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E4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C2 --------------- --------------- -A------------- --------------- --------------- ---------------
C2(T3) --------------- --------------- -A------------- --------------- --------------- ---------------
PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
|> 3' íåòðàíñëèðóåìàÿ îáëàñòü
735 750 765 780 792
B1 CCCAGGAGCGAGTCC ATCAAGAGGAGCGAG TGCACACTATTAGAT GCCtaaAGG . . . . . . . . .
E4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C2 --G----------T- --------------- --------------- ---taaG-CAAGAGA ATCCGTGTGATT
C2(T3) --G----------T- --------------- --------------- . . . . . . . . . . . . . .
PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ðèñ. 11. Íóêëåîòèäíàÿ ïîñëåäîâàòåëüíîñòü ïîëíîðàçìåðíîé êÄÍÊ êëîíà Â1 è ÷àñòè÷íàÿ ïîñëåäîâàòåëüíîñòü êÄÍÊ êëîíîâ Å4 è Ñ2 è ïðîäóêòà ÏÖÐ. Ïîñëåäîâàòåëüíîñòü êÄÍÊ êëîíà Ñ2 îïðåäåëÿëàñü ñ èñïîëüçîâàíèå ïðàéìåðîâ V1, V2, C1 (îáîçíà÷åíèå Ñ2) è Ò3 (îáîçíà÷åíèå Ñ2(Ò3)). Ïðî÷åðê îçíà÷àåò èäåíòè÷íîñòü â ýòîé ïîçèöèè ñ êÄÍÊ êëîíà Â1, FR - êàðêàñíûå ó÷àñòêè, CDR - ãèïåðâàðèàáåëüíûå ó÷àñòêè V îáëàñòè.
A
AruIgLBV QYTVTQTPAEKSVLPGDTVALNCKVNSAVLGNTYLHWYQQKPGEAPKLLIY*RASTLESGIPTRFSGSG**SGTDFTLTISGVQAEDEGDYYCVSV
IpuIgLVG -V-------V--A---E--TI--RT-P-Y*-H*-----------------K*F-NQ-H----A------**--S----------T--A-----Q-Y 71%
OmyIgLV1 -I------EM-AFQT--A-T-R-RF-KPSPPC**VA-------G--Q----*Y-T--Q--T-S------**--S-------------A-----Q-Y 63%
XIaIgLVR -VVL--S-DYV--S--E--TIT--AS-SS---**-------S-QT------*GT-NRYT-T-E------**----------RME---AA----QQY 58%
Rabbit kV IV ivm----ss---pv----ti--qasqs-ys-nr-a-f-----qp------*k----a--v-s--k---**---q------d--ca-aat---rva 57%
GciIgLVIII -M--S-PVL--GL-Q--TIT-TASQS-YS-**-A----RE-QK-S----*A-TNRYTEVSE------**---S-----RN--P--VA----QGT 48%
HulV2 -SAL--*-ASV-GS--QSITIS-TGT-SSYNL**VS----H--K----M--*EG-KRP--VSN-----K**--NTAS--T--L-----A----C-Y 47%
HfrIgLVII GTVL--*--SM-TSQ-K--KIT-TISGGGTYY**SS--W----S--VFVWRDYD--RG----D--T--RNT-SNVMH---TD--SR-TA-----AW 41%
XlaIgLIII -VSI--*-VSE--KL-E--RIS-TLSG-SGYH**VN-----A-NR-RY-LRFYSDSNK**HQD-----KDSPNNIGY---K-ALL--DA----ATW 35%
