Cloning and sequencing of an apparently recombinant...
Transcript of Cloning and sequencing of an apparently recombinant...
Ind ian Journ al o f Biochemi stry & Biophysics Vol. 3(), August 1999, pp. 22 1-226
Cloning and sequencing of an apparently recombinant promoter for napin gene from Brassica campestris genomic library and
its evolutionary significance
Somnath Bhattacharyya t and Radha Kanta Mandal*
Department of Biochemistry and Centre for PUnt Mo lecul ar Biology, Bose Institute, Calcutt a 700054 , West Bengal, India
Received 25 January 1999; revised 29 April 1999
Fwm a genomic lihrary of Brassica campeslris (brown sarson cv. B54), we have cloned and sequenced about 2 kb of ups tream regu latory rcgion from one o f the 25 albumin-cod ing gene famil y. The sequence has several seed-specifi c promo ter motifs. A sequcnce alignment o f the 5' n anking regions of the avail able B rassica 25 storage protein genes showed th at our sequence is a double crossover recombinant product o f the two members of the napin gene famil y. A possible explana tion o f th is fac t is th at I3 rassica species evolved th rough gene dupli cati on and recombinati on from a co mmon ancestor with fewer numbe r or chromoso mes and genes.
Among the va ri ous seed storage proteins, 2S storage prote in (napin ) is the maj or in the Brassica spec ies l
-3
.
apins are encoded by a multigene family with 4-7 numbers in B. rapa and 10-1 6 in B. llapus5
,6 . Nucleo
tide :-.equences of 2S coding regions showed about 9070 homology within different Brassica species7
.
With a view to construct a seed specific express ion syste m for tran sgenic pl ant , the upstream region of 2S prote in gene of BrassiclI juncea and Brassica oleracea have been amplifi ed from genomic DNA using o li gonuc leotide primers corresponding the napin gene'i (publi shed ) and consequently sequencedB
. We are aware of seque nce data for six different napin promoters. A mong the m three upstream sequences of napin gene (B. jUll cea , B. oleracea , B. carinata) showed c lose homology (over ' 90%) with that of a sequence of B. /lapus by J oseffson et af. 5. The upstream seque nces from B. rapa isolated by Kridl et al .
4 showed c lose homology '.vith that of another
napin promoter sequenced by Scofi eld and Crouc h6.
But in different Indian morphotypes (sarson, tori a) of B. cam/Jes t,-is (syn B. rapa ) thi s upstream regul atory region is not amplified by the above said oligonucleotide primers . Here we describe the isol ation sequencing and e voluti onary significance of a genomic napin c lone from o le ife rous B. campestris, one of the
* Author for correspondence ' Present addre~s : Regio nal Research Substation , 5ekhampur, Bidhan Chandra Krishi Vi swavidyalaya, P.O . Gadadharpur, Birbhum, West Bengal. India
diploid ances tral parents of both B. napus and B. Jun cea.
Materials and Methods Plant material
Seeds of Brassica campestris var. B54 was obtained from Pul ses and Oil Seeds Research Station, Berhampore, West Bengal , India.
Chemicals Enzymes were obtained from Bangalore Gene i, 1n
c;li a or Bethesda Research Laboratories (BRL) USA. Agarose, ethidium bromide, dNTPs, X-gal etc . were
obtained from Sigma, USA. [a-32PJ dATP and [a_35S] dA TP used for Southern blotting and sequencing were supplied by Board of Radiation and Isotope Technology , BARC, Mumbai , India. Hybond Wand Hybond N were obtained from Amersham, England. X-ray films used for autoradiography were from OROW or Agfa , India.
