Добавил:
Upload Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз: Предмет: Файл:

підручники. біохімія / Uchebnik_po_Biokhimii_E_S_Severin

.pdf
Скачиваний:
21
Добавлен:
25.02.2016
Размер:
44.13 Mб
Скачать
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

PMC. 4-62. nocnE!AOBaTenbHOCTb HyKneoTltAOB B AHK, yaHaeaeMaSI TpeMSI HaM6onee '18CTOMCnonbayeMblMM pecTpMKT838MM.

Pecrp111K4111py10u.i111e HyKnea3bl nonyYa10r 1113 pa3nlllYHblX 6aKrep111~: HPA I - Haemophilus parainfluenzae; Eco RI - 1113 Escherichia coli; Hind Ill - 1113 Haemophilus inf/uenzae. Pecrp111KTa3bl rnna 1 pacu.iennHJOT ,QHK c 06pa300aH111eM «cnenblX» KOH400, a Apyrne (r111na 2) c o6pa30B8H1110M no M0CTY pa3pb1Ba OAH04enO'i0'iHblX«mmKlllX» KOHL.108.

.6. .H)J.EHTH<l>HKAI.J,ml CIIEU,H<l>WIECKHX

IIOCJIE)J,OBATE.JlbHOCTEH

Ilp11 o6pa60TKe reHOMHOM 3yKap110T11qecKOM )],HK, B qacTHOCT11, )],HK qeJIOBeKa, pecTp11KTa3aM11 06pa3yeTC5I TaK MHOro <PparMeHTOB pa3JIIfqHOM M11Hbl, qTo 11X He y,nafac51 y,UOBJieTBOp11TeJibHO pa3,nen11Th c rroMOIUhIO 3JieKTpo<Pope3a B nOJrnaKpHJiaMH,UHOM HJI11 arapo3HOM rene. Ilocne 3JieKTpo<Pope3a peCTP11KIJ:11POBaHHOM reHOMHOM )],HK 11 npo51BJieH1151 c ·nOMOIUhIO 6pOMH,Ua 3TH,U1151 rronyqaeTC51 paBHOMepHoe OKpam11BaH11e no BCeM ,UJI11He reJI51. HLleHT11$11Kau1151 HY)l(HhlX <PparMeHTOB )],HK B TaKOM reJie B03MO)l(Ha TOJihKO nyTeM m6p11)l113aU1111 c MeqeHhIM11 )l,HK-30H,llaM11. C11HTe3 )l,HK-30H,llOB OCYIUeCTBmi:eTC51 B aBTOMaT11311poBaHHhIX MaIII11HaX, IT03BOJI5IIOIU11X C11HTe311poBaTh <PparMeHTbl O)lHOHITTeBOM )],HK M11HOH CBhIIIIe 100 ttyKJieOTH,UHhIX 3BeHheB co CTPOro onpe)leJieHHotl: rrepB11qHotl: CTPYKTYPOM. TaKHe MoneKyJihI MO)l(HO

11crroJih30BaTb ,UJI5I crreu11$11qecKoro CB513b1BaH115I c 11CCJie)lyeMblM11 yqacTKaM11 reHa.

1iJIOT-ru6pH,L1,H3aIUU1 no Cay3epuy

K.rracc11qecKHM MeTO)lOM H,UeHTHQll1Kau1111 11HTepecyiomHX yqaCTKOB )],HK CTaJI MeTO)l 6JIOT-m6- pH,U113aUIDT no Cay3eptty, npeAJIO)i(eHHhIH B

CYTh MeTOLla 3aKJiroqaeTC51 B TOM, 'ITOCnJIOIIIHa51 «JieCTH11Ua» <PparMeHTOB )],HK, no11yqttBIIIa5ICH B pe3yJlhTaTe HX )leJieH1151 no MOJieKyAApHotl: Macce B reJl51X, TTO)lBepraeTCH ,UeHaTYPau1111 11 nepeHOCttTCH c reJI51 Ha TTJIOTHblH HOC11TeJib (H11TpoueJIJIIOJI03HblM QlllJibTp ttJI11 HeMJIOHOBYIO MeM6paHy). IlepeHoc, HJI11 6JIOTT11Hr, OCY1UeCTBJI5IeTc5I 3a cqfa Kan11JIJ151pHbIX CHJI, 3JieKTp11qecKoro IlOJI51 11JI11 BaKyyMa (p11c. <1>11Kc11poBaHttyio Ha QlHJlhTPe )]HK m6p1mmy10T c )],HKHJI11 PHK-30H,llOM, co)lep)i(am11M MeTKy. MeTOLlOM paa11oaBTorpa<P1111 orrpe)leAAIOT rro11o)!(eH11e 11cK0Moro <PparMeHTa reHOMHOM )],HK Ha 3JieKTpo<PoperpaMMe. DJIOT-m6-

214

4-64).

pttl(H3au1-rn - BhICOKO'-IYBCTBttTeJihHhrtt MeTOfl. HL(eHTHcpHKaUHH crrem1cp11'-1eCKHXITOCJie)lOBaTeJih- HOCTeti.

K ttaCTOH:WeMy BpeMeHtt pa3pa6ornHo Mttoro MO)l11cpttKau11i1 3Toro MeTo)la. TaK, ,llHK-30H.Il He Bcer)la Menn pa.u11oaKTHBHhIMl1 H30TOnaMl1, Hepefl,KO 11CITOJTb3YJOT coe)lHHeHHH, KOBaJieHTHO CBH3bIBaJOlll,JlleCH c ,Il,HK; JllX MO)!(HO o6Hapy)!(HTh no o6pa30Batt11JO OKparnettttoro npo!lyKTa HJTJll cpJT!OOpecueHUH11. ,Il,mrna OJTl1fOHYKJieOTl1).lOB B )lHK-30H,IJ,ax TaIOKe MO)l(eT CHJihHO BaphHPOBaTh, 6yf1,y•m JllHOr)la O'-leHhKOpOTKOH - B 15-20 nap HyKJTeOTH)lOB. Orr11catthI MeTOL(hI L(OT- (rnntto) HJIJll CJTOT- (IIOJTOCKa) rtt6pHL(H3aUHH, KOrL(a Ha rnep- L(hIH HOCHTeJTh HaHOCHT rrperrapaTbI JJ,HK HJTH PHK 6e3 rrpe,11,BapHTCJThHOtt peCTpHKIJ,MH HJTH 3JTeKTpocpope3a JI[ rn6pH,11,H3YJOT l1X c MeL\eHblMJll JJ,HK-30HL(aMI1.