HfrIgLV1 VPVLN---ISDP-SA-E-SE-K-AMQNGGSYY**MS--R-R-----VFVL**YQ--SG-IYRD--KP-RDT-SNSHI---GSLEPG-SAV---AAN 31%
Á
AruIgLCB GSPTAPSSVSLLPPSKLELDSKGKATLVCLVNNFYPDVVDIKWTVDGVAQSSG*VLTSTMKQK**DGKYSASSSLTLTKAVWNSKETYTCTVKHEAVSTPRSESIKRSECTLLDA
CplIgLCII -RS*PT--V----SDQITA-NM-------SG-V-GAAE-E-----SVRGN-*-E--RIQ-EA*-NTF-V--Y---SASD---H-L-S-V-K-ETQAN-LQT--S--S-M 44%
HfrIgLCIII EKSQPTLT-M---PE-VKA--T------ADH----E-GVE-KK--A-I-A-*-Q--NYLRAS*-ST--C--L---SGSD-E-NARFS-ALT-ETL-S-L-K-VS----V 42%
IpuIgLCF G--VKP-----L--S-Q-S*E-S-S-L--LPAYS-QGALVS-----SEVKD-*----AEER-**TDG-TR--T---S--L-EKG-EFV-K-S-DN-DH-VT**FRK-Q-EV 41%
Hu kappa TV-AP-VFIF---*D-QLKS-T-SV---L-----REAKVQ-K--NAL--GN*SQE-VTE-DSK-ST--L--T---S--DYEKHKV-A-E-T-QGL-S-VTK-FN-G-- 39%
XlaIgLC3 GDVK--*---YF---*V-EIATK---V--SLSD-T-RGATVK-L---KD-TDS*-QS-GLSKQS*-NL-ME--Y-S--ADQ-LRH---S-K-S-Q**GKEIIQTL-----V 38%
Hu lambda1 Q-K-NPT-T-F---*S-ELQAN-------ISD---GA-TVA-KA--SPVKA-*-E-TKPSKQS*NN--A---Y-S--PEQ-K-HRS-S-Q-T-E**GSTVEKTVAPT--S 37%
HfrIgLCI SEDRKP-VL-----*S-EIDS-W---S---SR-K-GF-RVL-R--DKETD--*-T-G-VSTDS*-QS--L--YLRVPATA--KGSS---S-D-GSL-S-LLKT-SSTA-SD 37%
XlaIgLCS ND-KPA-FIFK--*D-QVKE-NP-A---I---F-RDLTVT-K--SQDV--SD-K--DFM-ES*-ST--Q--M-T---DK-DKADKFE-L-K-K**TAQLTQSFSK-Q-S 34%
IpuIgLCG LTQP--TV----SV--Q*QE-V-----AYKGF-SDWRLS-K---SSW---*ESR-SAVLQA*--L--W--T-S-HPEQ-RN-*VV--EASKDN*QP-VVSTVNTEQ- 33%
Ðèñ. 12. Ñðàâíåíèå àìèíîêèñëîòíûõ ïîñëåäîâàòåëüíîñòåé V (À) è Ñ (Á) îáëàñòåé L-öåïè Èà ñòåðëÿäè ñ ïîñëåäîâàòåëüíîñòÿìè äðóãèõ âèäîâ ïîçâîíî÷íûõ. Äåôèñ îáîçíà÷àåò èäåíòè÷íîñòü â äàííîé ïîçèöèè. Çâåçäî÷êà îáîçíà÷àåò äåëåöèþ àìèíîêèñëîòíûõ îñòàòêîâ. Ñëåâà óêàçàíû ïðîöåíòû ñõîäñòâà. Âèäîâûå îáîçíà÷åíèÿ: Aru - ñòåðëÿäü, Ipu - ïåùåðíûé ñîìèê, Omy - ðàäóæíàÿ ôîðåëü, Xla - øïîðöåâàÿ ëàãóøêà, Gci - àêóëà-íÿíüêà, Hu - ÷åëîâåê, Cpl - ïåñ÷àíàÿ àêóëà, Hfr - ðàçíîçóáàÿ àêóëà.
Ðèñ. 13. Ñàóçåðí áëîò ãèáðèäèçàöèÿ. Ãåíîìíàÿ ÄÍÊ áûëà âûäåëåíà èç ïå÷åíè è îáðàáîòàíà ýíäîíóêëåàçàìè ðåñòðèêöèè Pst I Pvu II. Ãèáðèäèçàöèþ ïðîâîäèëè â ìÿãêèõ óñëîâèÿõ ñ èñïîëüçîâàíèåì çîíäîâ, ãîìîëîãè÷íûõ VL (à) è CL (á) ãåííûì ñåãìåíòàì L-öåïåé Èà ñòåðëÿäè. Ñ V-çîíäîì ãèáðèäèçîâàëîñü áîëåå 20 ôðàãìåíòîâ ÄÍÊ, â òî âðåìÿ êàê ñ Ñ-çîíäîì ãèáðèäèçîâàëîñü òîëüêî 3-4 ôðàãìåíòà.