DNA isolation, genomic library construction and screemng
The high molecular nuclear DNA of B. campeslris cv. B54 was isolated following a rapid and simple method9
. The same nuclear DNA was partially digested with EcoR1 and ligated into the EcoRI site of EMBL3 phage vector. Recombinant bacteriophage were transfected into P2392 E. coli cells as described 10 . Plaques were lifted onto Hybond N membrane in duplicate and screened with a nick translated Pst1 fragment of the napin eDNA (pN2) c1one5 la-
222 INDIAN J. BIOCHEM. BIOPHYS, VOL. 36, A UG UST 1999
be lled w ith [a _12PJ dATP. Two positive recombinant ph age c lones were obtained and puri fied accordin g to a standard protoco l 1 I . They were subsequently found to conta in overl apping genomic inserts and des ignated as BcGL5 and BcGL6. Using proper res triction enzymes, the BcGL6 derived overl apping genomic frag.'nents were subc loned into the pUC 18 vec tor .
Restriction J//(/ppillg alld DNA sequence analysis Approx imate ly a 6 kb recombinant phage clone
was subc lo ned in to the EcoRI s ite of pUC 18 plasmid vec tor. T hi s 6 kb fragment was restricti on di gested by Hill dIlI , HaelIl and Xlw I separate ly and b lotted on Hybond N+ membrane according to standard procedu re. Bl ots we re hybrid ized with the labe lled 537 bp Xlwl/Xha l DNA fragment containing coding region and 1137 bp Eco RIIHilld III fragment containing promoter reg ion of nap in gene from B. juncea7
. Blots were prc hybri dized and hybridi zed in solutions conta inin g :iX SSPE. 0 . 1 % sodiu m pyrophosphate, 0.2.')';'r SDS. (J.027r po lyv iny l pyroli d ine, 0.02 % BSA
(frac ti on V) . 10 /l giml yeas t (RNA, 50% de ionised
formamide at -+O°c. Was hes were in 0 .1 % SSC and O. I C/r S DS a t 40°C.
A 2kb HiJ/ d llI fra gment was chosen as a promoter
2 3
1kb H Ha Ha I I I
region for sequenc ing. T his fragment was subc10ned into various smalle r p ieces through ExoIII-mung bean nuclease treatment according to supplie rs manual (Stratagene) into the pBsk( +) vector. Sequence of this upstream regulatory region was determined by the c hain te rminating dideoxy method using sequanase ve rsion 2 .0 (mod ifi ed T 7 pol ymerase) DNA sequencing ki t (USB , Cleave land, USA) and computer a ligned by UW-GCG program.
Results and Discussion Isolation alld characterization of a promoter f rom B. campestris genumic library
Us ing napin cDNA (pN2) as a probe, 6 .2 kb genomic clones fro m EMBL3 phage library of B. campestris was iso lated . T o identi fy the 5' upstream region fro m the genomi c c lone this 6 .2 kb fragment was restricti on digested by HindIII and HaeIIJ and Southem hybrid ized separate ly by napin promoter
g and coding
reg ion 7 (from B. j lll/cea) as probes. From the autorad iogram (Fig. I a. b) it is found that onl y a 2 kb HilldIIl fragment and 1.4 kb HaeIII fragment a re present when hybridi zed with promoter reg ion. But two Hin dlII frag ments (2.2 kb and 1. 1 kb) and same size of a HaeIII fragment ( 1.4 kb) a re present when hy-
2
H Ha H
I I
I , I
C
3 kb
23 .1
9.6 6.6
4 .3
2.3 2.0
Fi)! . I--Sout he rn hl\)( analysis of a () kb genu mic fragment isulated t'rum cl one I3cG L6. I Undiges ted fragment (lanes I ): d igested with Himllli (lanes 2): and Hac- II I (lanes 3). The blot was probed wit h (a), EcoRIIHilldl1l fragmen t (1 137 bp) cncompassing napin promotcr of
n. j UIICl'(/: (b). PSI I fra)!mcllt o f the nap in coding regiun (537 bp) of B. j Ull cea7. T he numbers indi cate IJH ind ll1 DNA s ize markers in kb. (e). The restriction Illap of c lone as derived fro m above Southern bl ut analys is results is depicted (a and b). T he fi ll ed box indicates the coding region. H. Hilld ll l: Ha. Hac- II I. The size of 1 kh is indic:lted l.
