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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