ÎÁÑÓÆÄÅÍÈÅ ÐÅÇÓËÜÒÀÒÎÂ
 ðåçóëüòàòå ïðîâåäåííîé ðàáîòû èäåíòèôèöèðîâàí ëîêóñ ãåíîâ, êîäèðóþùèõ L-öåïè Èà ñòåðëÿäè, ïðåäñòàâèòåëÿ ïîäêëàññà êîñòíî-õðÿùåâûõ ðûá. Ñðàâíèòåëüíûé àíàëèç ñòðóêòóðû V ãåíîâ ýòîãî ëîêóñà óêàçûâàåò, ÷òî îí îòíîñèòñÿ ê êàïïà òèïó. Ðåçóëüòàòû ñðàâíèòåëüíîãî àíàëèçà Ñ îáëàñòåé ìåíåå èíôîðìàòèâíû, ÷òî ñàìî ïî ñåáå íå óäèâèòåëüíî, òàê êàê Ñ-ãåíû L-öåïåé Èà çíà÷èòåëüíî ìåíåå êîíñåðâàòèâíû ÷åì V-ãåíû. Òåì íå ìåíåå, òîò ôàêò, ÷òî íà íóêëåîòèäíîì óðîâíå Ñ-ãåí ñòåðëÿäè èìååò íàèáîëüøåå ñõîäñòâî ñ êàïïà-ïîäîáíûìè ãåíàìè III êëàññà õðÿùåâûõ ðûá, òàêæå ñâèäåòåëüñòâóåò î òîì, ÷òî îáíàðóæåííûé ëîêóñ îòíîñèòñÿ ê êàïïà òèïó.
êÄÍÊ êëîíîâ Â1, Ñ2, Å4, à òàêæå ôðàãìåíò, ïîëó÷åííûé ñ ïîìîùüþ ÏÖÐ, ñîäåðæàò áëèçêîðîäñòâåííûå V-ãåíû. Ðàçëè÷èÿ ìåæäó íèìè ñêîíöåíòðèðîâàíû â îñíîâíîì â îáëàñòè 3-ãî ãèïåðâàðèàáåëüíîãî ðàéîíà. Ïîñëåäîâàòåëüíîñòè äâóõ îáíàðóæåííûõ J-ñåãìåíòîâ îòëè÷àþòñÿ ïî 10 íóêëåîòèäàì, ò.å., îíè ïðåäñòàâëÿþò ñîáîé ðàçëè÷íûå ãåíîìíûå ñåãìåíòû. Íåçíà÷èòåëüíûå ðàçëè÷èÿ îáíàðóæåíû òàêæå ìåæäó òðåìÿ ïîñëåäîâàòåëüíîñòÿìè Ñ-ãåíîâ. Ñ-ñåãìåíò êëîíà Â1 îòëè÷àåòñÿ îò ïîëíîãî Ñ-ñåãìåíòà êëîíà Ñ2 ïî 9 ïîçèöèÿì, èç êîòîðûõ 5 âåäóò ê çàìåíå àìèíîêèñëîòû. Ïî-âèäèìîìó, ýòè ïîñëåäîâàòåëüñòè òàêæå ïðåäñòàâëÿþò ñîáîé ðàçíûå çàðîäûøåâûå ãåíû.
Àíàëèç ñòðóêòóðíîé îðãàíèçàöèè ëîêóñà ñ ïîìîùüþ Ñàóçåðí áëîò-ãèáðèäèçàöèè óêàçûâàåò, ÷òî ó ñòåðëÿäè èìåþòñÿ ìíîæåñòâåííûå VL ãåíû. Òî÷íàÿ îöåíêà èõ êîëè÷åñòâà íåâîçìîæíà, íî íàëè÷èå áîëåå 20 ïîëîñ ãèáðèäèçàöèè ñ ðàçíîé èíòåíñèâíîñòüþ ñèãíàëà, ïî àíàëîãèè ñ ìíîãî÷èñëåííûìè ëèòåðàòóðíûìè äàííûìè, ïîçâîëÿåò ãîâîðèòü î íåñêîëüêèõ äåñÿòêàõ êîïèé.
Ïðè èñïîëüçîâàíèè Ñ-çîíäà êîëè÷åñòâî ïîëîñ â ìÿãêèõ óñëîâèÿõ ãèáðèäèçàöèè íå ïðåâûøàåò 4-õ. Ó÷èòûâàÿ âîçìîæíîñòü àëëåëèçìà ìîæíî ãîâîðèòü î íàëè÷èè â ýòîì ëîêóñå 2-4õ ãåíîâ.