3THX L(aHHhIX ycrnttaBJT11Ba10T nocJiefl,OBaTeJihHOCTh ttyKJieOTHL(OB BO BHOBh Cl1HTe311poBaHHhlX cpparMeHTaX, KOMITJieMeHTapHblX JJ,HK-MaTµl1IJ,e (p11c.

B ttacToH:mee BpeMH c03).laIOT np116oph1 L(JlH aBTO- M aT11'-lecKoro 0.IlHOBpeMeHHOro ceKBeHHPOBaHH5l 60JihIIIOro '-IHCJiarrpo6 c HCI10Jlb30BaHHeM Me'-leH- HhIX pa3HhlMH cpJIIOOpoxpoMaMl1 L(H)le30KCl1HYKJTeoTHL(OB.

B To )l(e BpeMH pa3pa6aThIBaIOT HOBhre, 6onee 3cpcpeKTJ1BHbie 11 3KOHOMH'-IHhICMeTOL(hI ceKBeHl1poBamrn. CymttocTh o,llttoro 113 HHX 3aKJIIO'-laeTCH B cneL(yIOmeM: H3 4 HYKJie03H/lTp11cpoccpaTOB (.uAT<D, fl.IT<l>, L(TT<l>, L(UT<l>) co3fl,aeTcH tta6op OJI11rottyKJieOTl1L(OB (HanpHMep, OKTaHyKJTeOTHL(OB), BKJIIO'-laIOl.l(HH Bee B03MO)l(Hhie BapttaHThI nocJTe- L(OBaTeJThHOCTett. 3TH OKTaHYKJieOTHL(hI HMM06HmnyTOT (npl1llIHBaTOT) B Hl.Jet1Kax, B pe3yJThTaTe L\ero C03L(aeTC5l TaK Ha3hIBaeMaH OJTl1fOHYKJieOTJ1L(HaH

B. y CTAHOBJIEHHE nEPBHqHoM CTPYKTYPbl ,Il,HK-<I>PArMEHTOB

( CEKBEHHPOBAHHE ,II.HK)

Ha1160JTee qacrn JJ)UI ycrnHoBJTeHHH rrepBH'-IHOtt CTPYKTyphr JJ,HK ttcnoJTh3YJOT llH)le30Kc11ceKBett11poBaH11e. B peam110HHhlX rrpo6ax, co,11,ep)!(am11x L(eHaTYPHPOBaHHYIO Ofl,HOHHTeByJO JJ,HK, JJ,HKITOJIHMepa3y' fl,e30KCl1HYKJie0311).lTpmpoccpaThI (,11,HT<l>) - !lAT<l>, ,11,UT<l>, L(fT<l> J11 ,11,TT<l>, OL(HH 113 KOTOpbIX HBJTHeTCH pa,11,HOaKTHBHbIM, J1 npattMep HHHUY!HPYJOT Cl1HTC3 )],HK B rrpJ11CyTCTBl1H cne- UHQH1.'-leCKHX L(H)J,e30KCHHyKJie03HL(Tp11¢occpaToB (MHT<l>), J1JIJll TepMJ11ttarnpoB, - llllAT<l>, MUT<l>, MIT<l> J1JIH MTT<l>. Cttme3 Ofl.HOBpeMeHHO Befl.YT B L\eThrpex napaJIJTeJihHhix npo6ax, B K~YIO 113 KOTOpbIX Hapw c KOMITOHeHTaMH peaKIJ,HOHHOH cMeCH npJ116aBJTHIOT Ofl.l1H 113 4 MHT<D. MHT<D 6y-

.IlYT KOHKypttpoBaTh c ttopMaJihHhIMH llHT<D 3a BKJIIO'-leHHeB paCTylll,YJO IIOJTHttyKJTeOTH)lHYJO uerrh. Ilp11 BcTµaJ11BaHJ11H MHT<D BMecrn coorneTCTBYJO- w:ero HyKJieOTl1fl,a CHHTe3 JJ,HK npeKpaw:aeTCH.

B pe3yJThTaTe B Ka)l(L(OH H3 npo6ttpOK rronyqaeTCH tta6op pa3JIHL\a10m11xcH no )J,JTHHe MeL\ettbIX cpparMeHTOB JJ,HK c 0)1,HHM H3 crreu,HcpH'-IeCKHX .Ill1- L(C30KCHHYKJTeOTH)lOB Ha KOHUe. CTocJie OL(HOBpeMeHHOfO pa3L(eJTeHl151 3THX cpparMeHTOB B 3JTeKT- PH'-leCKOM none Ha 4 coce)lHHX llOPmKKax 11. pa.unoaBTOrpacpHH pa3Mep CHHTe311pOBaHHbIX MOJTeKyJT MO)l(eT 6bITh ycrntto:rureH, a 3TO 3Ha'-IHT, '-ITO MO)l(eT 6hITh orrpe,11,eJTetta JTOKaJIH3aIJ,115l TepMHHl1PYJOlll,HX fl.H)le30KCHHyKJieOTHL(OB. Ha ocHoBaHHM

AHK f

D

l

m-

AHK-30HA l

l=l LJ

P1o1c. 4-63. 6noT-rM6pMAM381.1MSI no CayaepHy. <DparMeHTbl L\HK pasAen5lt0T c noMOll.lbtO aneKrpo<j:Jopesa, AeHaryp111pyt0r, ne· peHOC51T Ha HlllTpo4ennt0n03Hbllli QJlllflbTp Ill r116p111A1113YIOT c L\HK- 30HAOM.