BH ATIACHARYYA & MANDAL: CLONI NG AND SEQUENCING OF PROMOTER FOR NAPIN GENE 223
bridi zed with coding region (Fig. I b). So from the banding patte rn it is asce rtained that both the enzymes have atl east one restriction s ite eac h in 5' up
stream regio n and coding region. As evidenced from the autoradi ogram, one HaellI fragment from 5' upstrea m region is mi ss in g ( lane3 of Fig l a). The reason be hind thi s is di scussed late r on after compl ete sequenc ing o f the 2 kb 5' upstream regulatory region. The restr ic ti o n map of the c lo ne BcGL6 derived from the Southern bl o tting result s is depicted in F ig. Ic .
T he 2 kb Hilld lII frag ment and a 1.4 kb HaeIII fragment were subc lo ned into pBsk( +) vec tor for gettin g 5' upstrea m regul atory region of B. campestris. A 2- kb H illd III fragment was further fragmented into fi ve smal ler pi eces by E..w IlI-mungbean nuclease treatment and c loned int o the same pBsk(+) vec tor fo r seq ue nc ing. T he co mple te sequence of 2084 bp upst ream reg ion was subm itted to the EM BL data bank under the access ion numbe r Y 13 108 and is g i ven in
Fig. 2 (upper line). The sequence has severa l unive rsal and seed spe
c ific transcription regul ato ry moti fs. A poss ible TATA box lies from nl. 2020-2026 and a putati ve CAAT box from nl. 2001-2005. The hexameric, Gbox moti f (CACGTG) and seed spec ific a lte rnate purine pyrimidine motif (CATGCA) we re approx . 65 and 77 nL upstream respec ti ve ly from 'the tran scription start point deduced by ana logy with that of napin gene5
. A four base repeat CA TT is found ten times as shown by double underlin ing.
Comparison of B. call1pestris with other Brassica species and their evolutionary sign!ficance
One hundred per cent DNA ho mo logy upto 68 bp upstream from the transcripti on start site was obse rved among the three seq uences (Fi g. 2) . Moderate amount of homo logy (-70%) was found upto 200 bp upstream aft e r the above menti oned regi on i.e. upto
Bc2sP AAGCTTATTT CT .TTTTCGA CACTCTAATT GAGACCGTGC GCTCTATCTA GACCAATTAG AATTAGATGG AGCTCTAAAG GTTTGAATGG CTGTTTTCTT 100
Br2sP AAGCTTATTT CTCTTTTCGA TACTCTAATT GAG.CCGTGC GCTCTATCTA cACCAATTAG AATT.GATGG AGCTCTAAAG GTT . . GCTGG CTGTTTTCTT