ßðêî âûðàæåííîå êîëè÷åñòâåííîå ïðåâîñõîäñòâî V ãåíîâ íàä Ñ-ãåíàìè ÿâëÿåòñÿ õàðàêòåðíûì ïðèçíàêîì ñåãìåíòàðíîé ôîðìû îðãàíèçàöèè ëîêóñà. Ó õðÿùåâûõ è êîñòèñòûõ ðûá ñ êëàñòåðíîé îðãàíèçàöèåé ãåíîâ L-öåïåé êîëè÷åñòâî V è Ñ ãåíîâ î÷åíü âåëèêî è ïðèáëèçèòåëüíî îäèíàêîâî, ÷òî îòðàæàåòñÿ â ñõîäíîé êàðòèíå áëîò-ãèáðèäèçàöèè. Ïîëó÷åííûå íàìè ðåçóëüòàòû ÿâëÿþòñÿ ñåðüåçíûì îñíîâàíèåì äëÿ òîãî, ÷òîáû óòâåðæäàòü, ÷òî ó ñòåðëÿäè îðãàíèçàöèÿ ãåíîâ L-öåïåé êàïïà-ïîäîáíîãî òèïà èìååò ñåãìåíòàðíûé õàðàêòåð, ïîäîáíûé îðãàíèçàöèè êàïïà ëîêóñà ìëåêîïèòàþùèõ.
Ýòè äàííûå óêàçûâàþò íà òî, ÷òî ïåðåõîä îò êëàñòåðíîé ê ñåãìåíòàðíîé îðãàíèçàöèè ïðîèçîøåë â ôèëîãåíåçå ïîçâîíî÷íûõ î÷åíü ðàíî, âîçìîæíî óæå ó äðåâíèõ ïåðâè÷íî-êîñòíûõ.
ÂÛÂÎÄÛ
1. Ñêîíñòðóèðîâàíà áèáëèîòåêà êÄÍÊ ëåéêîöèòîâ ñòåðëÿäè ïðåäñòàâèòåëüíîñòüþ 6õ106 íåçàâèñèìûõ êëîíîâ. ÏÖÐ ÄÍÊ áèáëèîòåêè ñ ïîìîùüþ âûðîæäåííîãî ïðàéìåðà, ñîîòâåñòâóþùåãî J ñåãìåíòó ãåíîâ ÈÃ, ïîçâîëèë âûÿâèòü ôðàãìåíò ÄÍÊ äëèíîé 370 ïí, ãîìîëîãè÷íûé V ãåíàì Èà ïîçâîíî÷íûõ.
2. Èç áèáëèîòåêè êÄÍÊ ñ èñïîëüçîâàíèåì ïðîäóêòà ÏÖÐ â êà÷åñòâå çîíäà âûäåëåíî ïÿòü êëîíîâ, ñîäåðæàùèõ âñòàâêè êÄÍÊ ãåíîâ L-öåïåé ÈÃ. Ñðàâíèòåëüíûé àíàëèç ïåðâè÷íîé ñòðóêòóðû êÄÍÊ êëîíîâ ïîêàçàë, ÷òî îíè ÿâëÿþòñÿ ÷ëåíàìè îäíîãî ñåìåéñòâà ãåíîâ è èìåþò íàèáîëüøåå ñõîäñòâî ñ ãåíàìè êàïïà òèïà.
3. Ñîãëàñíî ðåçóëüòàì ãåíîìíîãî áëîò-àíàëèçà èäåíòèôèöèðîâàííîå ñåìåéñòâî ñîäåðæèò íåñêîëüêî äåñÿòêîâ V ãåíîâ è òîëüêî 2-4 Ñ ãåíà, ÷òî óêàçûâàåò íà ñåãìåíòàðíóþ ôîðìó åãî îðãàíèçàöèè.
4. Ïîëó÷åííûå äàííûå óêàçûâàþò ÷òî ïåðåõîä îò êëàñòåðíîãî òèïà îðãàíèçàöèè ãåíîâ ÈÃ, õàðàêòåðíîãî äëÿ õðÿùåâûõ ðûá, ê ñåãìåíòàðíîìó, ïðèñóòñòâóþùåìó ó âñåõ ìëåêîïèòàþùèõ, ïðîèçîøåë â ýâîëþöèè â ïåðèîä ïîÿâëåíèÿ ïåðâè÷íî-êîñòíûõ ðûá.
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