215

5'-npaillMep

 

HyK11eOT11Abl: 5'-npaillMep-10-11-12-13-14-15-16-17-3'

 

 

 

 

 

AHK-norn1Mepa3a+AAT<t>, ArT<l>, ALF<!>

Ecrm c1-1Hre3

-

 

 

1

ATT<l> "1 OA"1H "13 4 AAHT<l>

 

 

 

 

 

11

 

 

3aKaHY"1BaJlCf!: .QAAT<t> AQrT<t>

AQ L.J,T<t> AQIT<!>

 

 

10

 

 

12

-

 

 

 

 

 

 

 

 

 

 

-

 

 

 

 

Pa3Mep npoAyKTa

 

-

 

 

 

 

 

 

 

 

 

 

 

-14

 

 

13

(no 1.11-1cny HyK11eornAoB)

 

15

 

 

-

 

 

 

 

 

 

 

 

16

3neKTpocjJopea

 

17

"-

 

 

 

 

-

a nom1aKp1-1naM"1AHOM rene-

'\o

\

 

 

/

 

 

 

 

 

 

A

G

C T

 

 

 

 

 

18

 

 

 

 

 

 

 

 

 

17

 

 

 

 

 

 

PaaMep AHK-npOAYKTa

rn

 

 

 

 

 

(no '"1"1CJlyHyK1leOT"1AOB)

14

 

 

 

 

 

nocneAOBareJlbHOCTb BHOBb

 

 

 

13

 

 

 

 

 

C"1HT93"1posaHHOill L1en1-1

 

 

 

12

 

 

 

 

 

5'-npail!Mep-ACGTGATA-3'

 

 

 

11

 

 

 

 

 

10

9

8

PMC. 4-64. ,llMAe30KCMCeKeeHMpoeaHMe nocneAOBaTenbHOCTM ,llHK. Vlcnonb3YIOT 4 npo6bl, Ka>KAaR 1'13KOTOpblX COAep)f(1'1T,QHKMaTp11l.lY, npai'i!Mep,,QHK-non11Mepa3y, 4 AHT<P (p,AT<P, ArT<P, AUT<P, ATI<P). npai'i!Mepn1160 OA1'1H113 HyKneornAOB c0Aep)f(11T paA11oaKr11sHy10 MeTKy, 6naroA8PR YeMy nonoCbl MOryT 6b1Tb o6Hapy)f(eHbl B rene c nOMOl.L1blO P8Al108BTOrpaqm11. 0A1'1H1'134 A1'1Ae30KC1'1P1'160· HyKneOrnAOB (AAHT<P) A06aBnRIOT B K8*AYIO npo611pKy. OcraHOBKa C1'1HTe3anp01'1CXOA1'1TB TOM cnrme, KOrAa AAHT<P BKnlOYaeTCR B pacryL11y10 on11roHyKneornAHY10 L1enb.

MaTpttua. CeKBem1pyeMhii1 cf>parMeHT JJ,H K MenIT ITO cf>occf>aTHOMY OCTaTKY H ,LJ,06aBJUIIOT B 51qei1KM MaTPHUbI. <l>parMeHT JJ,HK rn6pw:unyeTC51 TOJibKO c TeMH OKTaHyKJieOTH)J,aMH, ITOCJie,LJ,OBaTeJihHOCTH KOTOPhIX KOMTIJieMeHTapHbI era yqacTKaM. TaKHM o6pa3oM, onpeL1,eJI5leTCH tta6op Bcex B03MO)!(HhIX OKTaHYKJieOTH,LJ,OB, rrpHCYTCTBYJOIUHX B HCCJie,LJ,yeMOM ¢parMeHTe JJ,HK. JJ,an:ee nptt ITOMOIUH cneIJJiall:bHOM KOMITbIOTepHOM o6pa60TKM ynop5l)J,oqHBaeTCJI paCITOJ10)KeHHe OKTaMepoB B HCCJie,LJ,yeMOM cf>parMeHTe JJ,H K.

f. fioJiyqEHME PEKOMI>MHAHTHhIX ,11,HK

qeJIOBeKa. Il03TOMY HCCJie;::i:yeMble cf>parMeHTbl JJ,HK 06bI'IHO rrpe,ll,BapHTeJibHO aMITJIHcPH11HPYIOT (yseJIH'IHBaJOTKOJIH'!eCTBeHHOB MHJIJIHOHbl pa3), MH Toro '!T06bJIIOJiyqaTb HX B mo6oe speM5l H B HeOrpaHtt'leHHOMKonwrecrne. HcKJIIO'!ttTeJibHOueHHhIM HHCTpyMeHTOM B perneHHH 3TOM rrpo6JieMbI OKa3an:OCb H:CITOJib30BaHHe peKOM6J'JHaHTHblXJJ,HK (T.e. JJ,HK, ITOCTpOeHHbIX H3 yqacTKOB pa3HOfO npoHCXO)!(,ll,eHH5I).

1. II0Ay1tenue peKoMounanmHblX J(HK

J],JIH rronyqeHJ15l TaKHX MOJieKyJl rrepBOHa'!aJibHO BbI,LI,eJIHIOT JJ,HK H3 2 pa3HbIX H:CTO'IHHKOB(pttc.