GTTCATATGA TTAACTTCTA AACTT GTGTA TAAATATTCT CTGAAAGTGA CTTCTTTTAG GCATATGTAG GTTGCATGAC AAAACTGAGG AAGATTAACC 200
GTTCATATGA TTAACTTCTA AACTTGTGTA TAAATATTCT CTGAAAGTG. CTTCTTTT .G GCATATGTAG GTTG ... GGC AAAAACGAGG AAGATT .. GC
TTCTCAATTT AAGGAAGAGG AGGAACAGCC GAAGAAG .AA ATAAGAATAG TCAGTe. ..... TGCATCTC ATGACTCAGC TTAACGGTCG TCGTCCTCAT N3
TTCTCAATTT . • GGAAGAGG ATGAACAGCC GAAGAAGAAA ATAAGAATAG GCAGTCCTGC TACTCAAT~ ATCTCAGTCT ATAACGGTCG TCGTCCCATG
GAACAG ..... ATACATTTT TGTCATATAC ACTTGAAAGT TCCTTCACTA ACTGTGTAAT CTTTTGGTAG ATATCACTAC AATGTCGGAG AGACAAGGCT 388
AAACAGAGGT AAAACATTTT TTGCATATAC ACTTTGAAAG TTCCTCACTA ACTGTGTAAT CTTTTGG1AG ATATCACTAC AATGTCGGAG AGACAAGGCT
GCGCCAGCAT ATACAAAAGG GAAATGAAGA TGGCCTTTTG ATTAGCTGTG TAGCATCAGC AGCT .ATCTC TGGGCTCTCA TCATGGATGC TGGAACTGGA 487
GCGCCAGCAT ATACAAAAGG GAAATGAAGA TGGCCTTTTG ATTAGCTGTG TAGCATCAGC AGCTAATCTC TGGGCTCTCA TCATGGATGC TGGAACTGGA
TTCACTTCTC AAGTTTATGA GTTGTCACCG GTCTTCCTAC ACAAGGTAAT AATCAGTTGA AGCAATTAAG AATCAATTTG ATTTGTAGTA AACTAAGAAG 587
TTCACTTCTC AAGTTTATGA GTTGTCACCG GTCTTCCTAC ACAAGGTAAT AATCAGTTGA AGCAATTAAG AATCAATTTG ATTTGTAGTA AACTAAGAAG
AACTTACCTT ATGTTTTCCC CGCAGGACTG GATTATGGAA CAATGGGAAA AGAACTACTA TATAAGCTCC ATAGCTGGTT CAGATAAC.G GAGCTCTTTA 686
AACTTACCTT ATGTTTTCCC CGCAGGACTG GATTATGGAA CAATGGGAAA AGAACTACTA TATAAGCTCC ATAGCTGGTT CAGATAACGG GAGCTCTTTA
GTTGTTATGT CAAAAGGTTA GTGTTTAGTG AATAATAAAC TTATTACCAC AAAGTCTTCA TTGACTTATT TATATACTTG TTGTGAATTG CTAGGAACTA 7f!6
GTTGTTATGT CAAAAGGTTA GTGTTTAGTG AATAATAAAC TTA . TACCAC AAAGTCTTCA TTGACTTATT TATATACTTG TTGTGAATTG CTAGGAACTA
Bc2sP CTTATTCTCA GCAGTCATAC AAAGTGAGTG ACTCATTTCC GTTCAAGTGG ATAAATAAGA AATGGAAAGA AGATTTTCAT GTAACCTCCA TGACAACTGC 886
Br2sP CTTATTCTCA GCAGTCATAC AAAGT GAGTG ACTCATTTCC GTTCAAGTGG ATAAATAAGA AATGGAAAGA AGATTTTCAT GTAACCTCCA TGACAACTGC
Bj2sP .. . ....... ......... . ....... t cttcatcggt gattgattcc
TGGTAATCGT TGGGGTGTGG GTAATGTCGA GGAACTCTGG CTTCTCTGAT CAGGTAGGTT TTTGTCTCTT AATTGTCTGG TTGTTTTTAT TTTCCCCTGA 9B6
TGGTAATCGT TGGGGTGT.G GTAATGTCGA GGAACTCTGG CTTCTCTGAT CAGGTAGGTT TTTGTCTCTT .ATTGTCTGG .TGTTTTTAT TTTCCCCTGA
tttaaagact tatgtttctt atcttgct tc tgaggcaagt attcagttac cagttaccac ttatat tctt gactttctga ctgatcctca tttttccaac
(COil/d.)
