Plant Molecular Biology 19: 391-399, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

391

The isolation and characterisation of a cDNA clone encoding L-asparaginase from developing seeds of lupin (Lupinus arboreus) Tony J. Lough, Brett D. Reddington, Murray R. Grant 1, Diana F. Hill, Paul H.S. Reynolds I and Kevin J.F. Farnden* Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand (* author for correspondence)," 1Fruit and Trees, D.S.I.R., Private Bag, Palmerston North, New Zealand Received 16 August 1991; accepted in revised form 31 January 1992

Key words."L-asparaginase, glycosylasparaginase, Lupinus arboreus, nitrogen metabolism, seed development

Abstract

An L-asparaginase cDNA clone, BR4, was isolated from a Lupinus arboreus Sims developing seed expression library by screening with polyclonal antibodies to the seed asparaginase. The cDNA hybridised with an oligonucleotide probe designed from amino acid sequence data and was found on sequencing to be 947 bp in length. Six polypeptide sequences obtained previously could be placed along the longest open reading frame. Computer-aided codon use analysis revealed that the cDNA sequence was consistent with other plant genes in terms of codon use. The cDNA insert was used to analyse asparaginase transcription in various tissues by northern blot analysis. A transcript size of approximately 1.2 kb was detected in L. arboreus seed total and poly(A) + RNA. The level of this transcript declined from 30 days after anthesis to an undetectable level by day 55. Furthermore, under the high stringency conditions used, the seed asparaginase cDNA did not hybridise with total or poly(A) + RNA isolated from root tips, suggesting that the asparaginase known to be present in this tissue may be the product of a different gene. Southern analysis suggested the seed asparaginase is a single-copy gene. The plant asparaginase amino acid sequence did not have any significant homology with microbial asparaginases but was 23 ~o identical and 66?0 similar (allowing for conservative substitutions) to a human glycosylasparaginase.

Introduction

In temperate legumes, including lupin, asparagine is the major compound formed from the assimi-

lation of ammonia [20, 34]. Asparagine has the dominant role in the transport and metabolism of nitrogen in lupin [2, 27] and other legumes [42]. Asparaginase (EC 3.5.1.1) hydrolyses the amide

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X52588.

392 group of asparagine to produce aspartate and ammonia and provides a route for the utilization of asparagine for the synthesis of amino acids and proteins [2, 5, 19, 34]. A second route for asparagine degradation has been proposed and involves transamination of the amino nitrogen by asparagine:2-oxoacid transaminase (see [1, 17, 34, 40] for discussion). Asparaginase has been found in developing seeds of lupin (see [21, 34] for review), pea [17, 25, 35] and developing roots, leaves and flowers of lupin [5] and pea [17]. Asparaginase activity has also been reported in Glycine max [40], Zea mays [24] and Phaseolus vulgaris [15]. These studies support the view that asparaginase is a key enzyme involved in asparagine utilization in lupins and plants in general. Sodek et al. [35] demonstrated potassium dependent asparaginase activity in cotyledons of Pisum sativum, Vicia sativa and Lupinus albus as well as seeds of Zea mays. Chang and Farnden [5] identified a potassium dependent asparaginase in young leaves, root tips and flowers of Lupinus arboreus and L. angustifolius that also requires sulfhydryl protection during extraction. The potassium-independent asparaginase has been identified and purified from maturing seeds of L. polyphyllus [ 19] and both L. angustifolius (cv. Uniwhite) and L. arboreus [ 5 ]. Antiserum has been produced against the potassium independent asparaginase from L. arboreus developing seeds and the protein has been partially sequenced [21 ]. In this study, we have isolated and sequenced a 947 bp partial cDNA encoding L. arboreus seed asparaginase and have aligned 6 peptide sequences along a translation of the longest open reading frame. This is the first report of the isolation and characterisation of a plant asparaginase cDNA sequence. Materials and methods

Plant material

Lupin (Lupinus arboreus Sims) developing seeds (ca. 40 days after anthesis, DAA) from St. Kilda

beach, Dunedin, N.Z. were harvested directly into liquid nitrogen.

RNA preparation

Total RNA was prepared from liquid nitrogen frozen seeds ofL. arboreus as described by Grant et al. [ 12].