M MX AMIIJIM<l>MKAQMJI

JLn.51 pa60Tbl c HyKJieOTHLl,HbIMH ITOCJIC,ll,OBaTeJibHOCT51MH B rettax 11 ,LJ,pyrnx yqaCTKax JJ,HK tteo6XO,LJ,HMO HMCTb Ll,OCTaToqHoe KOJIHqecTBO MaTepHa-

Jia )IJI5l HCCJie,LJ,OBaHH5l. 3TO HenpocTaH Ja,LJ,a'fa, oco6eHHO CCJIH HCTOl.fHHKOM JJ,HK CJI~aT TKaHH

4-65).

Ka)!(,ll,YJO 113 HJIX B OT,LJ,eJihHOCTH ¢parMeHTttpyIOT, HCIIOJib3Y5l O,LJ,Hy H Ty A<.e pecTpHKTa3Y, pacmerrJ15IIOIUYIO JJ,H K c o6pa30Batt11eM «JlHITKJIX» KOHUOB. ITocJie rrpoue,LJ,ypbI ttarpesamu:i H MMJleHttoro OXJia)!(,ll,eHmI (OTA<.Hra) HapHLJ:Y c HCXO,ll,HblMH

216

~10;1eKy.'Ja\tVi ;JHK\ 11 ilHK, Moryr ofipamBbJBaTb-

c'r peK01'l6HIJaHTHble \IOJICKY.lbl, COCTOHIU,Me Hl

'tipa f\l\:HTOB ~1HK\ 1-1 Ll HK,

cmna1-11-1 blX \H:~)KJtY

~~o6mi

".HJnKll\IH» KOllLiaMlL

Kona,1e1-111-1oe cw11-

ua Ii HC

rj:lpdl\ICHTOR OCYll lCCTIFIH I{)T c no~10Wbl0

JJl K-,1,ffaJbl B r1pHcyTcnivrn

AT<D KaK 11cTO'JH11-

Ka .rncpr111i.

B 1exHoJ10r-irn peKoM61rnaHTHbIX ilHK, KpoMc ct11x1L\H~HTOB n.H K. llbll!C.'lCl!HbIX HJ KJIC10K, co;1ep-

/KliUJlD. >upa, 1Krio.1b1y10T LlH K. no;ry 11eHHy10 c flOMOLL[blO o6paTHOfi TpaHCKpM m (l3bl. nPM 110forn-

_'JClHlH B peaK!Il10111lYIO cpcny 4 pa3HbIX Ll.t:'..!OKCMpl 16m1yICteo:rn;rq1l!~)OCCjJaTOB Q)CpMCHT Ha MaTpl1-

ue :-.1 PH K rm i1pH11u1111y Ko\1nJTeMeHrapHocn1

u11rreJ11pyn .JHK-Ko11mo. ~LIM KllHK. T:iK KaK l1CT(HHMKOM HH<!iop'>trnt}H1 npH 06paJoJJa111111 KLl,H K cny>Kin ipe.'lMI LU!TOI l.'Ja>~mn1'iccK<rn MPH K, TO TaKa51 ;JHK. B or1114i1c 01 IlHK c})parMcHTOH, no-

JIY'ICHHbI\ np11 rx1nuef1jJCHM 11 rc1-JOMHol1 Ll,H K :JyKap1m1ori, Ht co;1ep>KMT IJHTpOHOB.

2. K1011upo6a11ue //,HK

J.l,151 flOJlytiCH~Hl ·rna'll1TCJlbHblXKOJIH4CCTH l1H-

rcpccy1nu1cro Hae Ma1ep11aJla 11pono1rnT KJl1JH11po-

BaHHC D.H K. 11pe;1r10.11a1aioll1ce 1>npmrnaHMC Hy)K-

1wrn HaM cpparrv!CHTa IlHK B BCKTOPHYIO MOJlCKYJTY

JJ,H K I 11.:rn ReKTop). BeKTop 06ccne'IH1Jaer 11po-

1-ll1x11onet111e

:HOl~f pCK0\1611llaHTllOK,

lFll1 l(l1MCp-

l 1oi1, ,JH K B

6aKTqmaJlbHblC KnerK11.

B Kil'WCTBC

BCKTOPOB llCl10,1b1YfOT n:Ja1\1HJlbl, ¢ar11, pcTpO- H <UtHOB11pycbL Oco6eHHO 'lac10B Ka'ltCTBeBCKTO- pa CJ1Y>l<IH rrnaJMIUHa~I llHK.

n,1a.lMHjl,bl -- I ICOO.!lbUH1C KOJlbUCBblC D,ByxueflOLJt'lllb!C MOJJtKy:1hr nHK, n p11cyrcrny1C1IHl1C H

6::\KTCpiHrnhHhlX KJICTKax B pa3JIH'IHOM KOJlH'lCCTBC

Korrni~. Chm 11\1e10T aBTOHov111y10 c11c1-eMy KOllT- po.ri>1 pC!Lil1K3Ul1l1, KOTOp::rn 11om1ep>Kl1HaeT l1X KO-

;lWICCTBO H K.:IeTKax Hil onpe)1e,1eHHOM ypoBHC' ·-

OT HCCKO..'lhKl!XCllMHHL!. JlO HtCKO;IbKMX COTCH KOl111lt retlO~Wll Ila KJICTKy.

