Planta (1983)159:445~53
Planta 9 Springer-Verlag 1983
Cloning and characterization of complementary DNA for convicilin, a major seed storage protein in Pisum sativum L. C. Domoney and R. Casey John Innes Institute, Colney Lane, Norwich NR4 7UH, UK
Abstract. A complementary DNA (cDNA) clone for convicilin, a major storage protein, has been isolated from a cDNA library prepared in the plasmid vector pAT 153, using poly(A)+RNA from developing seeds of Pisum sativurn L. The clone was identified by hybrid-selection with poly(A) + RNA, translation of selected RNAs and immunoprecipitation of the translation products by antibody raised against purified convicilin subunits. The size of the m R N A coding for convicilin polypeptides has been established using this convicilin cDNA clone and has been found to be appreciably longer than the mRNAs coding for the polypeptides of vicilin, a related but separate storage protein. The insert from this convicilin cDNA clone has been compared with the insert from a vicilin cDNA clone by restriction enzyme analysis. Key words: Complementary DNA cloning - Convicilin - Hybrid selection - Pisum (protein) - Polyadenylated RNA - Storage protein.
Introduction The storage proteins of pea (Pisum sativum L.) seeds have been divided into two major groups on the basis of solubility (Osborne and Campbell 1898) and sedimentation coefficient (Danielsson 1949): these are the legumin (11S) and the vicilin (7S) fractions which together may comprise 80% of the total seed protein. Legumin has been shown to be the simpler fraction of the two in being composed predominantly Abbreviations: cDNA=complementary DNA; IgG=immunoglobulin G; PAGE=polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate; Poly(A) +RNA = polyadenylated RNA
of only two classes of subunits of M r approx. 40,000 and 20,000. Heterogeneity and inheritance patterns have been well-documented for these subunits (Casey 1979a, b; Croy etal. 1979; Matta and Gatehouse 1982). The 7S fraction is more complex in being composed of many classes of subunits of M r ranging from approx. 70,000 to 12,000 (Thomson et al. 1978; Croy et al. 1980; Gatehouse et al. 1981). It has been demonstrated that a native protein containing only subunits of M r approx. 70,000 is separable from other 7S protein molecules under non-dissociating conditions (Casey and Sanger 1980; Croy et al. 1980) and this protein has been named convicilin (Croy et al. 1980). It has been shown that convicilin and vicilin (7S protein molecules containing subunits in the M r range 50,000 to 12,000, approx.) exhibit reactions of serological identity in Ouchterlony immunodiffusion tests (Croy et al. 1980). The amino-acid compositions of convicilin and vicilin appear to be quite similar (Casey and Sanger 1980), although the glutamic acid: aspartic acid ratio is higher for convicilin than for vicilin; the former protein also contains a small amount of the sulphur-containing amino acids in contrast to the latter which contains none (Croy et al. 1980). Genetic variants have been described for convicilin subunits and the inheritance of variant subunit forms has been shown to follow a simple Mendelian pattern (Casey and Sanger 1980; Matta and Gatehouse 1982). In addition, convicilin subunits have been shown by these authors to segregate independently from the major acidic subunits of legumin and the convicilin gene(s) has been mapped by genetic analysis to chromosome 2, close to the k locus (Matta and Gatehouse 1982). A lack of useful variation in major vicilin subunits has so far hindered their genetic analysis and, therefore, the physical relationship between the genes coding
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
for these two biologically related proteins within the genome remains to be elucidated. In this paper, we report the isolation and characterization of a cDNA clone for convicilin and present evidence that mRNA coding for convicilin subunits is present in a distinct mRNA size class which differs from that containing mRNAs coding for the major vicilin subunits.
447
Materials and methods
(w/v) SDS at 37~ for 20 h (Weber and Osborn 1975). The gel pieces were removed by centrifugation and re-eluted in a further 10 ml buffer. The eluates were combined and freezedried. The freeze-dried material from each gel was acetone-precipitated, dissolved in 1 ml 0.3 M NaC1, 0.02 M sodium phosphate, pH 7.0 and used to raise antibody in a rabbit, as previously described (Casey 1979a); three injections of approximately 300 ~tg were given at weekly intervals. Antisera to legumin and vicilin were raised in rabbits; IgG fractions were prepared and purified by affinity chromatography on columns of both legumin- and vicilin-Sepharose 4B, all as previously described (Casey 1979 a).