Construction and probing of cDNA library

First-strand cDNA was prepared essentially as described by Maniatis et al. [22]. Second-strand cDNA synthesis used the RNase-H procedure of Gubler and Hoffman [14]. The cDNA was ligated into the expression vector 2gtl 1 (Promega) by the addition ofEco RI linkers as described by Huynh etal. [16]. The library was packaged in vitro using a packaging mix made by a procedure based on that of Scalenghe et al. [32] and amplified in E. coli Y1090. An antisense oligonucleotide probe 5'-AACATICCG/ACTIGTG/ ATTG/AAAIGGCATIGCIAT-3' was synthesized based on the back-translation of an asparaginase peptide IAMPFNTSGMF [21]. End labelling of the oligonucleotide probe was by the method of Maniatis et al. [22]. Other probes were labelled by either nick translation [30] or random priming [ 10]. Plasmid isolation was based on the method of Birnboim and Doly [4] and other recombinant DNA techniques were essentially as described by Maniatis et al. [22].

Plant DNA preparation, northern and southern blotting

Plant DNA was prepared by the method of Evans et al. [9]. Southern blotting [36] to Hybond-N (Amersham) and Zeta Probe membranes was performed according to the manufacturer's instructions and by Reed and Mann [29] with a final wash in 0.1 x SSC, 0.05~o SDS at 59 °C. Northern blotting to Hybond-N (Amersham) was

393 performed using the manufacturer's instructions with stringent hybridisation conditions (hybridisation buffers containing 50~o formamide [22] and 42 °C were used, with a final wash in 0.1 x SSC, 0.1% SDS at 60 °C).

DNA sequencing and computer analysis Nucleotide sequences were determined by the dideoxy chain termination method [ 31 ] using the M13mp series of vectors [23]. The sequencing strategy employed the methods of Deininger [7] and Bankier et al. [3]. The insert DNA, viz. the 950bp fragment released from pUCBR4 by Eco RI digestion, was concatenated by ligation and random fragments generated by sonication. These were end-repaired, sized and ligated into the bacteriophage M13. DNA and protein sequence analysis employed the programs of Stockwell [38, 39].

Results

Strategy for the isolation of an asparaginase coding sequence from lupin We based our search for plant asparaginase cDNA clones on two approaches. The first involved the detection of fusion proteins expressed by clones in a cDNA library. The second involved confirmation of putative clones by oligonucleotide probing. Probes were synthesised based on a back translation of asparaginase amino acid sequence data.

The cDNA library and antibody screening RNA from L. arboreus seeds 40 DAA was fractionated on an oligo-dT column. Poly(A) + RNA (15 #g) was used to synthesise cDNA which was cloned into 2gt 11. The original library, containing ca. 1 x 105 recombinants, was amplified and subsequently screened with antibodies against L. arboreus developing seed asparaginase. Ten plaques

out of 120000 screened gave positive signals. After three more rounds of screening at low density, four clones (BR1, 4, 6, 8) continued to yield a positive antibody reaction.

Confirmation of putative clones with an oligonucleotide probe Lambda DNA from BR1, 4, 6 and 8 was prepared but was found not to cleave with Eco RI. As a consequence a Kpn I/Sac I double digest was performed releasing a 2.08 kb vector fragment containing the 2gtl 1 Eco RI cloning site and cDNA insert. A 32mer oligonucleotide asparaginase probe (see Materials and methods) hybridised to the insert from clone BR4. In an alternative strategy a Pvu II digest released a 355 bp fragment containing the Eco RI cloning site and insert. Again the insert from BR4 hybridised with the probe. These restriction analyses revealed the presence of inserts 320, 950, 760 and 360 bp in length for clones BR1, 4, 6 and 8 respectively. It seemed that the original 2gtl 1 BR4 Eco RI cloning site was methylated. The site was subsequently demethylated however when subcloned into pUC and grown in Escherichia coli TG-1 as these plasmids were amenable to Eco RI digestion.