VI CJ!OJl hJYC'>1YfO LLJlH KJJOH I!pOB<.lH 11>1 IJjl(\J\'11IlHYK)

JIH K 11 111r1epecyJOu1yK) 11ac Ll,H K pac111ernrn10·1 no 011pe;1c:1eHHOMy y'laCTKY pecrpMKTa'C\OH. llOJly-

LJafOT peKoMG1111~111rny10 Ll,H K, noJ1Jpawa10T r116- p11rmy10 rrna3Ml1J1Y B K0,1blleByJO (POPMY H BBOJUlT

B 6aKTCpJ.la,1bllb!C KJlC'TKM, T.e. ocy111eCTB-15IIOT Tpa11cc:PopMau1110 6a1<1ep11i1. llp11 pa3~rno1KeH11H TpaHctj)opMHpOll::HIHhIX 6aKTepMi1 11pOHCXOJJ,11T yBeJlH'ICHl1e '111C.'l'1KOlll1i~ BBC/!tHHOIO 13 IL1;13MHD.Y cj:iparMCHTa Lll-IK, r.e. T3KHM cnoco6oM Yyil<.cpcm-·

CafiT peCTpV1KL\f1'1

(' ,...-""- -··~:,',--·MeCTO parn~enneHV1'1

C•lMMF.:Tp"l'i

MeGTO

BToporo nop>i,llKCI

pact1lenneH11>i

3HL\OHYKf18830~1 c 06pa3osaK1AeM

Bb!CTynafOil-('1X

l<OHL\08

,QHKy

1 OTiKHr G o6pa3osaH11eM i peKOMfomaHTOB,

l C08,UVIHeHHb!X 38 cc1E\T

nv1!1Kl!IX f:OHL(OB

L\HKA --~·~:11~:~::

,[\HK- ,CUJVl88Hl!le

1

f1Wa33 rnmKl1X KOHqOB

KoeaneHTHO

C08A1'1HeHHblB

peKOM6w-1aHTHbl8

)J.HK

PHC. 4-65. nony'teHHepeKOM61/iHaHTHblX JlHK. L:(sa o6pasqa /]HK:

.QHK, 111 .QHK{ pacll.lenmnoT 0/1H0'1111 ro~ >Ke pecrp111e:ra3ofi 1-1 nonyYaIOT q:iparMeHTbl c «ilV1nK111MV1>• KOHqaM111. np111 geHarypaq1-1~ 111 nocneAYIOll.IBM «ODK111re" ot)pa3y101c5-1 peKoM6m1aHTHb1e MoneKynb1 JJ.HK,

~1 .QHKb, nepBOHaYanbHO coe11111HeHHbl8 ppyr c ppyroM :;a C'-IBT«m1r1- n1X» KOHL(OB, 3aT8M CllJlllBaeMblX JlHK·nWa.!CJV'

HblK iV151 fiaKTepHll reHCTH'lCCKM~l MaTepit<Ll MO-

)KCT 6bITb llOJIY'lCH R 3!lil'lMTCJlbllblX KOJIWlCClll<lX

(pHC. 4-66).

B Ka'ICCTBC KI!Ol mpy1011rnx BCKTopou tJano MC- nOJib3yKrr cj:iarn, Fern )K 1oreH Hy10 llH K BBOil5JT H :JyKapl10TH'JCCKl1C K;JeTKH. TO Try llPOUCllYPY Ha-

3blBilKJT TpaHCDYKUJ1t'M.

3. flo.11u.IVtepa31wR 11,enuw pewa~uR

Merna nom1Mepa:311ofi uemmi1 pea1<111111 (f I UP), npe1VJO)KCHHhli1 B 1983 r. KappM My.ri;111co1v1 (Ho-

6e11eBcKaH npeMHJL 1993 r.), simrnrn ::Jnoxai1bHbI\.t

2t7

Y4aCTOK

Y4aCTOK

pa!111en11eHiR

,QHK, l.1CnOilb3yeMaR AilR KilOH1.1posaH1.1R

1

XpoMocoMa

0T6op KJ10TOK, COA0p>Ka1111.1x XlllM0pHbl0 nna3MlllAbl

PocT KYilbTypbl KJleToK

.Qm1 no11}'40HMR.QHK

PocT KYilbTYPbl s ycnos111Rx, np111 KOTOpblX 3KCnpecc1.1pyeTCR KJlOHlllPOBaHHbli::i reH

©©©

 

l Pac111en11eH1.1e

 

pecTp1.1KTa3oi:1

 

l 8b1AeneH111e KJloH1.1posaHHoi:1

 

.QHK

 

KflOH111pOB8HH8R ,QHK

6eflOK

 

PMc. 4-66. CxeMa KJlOHMpoeaHMll ,QHK e 6aKTepMBnbHblX KJleTKax.

 

OTKpb1Tl1eM xx BeKa B o6nacnt MOJieI<:ymlpHOH 6ttoJIOnrn. OH IT03BOJIHeT rroJJBepraTb crrell,mtmqHotf aMIUil1QJl1Kall,l1l1 B yCJIOBIDIX in vitro (B rrpo6ttpKe) yqaCTKlf ,IlHK )JJIHHOH OT HeCKOJibKHX )leCHTKOB .Il,O HeCKOJibKHX COTeH nap HyKJieOTH)JOB, HCITOJib3Y51 B Kaqecrne MaTpHll,hI mo6h1e o6pa3ll,hI ,IlHK. Heo6XO!lHMoe YCJIOBHe AJI5I rrpoBe.IJ,eHHH nuP - 3HaHHe

HyKJieOTH)JHOH ITOCJie.IJ,OBaTeJibHOCTl1 aMIUI11QJHll,H- pyeMOH o6nacTH. YqacTOK 11ccneJJyeMott )],HK ru6pH.Il,113YIOT c !lBYMH 11CKYCCTBeHHO CHHTe3ttpoBaHHbIMl1 rrpatfMepaMH - OJil1TO)le30KC11pl160ttyKJieOTH)JHhIMH ITOCJie)lOBaTeJibHOCTHMH )JJIHHOH OT 15

AO 30 nap HYKJieOTH)JOB, KOTOpbie KOMIUieMeHTapHbI 3'-KOHll,aM aMIIJil1QJHll,HpyeMoro yqacTKa Ha KO-