Materials. Radiochemicals were purchased from Amersham International plc, Amersham, Bucks., UK. Avian myeloblastosis virus (AMV) reverse transcriptase was from Dr. J.W. Beard, Life Sciences Incorp., St. Petersburg, Fla., USA. Restriction enzymes (HincII, TaqI, PstI) and $1 nuclease were obtained from Bethesda Research Laboratories, Cambridge, UK. The ribonuclease inhibitor from human placenta (RNasin) was from Biotec, Madison, Wis., USA. Proteinase K was purchased from The Boehringer Corp., Lewes, Sussex,UK. Restriction enzymes BstNI and RsaI were from New England BioLabs, CP Laboratories, Bishop's Stortford, Herts., UK. Sephadex and Protein A-Sepharose were purchased from Pharmacia (Great Britain), Hounslow, Middlesex, UK. Oligo dT and terminal transferase were products of P-L Biochemicals, Northampton, UK. BglII, HindIII and SalG restriction enzymes and the plasmid pBG6 were gifts from Mr. P.E. Dickerson and Dr. T.H.N. Ellis, respectively, of the John Innes Institute.
Preparation of RNA. Membrane-bound polysomes were prepared from seeds or cotyledons of P. sativum ev. Birte and from cotyledons of P. sativum BC1/1R and P. fulvum JI 224 Sp at selected stages of seed development, using a modification (Domoney 198l) of the method of Larkins and Davies (1975). Polysomes were digested with proteinase K; poly(A)+RNA was selected by two passages through a column of oligo dTcellulose and ethanol-precipitated. The RNA for cDNA synthesis was further purified by sizeselection on sucrose density gradients (10-40% (w/v) sucrose in 100 mM KC1, 10 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1, pHT.5; 18h at an average 110,000g, 2~ C). Gradients were fractionated and the RNA in each fraction identified by translation using messenger-dependent reticulocyte lysate as described below. Fractions which were enriched for convicilin mRNA were pooled, subjected to another cycle of oligo dT-cellulose chromatography and ethanol-precipitated.
Radioactive labelling of proteins by cotyledons in vivo. Cotyledons from seeds at the later stages of development (Domoney et al. 1980) were harvested from plants of Pisum sativum cv. Birte and placed on [14C]-labelled amino acids (Spencer et al. 1980). Extracts of slices from the labelled cotyledons were passed through columns of Sepharose coupled to anti-legumin immunoglobulinG (IgG) (Casey 1979a), Unbound and bound protein fractions were recovered by precipitation (Domoney 1981) and analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS) on 15% (w/v) gels (Laemmli 1970). Gels were stained, dried and autoradiographed.
Cell-free protein synthesis and immunoprecipitation. Polyadenylated RNA samples were translated in messenger-dependent rabbit reticuloeyte lysate (MDL; Pelham and Jackson 1976) using [3H]leucine as the label source. Incubations consisted of 18 gl MDL containing 3.77.104-9.43 910s Bq [3H]leucine, as appropriate, and up to 500 ng RNA in 2 gl water. Samples were incubated for 1 h at 30~ C and 2 lal removed for determination of the extent of incorporation into trichloroacetic-acid (TCA)-insoluble material. A portion of the incubation was diluted with an equal volume of twice-concentrated Laemmli (1970) sample buffer, boiled for 3 min and the total translation products were analysed by PAGE on 15% (w/v) gels (Laemmli 1970), followed by fluorography (Jen and Thach 1982). The remainder of the incubation was diluted with three volumes of 1 M NaC1, 1% (v/v) Triton X-100 and translation products reacted with specific antisera or IgG fractions, essentially as described by Martin and Northcote (1982). After a preincubation with 8 lal pre-immune serum (convicilin) or pre-immune IgG fraction (legumin and vicilin) plus protein A-Sepharose, the supernatant and washings (Martin and Northcote 1982) were incubated for 18 h at 4~ C with 8 gl antiserum (convicilin) or immune IgG (legumin and vicilin) plus protein A-Sepharose. After five washings, the pellet of protein A-Sepharose-IgG, translation products was dissociated in gel sample buffer and the radioactive proteins analysed by PAGE and fluorography.