Sequencing cDNA clone BR4 The insert of )~gt11 clone BR4 was released by digestion with Pvu II which cleaves 347 bp from the cloning site into the left arm of 2gtl 1 and 8 bp into the right arm of ztgtl 1. The approx. 1320 bp fragment was subcloned into pUC9 producing pUCBR4. Subsequent Eco RI digestion of pUCBR4 grown in E. coli TG-1 released fragments of 300 and 950 bp. The smaller fragment proved by sequence analysis to be the 347 bp fragment of 2gtl 1 and the larger fragment to be the putative asparaginase insert. The cDNA was sequenced completely on both strands. The nucleotide sequence and translation of the longest open reading frame is shown in Fig. 1. The underlined sequence between nucleotides 833

394 G CCG CCG GAG CGC CGA AAG P P E R R K CAAATC Q I

GGC GTT G V

GAA CTT GTT GTT E L V V GGA TCT G S

CCT CGG GAA GAA GGA CTC P R E E G L

GAA GCT CTC AAA E A L K CGT GAG TTG R E L

GTG TTG ACG AAT AGT V L T N S

GAT GGG AAG AGT ATG AAA D G K S M K

GAG AAT AAT E N N GGA ACA G T

TGT GGT C G

TCA TTA GCA AGA S L A R

AAT N

CCA ATT P I

CTT L

GCT TTC CAA GGA GCT CAG GAT A F Q G A Q D

GTA GAT V D

TCA AGT S S

CAC H

GCA ATA A I

GAA GCT AAT AGG E A N R

CAG ATT Q I

GTT GGG V G

CCT CTC ATA P L I

GTG TCT V S

GCA ACA A T

GAT GTG D V

GCT GCA CTC ATG A A L M

GAT D

TAT GTT Y V

GTG TCT V $

GCT A

GGC AAA G K

G T T G T A i00 V V 33 151 50

GAA GCG TCG ATA ATG E A S I M

202 67

TTG AGT ACA L S T

GAA AAA ACT E K T

GAT TAT AGC D Y S

CCT CAT ATG P H M

GAA AGA E R

GAG ACT E T

355 118

CTG L

406 135

TAT ACC Y T

457 152

GAA TTA CCA GTT E L P V

GCA AAT A N

GGT G

508 169

GTT GAT AGT V D S

CAA GGA AAC Q G N

GGT G

GAA CTT E mL

GCT GGG ACT A G T

TAT GCC AAT Y A N

GGT GAA GCA ATA ATC G Em A I I

CAG GCG ACG Q A T

GGA CTT TCT G L S

CCG AAA P K

GCA ATG A M

304 101

TAT AAC Y N

GTT V

GCA GGA GAA ATT A m G E .I

CTG 253 L 84 TAT Y

CTA AAG L K

GGT GGA TTA GTG AAC AAA ATG G G a L V N K M

GTA CAT GAG CGT ACA V H E m mR T

GTT V

CAG Q

TGT GTG GCT C V A

GAG TTC AAA E F K

49 16

GCG GGG ATA A ~ I

GTT V

GGA ACT G .T

GGT GAC ACA G D T

GAC D

GCA GAA AAT A E N

AGT S

TCC ACT S T

TTG L

CAA CAA GGA GTT Q Q G V

GAT GCT GAG AAG D A E K

GCA ACA A T

TGC CTC C L

TTT GCT AAA F A K

CAA GAT Q D

GCT TCT A S

CAC H

GAA CAT TTC AAT E mH F N

GCT GTT TCT GGT A V S G

CAA CCT GTC Q P V CAA ATT Q I

TCT CCT S P

GTG GAAATG V E m M

CTT GTT ATG L V M

TTT ATT ACT F I T GTC V

GCG CGAAAG A R K

CGC R

GGC ACT G T

CTC AAG L K GTT V

C T A 559 L 186

GGT CGA ATC G R I TGT C

610 203

G C A 661 A 220

GTA GCA AGA V A R

712 237

GAA GCT GCT 763 E .A A 254

GGT TTG ATT G L I

CCT TTT AAC ACA ACA P F N T T

GAC D

G C T 814 A 271

GGC ATG G M

TTC F

865 288

TGG CCT W P

CCT P

916 305

W AGA R

GCA TGT A C

GCT ACT A T

GCC TAA AAT AAA A *

GAA GAT GGC AAT Em D G N

TTG AGT

GGT TCG AAA

TCA GAG ATT S E I GCT G

GCA ATT A I

947 306

Fig. 1. Nucleotide and deduced amino acid coding sequence of the L. arboreus asparaginase cDNA clone. The nucleotide and amino acid sequence corresponding to the longest open reading frame in the Eco RI fragment of pUCBR4 is presented. The putative terminator (TAA) is indicated (*). A possible polyadenylation signal (between nucleotides 923 and 928) is underlined. The underlined sequence between nucleotides 833 and 864 is the site of probe hybridisation. Six L. arboreus seed asparaginase peptide sequences from [21] are shown by underlining. Capital letters in the underlined peptide sequences were definite assignments; the lower-case 's' is a probable assignment and a gap indicates no assignment. The non-aligned peptides from [21] were: G G E S S I A L - G G A G D I P F S L and IAIPDNKSMD.