218

,L1,Hpy10w.eit H tteKo,L1,11py10w.et1 HIIT51X ,Ll.H K. PaccTOHH11e Me)l(,ll,y rrpatfMepaMH orrpe,ll,eJrneT ,ll,Jll1HY CHHTe311pyeMbIX MOJieeyJI. B Ka'IecTBe MaTpm~hI ,ll,JIH c11HTe3a rrpo,ll,yKTOB ITUP HCITOJih3YIOT JI106o:H Tl1n ,LI.HK: reHOMHYIO ,LI.HK qeJIOBeKa, pa3JIWIHhIX BH- ,ll,OB npo11 3YKaPl10TOB, llHK, Bbl,ll,eJieHHYIO H3 KYJihTYP KJieTOK, «6tt6JI110TeK» reHoB H ,ll,pyrHX 11cTO'IHHKOB. MeTo,ll, He TPe6yeT 6oJihlllHX KOJIH'IeCTB 11CCJieeyeMOM llHK, B np11Hu11ne, ,ll,OCTaTO'IHO,ll,a)l(e O,ll,HOM MOJieeyJihl, CO,ll,ep)l(alll,eMC51 B O,ll,HOM BOJIOCe Ha fOJIOBe, O,ll,HOH KaITJie KPOBl1 HJil1 cnepMbl.

Ycrrex B pa3pa6oTKe MeTO,ll,a B 3Ha'IHTeJihHOH CTeneHl1 o6ycJIOBJieH HCilOJib30BaHHeM B Ka'IeCTBe ¢epMeHTa TepM0¢11JihHOM llHK-noJI11Mepa3hl, Bbl,ll,eJieHHOM 113 6aKTep11it, )l(HBYW.HX B rop51'Il1X 11CTO'IHHKaX, H ITOTOMY YCTOM'Il1BOM K ,L1,eMCTBl1IO BbICOKHX TeMneparyp.

PeaKU110HHaH CMeCh ,ll,JIH noJiyqemrn 11HTepecy10w.eit Hae ,LI.HK CO,ll,ep)l(HT 11CCJie,ll,yeMyIO llHK, cy6cTpaThI peaKU11H - 4 ,ll,HT<D, 2 rrpatfMepa, TepMocTa6HJihHYIO, HJIH Taq-rroJIHMepa3y 116y¢ep, co- ,L1,ep)l(aw.tt:H HOHhl Mg2+.

0,ll,HH UHKJI IlOJll1MepH3aUHH BKJIIO'IaeT3 3Tarra

(pHC. 4-67):

DJiaB.Tlemte: Ha 3TOM CTa,ll,1111 peaKU110HHYIO CMeCh HarpeBaIOT ,ll,O TeMrreparyphI 90-97 °C. l1ccJieeyeMAA ,ll,ByUenoqeqHaH ,LI.HK ,L1,eHaTyp11pyeT H nepeXO,ll,11T B O,ll,HOHHTeBYIO ¢opMy;

nt6plUJ.H3alJ,IUI H.1B1 OTIKHr ,LI.HK c npailMepaMH. B

pe3yJlhTaTe CHIDKeHHH TeMITeparyphI ,ll,O 50-60 °C npoHCXO,ll,11T KOMrrJieMeHTapHoe CB513hIBaH11e npaitMepOB c uenHMH MaTpl1'IHOMllHK H o6pa30BaHHe ,ll,Byxuerroqel.JHoro yqacTKa Ha Ka)l(,ll,OH m HHTeit llHK;

3JIOimllJ,IUI, YMHHeH11e HHTeM llHK, KOMITJieMeHrnpHblX MaTPH'IHotfllHK, KaTaJ111311pyeT TaqnoJIHMepa3a B HanpaBJieHHH OT 5'- K 3'-KOHUy.

3aTeM cttoBa ttacryrraeT 3Tan ITJiaBJieH1151, KOr,ll,a 3a cqfa IlOBhillleH1151 TeMneparyphI CHHTe3 }lHK npeKpaiuaeTC5f, H ,ll,ByttHTeBOH yqacTOK Me)l(,ll,y MaTPH'!- HbIMH H BHOBb CHHTe311POBaHHhIM11 MOJieeyJiaMH ,LI.HK ,L1,eHaryp11pyeT. Bo BTOPOM H IlOCJie,lJ,YlOlll,HX UHKJiax npaitMephl rn6pH,L1,H3HpyIOTC51 c 11CXO,ll,HOH MaTpwtHOM llHK H c BHOBh CHHTe3HpOBaHHblMH MOJieKyJiaMH llHK, KOJIH'IeCTBOKOTOPhIX HapacrneT B reoMeTpmecKoit nporpeccm1. B nocJie,ll,HeM CJiyqae CHHTe3 llHK 3aKaH'IHBaeTC5f He H3-3a 113MeHeHIDI reMneparypHoro pe)[(J1Ma, a no ,ll,OCTmKeHHH JlHKnoJIHMepa3oi1 rpaHHUhl aMITJil1¢HUHPOBaHHOfO yqaCTKa, '!TO onpe,ll,eAAeT CTPOfO onpe,ll,eJieHHbIM pa3Mep rrpO,ll,YKTa c TO'IHOCTblO,UO O,UHOfO HYKJieOTH,ll,a.

Ka)l(,!lhIH 113 3TaITOB UHKJia 11MeeT nponom1mTeJihHOCTh OT ,UeCHTKOB ceeyH,U ,ll,O 1-3 Ml1H, B pe3YJibTaTe ITOJIHhIM U11KJI ,ll,JIHTC51 OT 0,UHOH ,UO He-

CKOJihKHX MHHYT.