Purification of convicilin and antibody preparation. A salt extract was prepared from cotyledons of cv. Birte which were harvested from seeds representing the later stages of seed development (Domoney et al. 1980). Legumin was removed from extracts by passage through anti-legumin IgG affinity columns, as previously described (Domoney et al. 1980). A protein fraction enriched in subunits of M r 70,000 was obtained from the unbound protein by zonal isoelectric precipitation. The polypeptides present in this protein preparation were separated by preparative electrophoresis on slab gels (140 minx 160 minx 0.8 ram). Gels were composed of 5% (w/v) acrylamide and 0.14% (w/v) bis-acrylamide in the continuous phosphate-buffer system described by Weber and Osborn (1969). Approximately 2 mg protein (1 mg ml 1 sample buffer) was applied to each gel and electrophoresis was at 50 mA for 20 h. Gels were fixed in 10% (v/v) ethanol, 5% (v/v) acetic acid for 2-3 rain or until a white precipitate became evident in the upper part of the gel. This band, which contained the convicilin subunits of M r 70,000, was excised immediately, cut into small cubes and placed in water for 15 rain. The gel pieces were removed and macerated by forcing through a syringe and protein was eluted by shaking the gel pieces in 20 ml 0.05 M NH4HCO3, 0.05%
Radioactive labelling of protein standards for PAGE. Catalase and bovine serum albumin (BSA) were carboxymethylated in 10 M urea, 0.1 M Tris-HC1, pH 8.6 using 3.77.107 Bq iodo-[23H]acetic acid (BSA) or 1.89.106 Bq iodo-[2-a4C]acetic acid (catalase), to specific activities of 2.1.10 e counts per minute (cpm) rag-1 and 6.4.105 cpm mg -1, respectively. The labelled proteins were dialysed into gel sample buffer and stored at 20~ C. The apparent M r values of the subunits of the labelled proteins were 60,700 (catalase) and 70,500 (BSA). -
448
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
Synthesis and cloning of eDNA. The DNA complementary to poly(A) +RNA was synthesized in a 100-p-1reaction volume containing 50 mM Tris-HC1, pH 8.3; 5 mM MgCl2; 80 mM KC1; 1 mM dithiothreitol (DTT); 10 p-g ml- ~ oligo dT; 500 p-M each deoxynucleotide triphosphate; 1.89.105 Bq [~ 32p] deoxycytidine 5'-triphosphate (dCTP); 500 units ml- ~ ribonuclease inhibitor (RNAsin); 80 p-g ml- i RNA (prepared from cv. Birte), and 10 units p-g-1 AMV reverse transcriptase. Following a 1-h incubation at 42~ C this reaction mix was boiled, after addition of potassium phosphate, pH 6.9 to give 110 mM final concentration, for second-strand synthesis. The eDNA was rendered double-stranded in a 200-p-1reaction volume containing 6.5 mM MgC12, 10 mM DTT, 250 p-M each deoxynucleotide triphosphate, 7.45.105 Bq [e-32P]dCTP, 20 units p-g-1 DNA polymerase 1 (Klenow fraction), and a portion of the first-strand reaction mix (approx. 3 gg single-stranded cDNA, as estimated from the proportion of radioactivity incorporated into DNA when an aliquot was chromatographed on Sephadex G50). Following a 4-h incubation at 15~ C, the reaction mix was extracted with phenol, fractionated by gel filtration on Sephadex G50 and double-stranded DNA (approx. 4 p-g) recovered from the DNA fraction by ethanol-precipitation. The eDNA was then treated with S1 nuclease (100 units p.g-1) in a reaction containing 0.3 M NaC1, 30 mM Na acetate, 45 mM ZnClz, pH 4.5. Following a 1-h incubation at 370 C, this reaction mix was layered on an ll-ml 5-29% (w/v) sucrose gradient in 50 mM Tris-HC1, pH 7.5, 0.25 M NaC1 and centrifuged at an average 75,000 g for 40 h at 4~ C. The DNA was recovered from the leading fractions by ethanol precipitation and the average length of DNA molecules present in different fractions assessed by agarose-gel electrophoresis. Pst I-digested pAT 153 and size-fractionated cDNA were tailed with deoxyguanosine (dG) and deoxycytidine (dC) residues, respectively (approx. 