and 864 indicates the position where the 32mer oligonucleotide probe hybridised. Amino acid sequence data obtained from L. arboreus seed asparaginase by Lough et al. [21] were aligned with the predicted amino acid sequence and it was found that 6 of the 8 peptide sequences could be

aligned with the predicted amino acid sequence (Fig. 1). An analysis of the c D N A sequence of clone BR4 by the program C O D O N U S E (Stockwell, personal communication after Staden and McLachlan [37]) confirmed that the phase in which the peptide sequence data were aligned had

395 the highest likelihood of conforming to plant codon usage [13, 26].

Northern blot hybridisation The insert of plasmid pUCBR4 encoding asparaginase was labelled by random priming and used as a probe in a northern blot hybridisation (Fig. 2). Total RNA and poly(A) + R N A from developing L. arboreus seeds, roots and L. angustifolius nodules were fractionated by denaturing formaldehyde gel electrophoresis for northern blot hybridisation. Stringent hybridisation conditions were applied (see Materials and methods). Three stages of seed development were examined. The highest level of m R N A was observed in seeds 30 DAA, less in seeds 40 DAA and no asparaginase m R N A was detected in R N A from seeds 55 DAA. There was no apparent hybridisation to a root tip total or poly(A) + RNA sample nor to a nodule total RNA sample (Fig. 2). The m R N A species recognised by the probe was ca. 1.2 kb in length.

Genomic blot hybridisation The asparaginase c D N A insert from pUCBR4 was used to probe Southern blots of L. angustifolius genomic D N A digested with Barn HI, Eco RI, Hind Ill and Pst I (Fig. 3). Hybridisation was clearly obtained in each case to one fragment when L. angustifolius D N A was digested with Eco RI, Hind III and Pst I. There was also possibly one large fragment in the Barn HI digest (Fig. 3). This suggested that L. angustifolius seed asparaginase is probably a single copy gene.

Sequence homology with other asparaginases The plant asparaginase sequence reported here did not have any significant homology with any of the microbial asparaginase sequences (Bacillus subtilis (ansA), E. coli (ansB), Erwinia chrysanthemi, Saccharomyces cerevisiae (ASP3)) in the EMBL or GenBank databases. However it was found to have 23 ~o identity with a human glycosylasparaginase (EC 3.5.1.26) [ 11 ] (Fig. 4). The

Fig. 2. Northern analysis of asparaginase transcription in different L. arboreus tissues. A. Total RNA extracted from developing seeds, nodules and root tips was fractionated under denaturing conditions and ethidium bromide stained. B. The gel was blotted onto Hybond-N (Amersham), and the blot hybridised overnight with pUCBR4 asparaginase insert labelled by random priming, Washed mad exposed to X-ray film. Lane 1, seed 30 DAA total RNA (10/~g); lane 2, seed 40 DAA total RNA (10/zg); lane 3, seed 55 DAA total RNA (10/~g); lane 4, L. angustifolius nodule total RNA (10 #g); lane 5, root tip total RNA (10 #g); lane 6, seed 40 DAA poly(A) + RNA (2.5 gg); lane 7, root tip poly(A) + RNA (2.5/~g). The 23S and 16S rRNA bands in lane 4 are Rhizobium rRNA in the nodule total RNA.

396

Fig. 3. Southern analysis of L. arboreus DNA (20/~g/lane)

restriction fragments probed with pUCBR4 insert labelled by random priming. Lane 1, Barn HI; lane 2, Eco RI; lane 3, Hind III; lane 4, Pst I digests.

degree of similarity increases to 66~o if conservative substitutions are permitted (Fig. 4).

Discussion Asparaginase is a key enzyme involved in the utilisation of asparagine in amide transporting

ASN. GLASN. ASN. GLASN. ASN. GLASN. ASN. GLASN. ASN. GLASN. ASN. GLASN.