224 INDIAN 1. BIOCHEM . BIOPHYS., VOL. 36, AUGUST 1999
TAGTCTAATA TeAT .AACTC TGCGTTGTGA AAGGTGGTGG AGCTTGACTT TTTGTACCCA AGCGATGGGA TACATAGGAG GTGGGAGAAY GGGTATAGAA 1005
TAGTCTAATA TGATAAACTC TGCGTTGTGA AAGGTGGTGG AGCTTGACTT TTTGTACCCA AGCGATUGGA TAr.ATAGGAG GTGGGAGAAV GGGTATAGAA
attttaaatt tcactattgg ctgaatgctt cttctttgag gagaaaacaa ttcagatggc agaaatgtat caaocaatgc atatat acaa atGTAcctct
TAACATCAAT rn:.AGCAACT G.C.GATCAA GCAGC.TTCA TATTAAGCAT ACCAAAGCGT AAGAATGGTG GATGAAACTC AAGAGACTCT CCGCACCACC 1182
TAACATCAAT rn:.AGCAACT G.CGGATCAA GCAGCTTTCA TATTAAr.r..AT ACCAAA'XGT AAG.ATGGTG GATGAAACTC AAGAGACTCT CCGCACCACC
tgttctcaaa acatctatcg gatggttcca tttgctttgt catccaatta gtgactactt tatattattc act cctcttt attact at tl: tcatgcgagg
GCCTTTCCAA GTACTCATGT CAAAGGTTGG TTTCTTTAGC TTTGAAr.ACA GATTTGGATC TTTTTGTTTT GTl TCCATAT ACTTAGG.CC TGAGAGCTTT 1281
GCCTTTCCAA GTACTCATGT C.AAGGTTGG TTTCTTTAGC TTTGAA(~CA GATTTGGATC TTTTTGTTTT GTi TCCATAT ACTTAGGACC TGAGAGCTTT
ttgccatgta cattatattt gtaaggattg acgctattg • • •• • •• agcg tttttct t ca attttct tta tt t aagacat gggtatgaaa tgt gt g.tta
tggttctcga gattcccgtc aaa ••••••. gtgaattGCA TACCGTTCTC TAGTAAGATG ACCTACCATT CALICTTGAG ACAAATGTTI\ CATTTTAGTA 1374
tggttgattt ttttttcagg acaaatgggc gaagaatctg t acattgcat caat atgcta t ggcaggaca gtgtgctgat acacacttaa gcatcatgt g
gagttgggtt gaatgagata t acgttcaag tgaagt~ TACCGTTCTC GAGTAAG •• G ATGACCTCCT .ATTCTTGAG ACAAATGTTA CATTTTAG • •
TCAGAGTAAA ATGTGTACCT ATAACTCAAA TTCAGTATTG ACAATGTATC CATTCAAACT ATAAAATTAA AC .CAGCCTG CACTCGCATC CACATTTCAA 1473
gaaagccaaa .. .. gacaat tggagcgaga ctcagggt cg t ca t aatacc aat caaagac gtaaaaccag acgcaacctc ttaacct ctt tgtaatgaaa
TCAGAGTAAA ATGTGTACCT ATAACTCAAA TTC • • . GATT GACATGTATC CATTCAAACT • •. AAAATTA AACCAGCCTG CACTCGCATC CACATTTCAA
GTATTTT CAA A ... CGTTCG GCTCCTATCC ACCGGGTGTA ACAAGACGGA TATCGAATTT GGAGUT .. . .. . .. .. .. . . . ... .. ... ... .... TTT 1540
gggatgtgtc t ••. tgg t at gtatgtacga ataacaaaag agaagat gga attagtagta gaaatatttg ggagcttttt aagcccttca agtgtgcttt
GATATTTTTC AAACCGTTCG GCTCCTATCC ACCGGGTGTA ACAAGACGGA TTCCGAATTT GGAGGAT • • • . • •• ••••• • •• • •• ••• •• •• • • •• • TTT
GACTCAAATT CCCAATTTAT ATTGACCGTG ACTAAATCAA CTTTA . • • ••.. ACTT CTAT AATT CTGATT AAGCTCCCAA TT TATATTCC GAACOGCACT 1633
ttatct t att gat atcata atttgcgttg ttTAA tgcgt CTct agatat gt tatat at ctttctcag t .gtctgataa gtgaaatgtg agaaaaccaT
GACTCAAATT CCCAAT TTAT ATTCACCGTG ACTAAATCAA CT1TA • • ••• •. ACTTCTAT AATTCTGATT AAGCTCCCAA TTTATATTCC CAACrn:.ACT
A..'T.AAACCAA TATATTC. M TCTTATTTTA TA TAAT GTTC AATCAGT(X1; AGTTGCCACC TTCT GT~ AT"lGTGCTGA ATCTATCACA CTAGN#W\ 1730