14 G's or C's per 3' end group) in reactions containing 140 mM cacodylate; 30 mM Tris-KOH, pH 7.6; 1 mM DTT; 2 mM dGTP or dCTP; 7.45.105 Bq dGTP or 3.77. i06 Bq dCTP; 1 mM CoC12 and terminal deoxynucleotidyl transferase (5 units pmol- 1 3' end group) (Roychoudhury et al. 1976). Following a 0.5-rain (plasmid) or 8-rain (eDNA) incubation at 37~ C, samples were extracted with phenol, fractionated by gel filtration as previously and equimolar proportions of plasmid and cDNA co-precipitated with ethanol. Annealing was in 10 mM Tris-HC1, pH 8; 100 mM NaC1; 10 mM ethylenediaminetetracetate (EDTA) (5 ng DNA p-1 1) ; at 65~ C for 4 min followed by slow cooling to room temperature over a 20-h period. Annealed DNAs were made to 0.1 M CaC1z before addition of 200 gl competent Escherichia coli HBI01 cells. Following a 1-h incubation on ice and 2 min at 42~ C, 5 ml L-broth (1% tryptone, 1% NaC1, 0.4% glucose and 0.5% Difco Bacto Yeast extract, all w/v) were added and the cells incubated at 37~ for 90 min. Transformants were selected on L-agar (L-broth containing 1.5% Bacto Agar; Difco, Detroit, Michigan, USA) containing tetracycline (10 p-g ml-1) and recombinants identified by failure to grow on L-agar containing ampicillin (100 p-g ml- 1). Characterization of plasrnids containing cDNA inserts. A total of 120 recombinant clones were obtained by transformation when the longest eDNA fraction (DNA of 1.3 kilobase pairs mean length) from sucrose gradients was used. Plasmid DNA was prepared from mini-cultures of these clones by a modification (A.O. Jackson, Department of Plant Pathology, Purdue University, West Lafayette, Ind., USA, personal communication) of the method of Davis et al. (1980). Digestion by Pst I of plasmid preparations followed by agarose-gel electrophoresis allowed the determination of insert size for each clone. Colony hybridizations were performed essentially as de-
scribed by Grunstein and Hogness (1975). Individual nitrocellulose fiters were screened by hybridization with poly(A)+RNA which had been hydrolyzed and labelled with T4 polynucleotide kinase and [?~-32p]ATP(Bedbrook et al. 1980). The RNA preparations used were from different genotypes and were enriched for one or more RNA species as judged from their translation products. Hybridization was in 3 x SSC (SSC is 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 4 x Denhardt's mixture (Denhardt 1966) at 65~ for 16 h. Filters were washed in 2xSSC, 0.1% (w/v) SDS at 65~ for 10h and exposed to X-ray film at - 7 0 ~ C. On the basis of insert size and hybridization intensity, a number of clones were selected for identification by hybridrelase translation and immunoprecipitation. Plasmid DNA, prepared from litre cultures of selected clones, was digested with Pst I and, following phenol extraction and ethanol precipitation, bound to diazobenzyloxymethyl (DBM) paper (10 p.g per 1 cm z disc), as described by Christophe et al. (1982). Hybridization of poly(A)+RNA to plasmid DNA bound to DBM paper was at 42~ for 18 h in 50% (v/v) formamide (deionized); 0.4 M NaC1; 20 mM 1,4-piperazinediethanesulfonic acid (Pipes), pH 6.4; 0.2% (w/v) SDS; 1 mM EDTA (200 gl per disc) containing 25-100 p-gml 1 RNA (prepared from cv. Birte). Papers were washed five times with 1 ml 50% (v/v) formamide; 0.15 M NaC1; 10raM Tris, pH 7.4; 1 mM EDTA; 0.1% (w/v) SDS; at 42~ C for 15 rain. Bound RNA was eluted with two successive 200-p-1 aliquots of 90% (v/v) formamide; 20mM Pipes, pH6.4; 1 mM EDTA; 15p-gm1-1 calf-liver tRNA; at 68~ C for 10 min. The eluates were diluted 1:1 with water and RNA recovered by ethanol-precipitation. Hybridselected RNAs were dissolved directly in 22 p-1MDL containing [3H]leucine (1.89.105 Bq dried) as label-source. Following a 1-h incubation at 30~ C, 3 p-1of the translation mix was retained for analysis of products of selected RNAs, while aliquots of the remainder were used for immunoprecipitationwith different antibody preparations. Total products of selected RNAs and immunoprecipitated products were analysed by PAGE and fluorography. The RNAs complementary to various clones were further characterized by hybridization of 'Northern' transfers to nicktranslated plasmid DNAs. One p-g poly(A)+RNA and 0.5 ng of each of the DNA standard mixtures (see below) were denatured in glyoxal-formamide (Covey et al. 1981) and fractionated by electrophoresis on agarose gels. Reference samples for the determination of RNA sizes consisted of a TaqI partial restriction digest of pAT 153 and a PstI-SalG digest ofpBG 6 (Goldsbrough et al. 1982). DNA markers and RNA were transferred to nitrocellulose in 20 x SSC (Thomas 1980), hybridized to nicktranslated plasmid and the filters washed as above. Apparent RNA sizes were estimated from the autoradiographs by reference to the DNA standards (McMaster and Carmichael 1977). The cDNA inserts were further characterized by restriction analysis using various restriction enzymes which either were prepared in the laboratory or were obtained commercially.