- .......................

plants [2, 5, 19, 34]. This study, to our knowledge, is the first report of the cloning of a plant asparaginase cDNA. We focussed on the cloning of the seed asparaginase ofL. arboreus. The findings presented indicate that 2gtl 1 clone BR4 is a cDNA clone for L. arboreus seed asparaginase. However, the clone appeared to be truncated at the 5' end since the sequence had one complete open reading frame and no initiating codon could be identified (Fig. 1). The clone BR4 has been used to probe genomic D N A o f t . angustifolius by Southern blot analysis. A single hybridising band was observed in D N A digested with Eco RI, Hind III and Pst I (Fig. 3). This indicated that L. angustifolius seed asparaginase is probably a single copy gene or alternatively that different members of a multigene family are highly conserved. The transcription of asparaginase mRNA in different L. arboreus tissues was examined using the clone BR4 as a probe. The data in Fig. 2 show hybridisation to a transcript of ca. 1.2 kb present in 30 DAA and 40 DAA seed mRNA. The highest level o f m R N A accumulation was observed in seeds 30 DAA, lower in seeds 40 DAA and none was detectable by 55 DAA. This was consistent with the evidence of Lough et al. [21] obtained by direct assay of seed asparaginase activity in pro-

PPERRKPREEGLRHCLQIGVEALKARKSPLDWELV .: . . . . . , . . :: .

36 :.::.::

MARKSNLPVLLVPFLLCQALVRCSSPLPLVVNTWPFKNATEAAWRALASGGSALDAVESG

60

VRELENNE-HFNAGIGSVLTNSGTVEMEASIMDGKSMKCGAVSGLSTVLNPISLARLVME : ...... :.:. .. :.,...: ::::..:. :::..:.

95 .

:.:.°::

:.:

CAMCEREQCDGSVGFGGSPDELGETTLDAMIMDGTTMDVGAVGDLRRIKNAIGVARKVLE KTPHMYLAFQGAQDFAKQQGVETVDSSHFITAE-NVERLKLAIEAN-RVQIDYSQYNYTQ .:.: :. ..: .::. : . : : ...... : .

120 153 ..:

.

..

..

.:

HTTHTLLVGESATTFAQSMGFINEDLSTSASQALHSDWLARNCQPNYWRNVIPDPSKYCG

180

PVQDDA--EKELPV---ANGDSQIGTVGCVAVDSQGNLASATSTGGLVNKMVGRIGDTPL : . . . . . .: . . . . :. .:.: : .... :,.:..:::.:.

208 :.

::.::.:.

PYKPPGILKQDIPIHKETEDDRGHDTIGMVVIHKTGHIAAGTSTNGIKFKIHGRVGDSPI

240

IGAGTYANELC-AVSATGKGEAIIQATVARDVAALMEFKGLSLKEAADYV--VHERTPKG

265

PGAGAYADDTAGAAAATGNGDILMRFLPSYQAVEYMI~RGEDPTIACQKVISRIQKHFPEF

300

TVGLIAVSAAGEIAMPFNTTGMF ..: . . . . :. . .

..... :.

.

:

RACATEDGNSEIAIWPPA ..... : ......

FGAViCANVTGSYGAACNKLSTFTQFSFMVYNSEKNQPTEEKVDCI

306 346

Fig. 4. Alignment of the amino acid sequences of L. arbomus seed asparaginase (ASN) and human placenta glycosylasparaginase

[11] (GLASN). Identities ~ e indicted by (:), and conservative substitutions [6] are indicated by (.). The computer program ALIGN [28] was used to gener~e the alignment.