ACCAAACCAA AATATTCAAA TCT TATTTTT AATAATGTTG AATCACr(X1; AGTTGCCACC TTCTGTGCCA ATTGTGCTGA ATCTATCAO\ CTAGAAAAAA
ACC t ccaaaa ttTAt at a ..... .... .. ... . ....... . .. . .................................. GA CTCTcat ca: CT tttaaacc
.. \&JCTTC AAa:;TAATGA CTTGTGGACT A1GTTCTGAC TTCT\ALIAA GTTTTTATTT Tcr.cGAAGTT AAGTTT TTAC CTTCTGmr GAAATATATC 11'30
ACAITr m C AAa:;T AATGA CTTGTGGACT ATGTTCT GAA TTCTCATTAA GmTTATTT TCTGAAGTTT AAGTTTTTAC m CTGTn T GAAATATATC
aac t tagtaa acg ....... .. .... .. .. .. ...... .. ..... tt tt t t TTTTTAATT Tta t GAAGTT AAGTTTTTAC CTTGr. TTTT AAAAagaATC
GTTCATAAca t cgttCTCAC GCCAGGACca ttGCagctac atacgJ..tCJ: asx;atctacC ATtcgccatg ca4HXAGAT CAffiACGATT TGTCACTCAC 1930
GTTCATAA .... GATGTCAC GCCACJ:ACAT GAGCTAC ... .. ....... . ...... .. . N:. ATcgca:::ata gcATGCAGAT CAGGACr.ATT TGTCACTCAC
GTTCATAA .. .. GATGCCAT GCCAGAACAT TAGCTAc.acg ttacacat. •• •• agcatg: Agccgcggag aat t gtt ttt cttcgccacT TGTCACTCCC
TTCAAACACC TAAGAGCTTC TCTCTCACAG CGCACACA • .•• • • CA rATG W~ ... A TA. TTJI.~ !~TC . GCAT ~TCTCC ATTCTTCACC 2019
TTCAAACACC TAAGAGCTTC TCT CTCACAG CGCACACA .. .... CA rATG CATGCA ... A TATTTACACG TGATarrJ.T GCAAATCTCC ATTC. TCACC
TTCAAACACC TAAGAGCTTC TCTCTCACAC GACACACAta caatcacatg q;TGCAtgca ttaTTACACG TGATCGCCAT GCAAATCTCC • TTtat aIICC
TATAAATTAG AGCCTCGGCT TCACTCTTTA CT~CCAA AACTCATCAC TACAG .ACAT ACACAA 2004
TATAAA TTAG AGCL,CGGCT TCACTCTTTA CTCAAACCAA AACTCATCAC TAr.AGlIACAT ACN.AA
TATAAATTAA CTCATCCGCT TCACTCTTTA CTCAAACCAA AACTCATCAA U.CAaacaag attaauAACATA
rig . 2- t\liglllllt:llt of 'i" Il pstrea m sequ ences of 2S protein genes from B. C[]IIII ' ('s l r i s in the present study denoted by Bc2S P (upper lines). II. }"{III('" (Illiddle li ne, ) and H. j l/l/ (" (,U
K (lower li nes). [Capi tal letters indi ca te seq uence identi ty al leas t in three successive nucleot ides. H ~ I , e difkrc nce, ~ IJC ind icated hy small lette rs where atleast three consecut ive bases arc di ss imi lar. Dots represent gaps for mi ss ing nu· cleot lde:, lrum o Ill' ,c lj ucnL'e t() al low maximum homology a lign ment. The T ATA box (solid line). CAAT like box (dotted line), G·box (da , il e:u li ne ). ,eed , peci lic Ct\TGCA (aste risk under nt), four base repeats like CATT (double lines) and presumed transc ription start P()l llt (~ I rrow il e ~ ld ) arc indi cated . The seq uence o f Be2SP was ubmitted to the EMB L data bank under the access ion number Y 131 08] .
BHAlTACHARYYA & MANDAL: CLONING AND SEQUENCING OF PROMOTER FOR NAPIN GENE 225
-1141 -615 -330 -270 +1
5'~~A~d ( a
-1137 -614 -331 -270
5' f0Z?Z'7AV~! b
-2084
5' L.·,;:·:--:·:·:·:-:·:·:·:··-:· ...... . . .. . ...... .. .. . ... · t/'l'h~ ·· c
- 2142
- 2160 s' 1:·:-:-· :··
.. . . . . . .... . .