01mcaHHYIO rrpouenypy aMITJ111¢11KaUHl1 llHK npoBO,!l5IT B aBTOMaTH'IeCKOMpe)l(lfMe B rrp116ope -

UHKJIH3aTOpe, HJil1 TepMOUHKJiepe, aMITJll1¢HKaTOpe llHK. TaKotl np116op no3BOAAeT 3a,ll,aBa.Tu tty)l(HOe KOJIW!eCTBO U11KJIOB 11 Bb1611paTb OITTifMaJihHbie BpeMeHHbie 11 TeMnepazyptthie rrapaMeTphI. 3a 25-30

UHKJIOB 'Il1CJIOCl1HTe3HpOBaHHhlX KOITl1H JlHK ,UOCTMraeT HeCKOJihKHX MHJIJll10HOB.

c ITOMOlll,bID ITUP MO)l(HO ITOJiyql1Tb ,!lOCTaTO'I- HOe KOJIH'IeCTBOKOil11M yqaCTKOB ,LI.HK, B KOTOpbIX npe,!liloJiara10TcH np11CYTCTB11e MYTau11M:, noJI11Mop- ¢113M catfTOB, MO)l(HO npoBO,!ll1Tb llHK-,L1,11arHOC- ™KY 11H¢11u11poBaHHOCT11 nauHeHTOB B11pycHhIM11, 6aKTepl1aJibHbIMl1 11 rp116KOBbIMl1 B036y,Ul1TeAAMl1 6oJie3ttetf.

,II,. ,IJ,HK-)l.HAIHOCTMKA 3AEOJIEBAHMM

11crrOJih3YH TeXHHKY peKOM611HaHTHhIX llHK, ynafac5I 11CCJie,UoBaTh Bap11aHThl reHOB, OTBeTCTBeHHhIX 3a pa3BHTl1e MH0fl1X 3a6oJieBaHHM. 3T11M cnoco6oM 11neHTH¢11u11poBaHhI TO'!e'IHhieMYTaUHH, Bhl3BaHHbie 3aMeHOH O,!lHOfO a30Tl1CT0f0 OCHOBaHHH, ,UeJieUH51Ml1 11Jil1 BCTaBKaMl1, rrpHBO,UHlll.HMH K Il05IBJieHl1IO am1eJie:H, KO,U11py10w.11x ¢YHKUl10HaJibHO HeaKTl1BHbie 6eJIKl1. lle<t>eKTHbie «ITOJil1MOP¢hI» B03H11KaIOT KaK 3a cqfa 113MeHeHHM B KO,Ul1PYI0lll.11X yqacTKax retta, TaK 11 B pe3yJihTaTe MYTallHM B HeKO,UMPYIOlll.HX o6Jiacrnx, TeCHO np11MbIKa10w.11x K reHaM H Bhl3bIBalOW,11X HapY111eH11e HX pa60Tbl.

Pa3pa6ornHHhie TeXHOJIOfHH no3BOJI5IIOT BeCTH ueJiettarrpaBJieHHOe KapT11poBaHHe reHOB qeJIOBeKa B paMKaX Me)l(,ll,yttapO,UHOfO npoeKTa «feHOM qeJIOBeKa». 0¢11u11aJibHO 3Ta HayqHa5f nporpaMMa c yqacTHeM Benyw.11x MOJieKyJIHpHo-retteTl1l.JeCKHX Jia6opaTOpati CIIIA, cTpaH 3ana,ll,ttoti: EBponhl, a TaK)l(e Pocc1111 11 5lnoH1111 o¢opMHJiach B 1990 r. B xone pa6oThI HM npoeKTOM KapTHpoBaHhI 923 reHa, BhI3hlBaIOW,HX pa3Bl1THe MOHOfeHHblX 3a6oJieBaHl1H, 6oJiee 100 113 Hl1X ITOJIHOCThlO ceKBeHHPOBaHhl. K KOHUY 2001 r. pa60TaMH Jia6opaTOp11:H CIIIA, BeJI11Ko6p11rntt1111, 5lnott1111 11 pH,ll,a eBporrettCKl1X CTpaH c TO'!HOCTblO,UO 90% 3aBep111eHa pac111H¢poBKa reHOMa. 0)1(1f,ll,aeTCH, '!TO B Te'IeHHe6JI11)1(ai1.11111x 2- 3 JieT 6ynYT H3yqeHbI BCe reHhl, OTBeTCTBeHHhle 3a pa3B11Tl1e naTOJIOfH'IeC-

219

YYaCTOK ,[\HK, KOTOpbH.1 Heo6xo,o,111MO

aMnn111¢111u111poearb

L.(enb 1 3'

Ljenb 2 5'

c===

c=

HarpesaH1Ae, Bbl3btBalOli.\ee

pacn11ereH1-1e Hll!TeLil

OD1<111r, B XOA8 KOToporo

l np111coep1-1Hs:i10TCS'lnpal'.1Mepb1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

•·············-

 

 

 

 

 

 

 

 

 

 

 

 

 

:::..:::mt~"'••·~1··~3~n~o~H[r=aL1="1=S't=l1=8n=e=H=Te=p=~~~~;5~JlbHOLi!..

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

AHK-no11111Mepaa0Lil

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5'

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

;·~----

 

·-~··--

 

 

·~~···

 

 

 

=--=·..-=-·-=:====:],

 

5' [

...............................................

 

 

 

 

 

 

==....................

 

 

l ...

 

 

 

 

 

 

 

 

 

 

 

 

5'

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L.\1<1Kn 2

l

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.D.eHarypat.111151 AHK 111 0D1<1<1r

Uenb 1

3' C:::[..

.L:::"_..[:[=[[[=~=-•mmmmmmmc::::==···==-===-.-.J

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-----

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L.___......... ___,

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

l.lenb 2

5' c

.....-

................

 

 

=·-.[[]..·~~•r.-.-

....========·=:]·1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ljenb 1

3'

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fi

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LjGflb 2

! --

 

___

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

..

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

q111KJ1bl c 3-ro no 20-H 06ecneY111satoT o6paaosaHIAe 106 Komn1

P111c. 4-67. nom1Mepa3HaSI 4enHaS1 peaK4111si.

KHX npoueccoB y 'leJIOBeKa. 3To fl03BOJIHT Bb!BeCTl1 .[lHafHOCTHKY 11 Jie'leHtteMHOfHX 6oJie3Heti Ha HOBbIH ypoBeHb.