397 tein extracts from seed tissue differing in age. There was no hybridisation to a root tip total R N A or poly(A) ÷ R N A sample. This was somewhat surprising as root tips are known to contain a potassium-dependent, sulphydryl-dependent asparaginase [5]. However, this result is supported by the immunological evidence of Lea et al. [18] who failed to observe cross-reacting asparaginase in mature roots, stems or leaves of L. polyphyllus with antisera raised against L. polyphyllus seed asparaginase. Similarly, Lough et al. [21] did not observe cross-reactivity between antiserum raised against L. arboreus seed asparaginase and protein extracts of developing flowers and root tips by Ouchterlony double immunodiffusion. The Mr of the truncated protein predicted from the translation of the nucleotide sequence of BR4 is 32775. The Mr of native L. arboreus asparaginase was determined to be 75 000 by gel filtration with reference to protein standards [5]. Lea et al. [19] have purified L. polyphyllus asparaginase protein. The Mr of the native enzyme was determined by sucrose density gradient centrifugation to be 72000. The purified protein was resolved into two near identical subunits with Mrs of ca. 38000 on a 12.5~o polyacrylamide gel. If this is true then the simplest hypothesis would be to assume that L. arboreus asparaginase is composed of two subunits of the same size. The analysis by northern blotting (Fig. 2) did not allow a distinction to be made between two transcripts of approximately the same size, a single hybridising transcript or non-homologous transcript of different size. The alternative of non-identical subunits is thought less likely as all but 2 of the 8 known peptide sequences [21] could be aligned with the truncated sequence of the cDNA clone BR4. These data do not explain the observation of three seed asparaginase isoforms [21]. Characterisation of more c D N A and genomic clones may reveal the molecular basis for the differences in these isoforms, but if the seed asparaginase is a single copy gene then these isoforms are probably variants produced post-translationally. When Chang and Farnden [5] purified L. ar-

boreus seed asparaginase, polypeptides with Mrs

of 14000 and 15500 were observed by SDSPAGE. This result was confirmed by Lough et al. [21] who observed asparaginase polypeptides with Mrs in the 14 000 to 18 000 range. In the light of the current evidence it would appear that asparaginase as extracted and purified by both Chang and Farnden [5] and Lough etal. [21] was cleaved into several polypeptides. Whether this is the result of post-translational modification, protease action during extraction or fragmentation during SD S-PAGE is unknown. However, the peptide alignment data (Fig. 1) revealed how these peptides could be derived from the original translation product. One peptide would be the N-terminal portion of the protein, the other the C-terminal and cleaved at residue 173 with a sequence beginning TVG (Fig. 1). Sieciechowicz et al. [33] found that the diurnal decrease in asparaginase activity at the end of the light period could be prevented by treatment with the protease inhibitor leupeptin. It would be interesting to purify L. arboreus seed asparaginase in the presence of appropriate levels of leupeptin to see if this prevented the cleavage of the protein during purification. The L. arboreus seed asparaginase protein sequence reported here did not have any significant homology with any of the microbial asparaginase sequences in the EMBL or GenBank data-bases. This was not surprising since microbial asparaginases are quite different to plant asparaginases in terms of Mr, substrate specificity and K m (see [5] for discussion). However, it was particularly interesting to find that the plant asparaginase was 23~o identical to a human placenta glycosylasparaginase [ 11] (Fig. 4). Glycosylasparaginases are lysosomal enzymes that cleave the Nacetylglucosamine-asparagine bond which joins oligosaccharides to the peptide of asparaginelinked glycoproteins. This enzyme requires a free 2-amino and carboxyl group on the asparagine [11] but does not hydrolyse asparagine [41]. Plant asparaginases are cytosolic [34] and although the substrate specificity of these enzymes has been examined [ 5, 19] it is not known whether they can hydrolyse a N-acetylglucosamine-aspar-

398 agine bond. Of further interest to the work discussed above is the finding that human glycosylasparaginase is encoded as a Mr 34600 polypeptide and post-translationally processed to generate two polypeptides of Mr 19500 and 150OO [11]. A genomic sequence encoding the L. a/zgustifolius seed asparaginase gene has been isolated using the clone BR4 as probe and sequenced [8]. This sequence has revealed that the 2gtl 1 L. arboreus asparaginase clone isolated and sequenced here was probably truncated in the coding region by 56 bp. The probable initiating codon has been identified from the genomic sequence [8] and the peptide GGESIAL that could not be aligned to the cDNA sequence (Fig. 1) is the N-terminal sequence of the protein. No signal sequence was found which is in agreement with the cytosolic location of plant asparaginase [ 34]. Currently the 5' regulatory sequences of the L. angustifolius seed asparaginase gene are being investigated to identify tissue-specific and developmental regulatory sequences.

Acknowledgements This work was supported by a University Grants Committee scholarship to T.J.L. We gratefully acknowledge Mr Andrew Shelling for the gift of packaging mix during 2gtl 1 library construction. We thank Miss Diana Davidson for the synthesis of oligonucleotide probes. We thank Mrs Gillian Hughes, Miss Laura Smith and Miss Karen Rodber for technical assistance at various stages of this work.

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