- 762 - 40 7 - 270
.. ·'>l ·· . ~ .. ) .......... . ........ .. . . ......... d
-779 -412 - 270 ..... . .. >: .. ! :-:':-:. :1: -: .·.· ·.·.1. ·. e
Fig . .l-SChem:'llic diagram of li ve nap in promoters; [(a), 8 . napus (Joseffson el a /5 ); (b), 8 . juncea (Dasgupta el a/.R); (e), B. campeslris (present study): (d ), B. rapa (Kri d l el a l. ~) and (e), 8. napus (Scofield & Crouchl'). Common homo logy regions are filled wi th bl ack in all the sequences. Simi lars :Ire the cross hatched regions in (a) and (b) , and dotted regions in (d) and (e) . (c), is the sequence of 8. campeslris (present study) showin g the recombination between members of the two napin gene families. Unfill ed portions represent part of the coding region of the lupin gene i.
-270 bp among those sequences. After this region , the promote r under stud y showed high degree of ~omology (>95 o/c) with the B. rapa4 or B. napus6 promoter upto -420 bp (Fig. 3) and in this region , no homology is found with the B. juncea promoter8
. It is interesting to note that our c loned napin gene promoter has approx . 90% homology with the B. juncea or B. napus promoter pN36 (ref. 5) in the region 1312 to 1635 nt. From Fig . 2 it is c lear that -420 to -741 nt. region of the promoter shows c lose homology (-95 %) with that of B. jUl1cea from -33 1 to -648 nt. region. Remaining upstream region 1 to 13 11 i.e . the region beyond upstream -74 1 bp has more than 90% homology with the B. mpa promoter, BcNA I (ref. 4) or B. napus promoter gNa (ref. 6) but absolutely no homology with B. jUllcea promoterR (Fig. 3) . It has been found from restricti on mapping and sequence analysis that there are three Hae III sites on the napin gene; one at 3' end of the cod ing region, one at -784 nt. and other at -1665 nl. So when we used B. juncea promoter or codi ng region as probes only t~e 1.4 kb HaeIII fragment (-784 to 3' end of coding region) was lighted up in both the autoradiograms (Fig. la and b). This is due to the fact that B. juncea promoter has no homology above the -741 nl. region (Fig. 3) of B. campestris promoter. Therefore, it is concluded that 5' upstream region of B. campestris napin gene, we have sequenced mi ght be a double cross over recombinant product of the two members of the napin gene family. The recombinati on may have occurred during evoluti on of B. campesfris through interspecific or intraspec ific crosses among the wild species of Bras-
sica as evidenced from the cytological t2 or RFLP analysis 13
· '4. Three diploid species of Brassica evolved through two evolutionary pathways. One pathway gaves ri se to B. nigra and another common pathway to the diploid cultivated species, B. oleraceae and B. campestris. Thus, according to the previous study'2-'4 the diploid cultivated species, B.oleraceae (CC) and B. campestris (AA) are secondary polyploids and share common ancestral genome of chromosome number six. Conserved regions of the Brassica lineage (i .e . shared linkage between species) evolved through duplicated loci , and hence served as orthologous linkage blocks. These orthologous linkage blocks allow sexual recombinations between genomes. So being an essential gene, this recombinant member of the napin gene isolated from B. campestris evolved during change of ascending chromosome numbers from a common ancestor of basic chromosome number below nine. Thus the napin gene evolved much earlier than the time of divergence of cultivated diploid and amphidiploid Brassica species. It could be interesting to compare the napin promoter sequences isolated from B. nigra to shed further light on the origin of Brassica amphidiploids . Further studies along these lines are in progress.
Acknowledgement The research was supported by a UNDPIFAO
project (IND/87/0 17) and CPMB, DBT, Govt. of India. Thanks are due to Drs N Das and S Ghosh for providing the genomic library and to Distributive In-
226 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 36, AUGUST 1999
formation Centre, Bose Institute for help in sequence analysis.
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