OcrnHoB11MCH Ha HeKOTOphIX MeTonax, urnpoKo 11CfIOJih3yeMblX M5I H,UeHTIHPHKaUHH MOHOreHHhlX 6oJie3Heti.

1. Ilo.!lUMOprjJU3M iJ.!lUHbl pecmpUKU,UOHHblX r/Jpal.Menmo6 (IIJ(P<P)

Myrau1111, B03HttKa10m11e B y'laCTKaxY3HaBaHHH onpeneJieHHhIX pecTpttKTa3, neJiaIOT 3TH yqacTKH L(H K He'l)'BCTBttTeJihHhIMMK neiicTn1110 ¢epMeHTOB. 3TO MO)l(eT 6bITb JierKO o6Hap)')KeHOno 113MeHeH11IO MHHbl pecTpHKUHOHHbIX ¢parMeHTOB )],HK. Il)],P<l>-aHaJIH3 BKJilO'laeTcJienyrom11e 3Tanhi: BhI.[leJieH11e reHOMHOH )],HK, ee peCTpHKUHIO cneumptt'-IecKoti3H.IIOHyKJiea3oil, 3JieKTpo¢opern- 'leCKOe pa3.[leJ1eH11e o6pa3YIOilIHXC51 ¢parMeHTOB )],HK H H.[leHrn¢11KaUHIO 3TI1X ¢parMeHTOB nyreM 6JIOT-m6p11JJ,113aUHH no Cay3epHy. Ha 3JieKTpo¢o- perpaMMax np11 OTCYfCTBHH peCTPHKUHH B HCCJieJJ,yeMOH )],HK BhrnBJIHIOT OJlHH KpynHhIH ¢parMeHT, COOTBeTCTBYIOlUHM no MHHe fIOCJie,llOBaTeJibHOCTH )],HK Me)!(,Ily JlBYM51 coce,II,HHMH yqacTKaMH peCTPHKUHH M5i TOH )Ke 3H,llOHyKJiea3bl. IlpH HMM - 'lMH pecTpHKUHH B nOJIHMOPWHOM y'-IaCTKe Ha 3JieKTpo¢operpaMMe 6yneT npttcyrcrnonaTh MeHbwttti no pa3MepaM ¢parMeHT, paBHhlM paCCTOHHHIO Me)!(,Ily noJIHMOPcPHblM YlJaCTKOM pecTpHKUHH H O,!I,HHM 113 6JIH)l(aHWHX noCT051HHbJX y'-IaCTKOB peCTpMKUl1l1 (p11C. 4-68).

Il,l]P<l>-aHaJIH3 MO)l(eT 6hITb 3Ha'-IHTeJihHOynpo- lUCH B TOM cnyqae, eCJil1 B03MO)l(Ha cneutt<l>H'-IeC- KaH aMTIJIHcPl1KaQH51 y'laCTKa)],HK, CO,II,ep:xrnmero noJI11Mop¢HbIH catiT pecTpttKUHH. TecTttposaHtte COCT051Hl151 3TOfO JIOKyca B03MO)((H0 nyreM nposeneHl151 nUP 11 peCTPHKUMH aMJJJIMWMUMpOBaHHOro ¢parMeHTa. Ilp11 0Tcyrcrn11H catlrn Y3HaBaHttH B HCCJienyeMOH o6JiaCTH )],HK pa3Mepbl aMnJIHcPMUHPOBaHHOfO ¢parMeHTa He H3MeH51TC51 nocJie ero o6pa60TKM pecTPHKTa3oil. EcJIM yqacTOK y3HaBaHH51 He M3MeHeH, o6pa60TKa ¢epMeHTOM np11neneT K o6pa30BaHl1IO 2 MaJieHbKHX ¢parMeHTOB c TOM )Ke CYMMapHOM MHHOM, 'ITOH HCXO,!I,HbJM cf:>parMeHT.

IlpM o6cJie,II,OBaHHH nautteHTOB l1 'lJieHOB HX ceMeti Ha HOCI1TeJibCTBO naTOJIOfH'-IeCKHX reHOB W11pOKO HCTIOJib3YJOT 3TOT MeTO,ll, C nOMOillhlO KOTOporo:

11,II,eHTHWHUHPYIOT ,II,eJieUHl1 B reHe ,!I,HCTpo¢ttHa, Ha ,!I,OJIIO KOTOpb!X npHXOJl,HTC51 OKOJIO 60%

ncex Myrau11tl:, Bbl3bIBalOUIH.X MHO)lHCTPOcPHIO )],10weHHa;

n11arnocT11pyroT reMo¢HJittIO A, HeK0Topb1e TaJiacceMHH, peTHH06JiaCTOMY 11 rpaHyJieMaT03;

KOHTPOJIHPYIOT 3!.lOPOBbe ,II,eTeH B ceMMIX, B KOTOpblX pO,!I,MTeJIH HBJIHIOTCH reTepo3HroTaMl1 no reHy cepnOBH)lHO-KJieTO'-IHOH aHeMH11 11

JlPYrHM ne<t>eKTHbIM reHaM.

2. OnpeiJe!leuue Mymau.uii c nOMOUl,blO ll.!l.!le!lbcneu,urjJu1tecKux npo6

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,

 

'

<::i-::7

"XJ} . /

co.a.ep>Kalll,111x x111MepHb1e

 

 

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

BeKTop

b8KTepvianbH85'1

nna3M111Abl

 

 

xpOMOCOMa

 

l

l

CD

 

 

\j'\J\ . j

'v'\..F\_/

 

A·L\erlb

 

B·L\Onb

 

\

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

J

 

 

//

 

 

 

..--·,,..,.,/

 

 

 

 

i (4'\

 

 

 

 

..:

'j

 

 

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

Тут вы можете оставить комментарий к выбранному абзацу или сообщить об ошибке.

Оставленные комментарии видны всем.

Соседние файлы в папке підручники. біохімія