111 B-L1enelii 111Hcyn111Ha;

2 - ClllHTe3 A- 111 B-4enelii 111Hcyn111Ha

B npo4ecce Bb1palL(111BaH111H KynbTYPbl

TpaHccpopM111posaHHb1x Kfl8TOK E. coli; 3 - BblAeneH111e 111 OYlllCTKa A- 111 B-4enelii 111Hcyn111Ha; 4 -

npocTpaHcrneHHasi yKn8AKa A- 111 B-

4enelii lllHcyn111Ha Ill OKlllCneHllle OCTaTKOB 4111CT8111Ha.

 

 

 

AHaJ10fl11c!HbIM cnoco601v1 no11y1c1eH ropMoH poCTa, 11cnOJlb3yeMblM )lJUI Jie4eHl1}1 )leTeJ.i c He)lOCTaT01c!HOCTbJO 3TOro ropMotta. 6011ee CJIO)l(HbJe 6eJIKI1 noJiy4eHbl B KyllbType KJleTOK MJieKOnl1TaIOllU1X. TaK, nect>eKTbI u rette ¢aKTOpa VIII, Kon11py10w,ero OJlMH 113 6eJIKOB - Y'IacnmKoBcnepThl-

Ba10mei1 CMCTeMbl KPOlll1, OTBeTCTBeHHbl 3a B03Hl1KHOBett11e re1v10¢11J11111. Ll,o rnro KaK ¢aKTOp VII I

6bUT nOllY'IeHMeTO)laMM reHHOH JUDKettep1111, 60JlbUJOe KOJll14ecrno 6011hHbJX norn6ano OT CDl1Ll,a l1Jll1 remtTMTa, KOTOpb!Ml1 OHM 3apa>Kafll1Ch B peJyJlbTaTe BBe)leHM}I Bbl)leJieHHOro 113 KPOBI1 ¢aK-

223

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