Results T a b l e 1 s h o w s the a m o u n t s o f c o n v i c i l i n , e s t i m a t e d as a p e r c e n t a g e o f t o t a l p r o t e i n , i n five d i f f e r e n t p e a g e n o t y p e s . A p a r t f r o m JI 224 Sp. (a P. f u l v u m line), t h e r e w a s l i m i t e d v a r i a t i o n i n the p e r c e n t a g e c o n v i c i l i n ( T a b l e 1). I n this s t u d y , c o n v i c i l i n w a s p u r i f i e d f r o m c o t y l e d o n s o f cv. Birte, w h i c h , although potentially yielding smaller amounts of c o n v i c i l i n p e r u n i t p r o t e i n t h a n JI 224 Sp., was
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
449
Table 1. The amounts of convicilin in five genotypes of pea, estimated by densitometry, as a percentage of the total Coomassie-Blue staining material when 25 I~g total protein were analyzed by PAGE. Each value (+SE) represents the mean of three determinations Genotype
JI 224 Sp cv. Dark skinned perfection cv. Birte BCI/IW BCI/1R
Convicilin
(%)
13.4_+0.6 8,3 + 0.4 6,2_+0.6 5.8_+0.4 5.9_+0.4
chosen since it had been the subject of earlier studies (Domoney et al. 1980; Domoney 1981). Stages of seed development are best defined in terms of the pattern of current protein synthesis and the cotyledons used in this study were defined by pulse-labelling with 14C-amino acids in vivo (Spencer et al. 1980). When extracts of these cotyledons were passed through anti-legumin IgG-Sepharose columns (Domoney 1981), unbound 14Clabelled material was shown by PAGE and autoradiography to consist almost exclusively of subunits of M r approx. 70,000 (Fig. I a). Stained gels of similar material (Fig. 1 b) revealed vicilin subunits in addition to convicilin. Analysis of bound ~4C-labelled material from these columns showed that legumin was also being actively synthesized by these cotyledons as evidenced by the presence of labelled precursor polypeptides of M r approx. 60,000. It has been previously shown that the bulk of convicilin and legumin synthesis occurs relatively late in seed development compared with the major period of vicilin synthesis (Millerd et al. 1978; Spencer etal. 1980; Croy etal. 1980). The lack of coincidence observed between the in-vivo ~4Clabelled and the stained bands of M r 70,000 is in agreement with the observations of others (Higgins and Spencer 1981) that post-translational modification of these polypeptides takes place (assuming that the polypeptides of M r 76,000/75,000 described by these authors are equivalent to those referred to as M r 70,000 in this study). These putative precursors (Fig. i a) must be relatively longlived, since pulse-labelling was carried out for a period of 2 h. The relative purity of convicilin prepared by elution from Weber-Osborn gels (Weber and Osborn 1969) as assessed by PAGE (Laemmli 1970) is shown in Fig. 2. A sample of the vicilin-convicilin preparation used for preparative gel electrophoresis is shown in Fig. 2a. It can be seen that the subunits of M r approx. 70,000 purified by this
Fig. 1. An analysis by PAGE and autoradiography of unbound protein from anti-legumin IgG columns of extracts from cv. Birte cotyledons 14C-labelled in vivo. a, autoradiograph of the gel track shown in b, b, stain: the stained polypeptide in the region M r 70,000 did not correspond with either of the polypep~ tides in the same region of the autoradiograph
method were free from all other subunits (Fig. 2 b); it was estimated that approx. 20% of the protein applied to each gel was recovered as convicilin. Antiserum raised against purified convicilin subunits was used to identify convicilin polypeptides among the translation products of poly(A)§ When total translation products (Fig. 3 a) were challenged with anti-convicilin, polypeptides of Mr approx. 70,000, 50,000 and 47,000 were selectively immunoprecipitated (Fig. 3 b), in agreement with the serological cross-reactivity of convicilin and vicilin previously reported (Croy et al. 1980). The cell-free translation products immunoprecipitated by anti-vicilin are shown in Fig. 3 c; longer film exposures of such immunoprecipitates would also show polypeptides of M r approx. 70,000. A direct comparison of the proportion of cross-reactivity exhibited by each antibody preparation is not possible, however, since antibody was raised against the whole protein in one instance (vicilin) and against denatured subunits in the other (convicilin). It might be expected that, in the latter case, affinity for primary translation products would have been greater.
450
C. Domoney and R. Casey : Complementary DNA clone for Pisum convicilin
Fig. 2. An analysis by PAGE of a, the convicilin-enrichedprotein fraction obtained by zonal isoelectric precipitation column chromatography of unbound protein from anti-legumin IgG affinity columns; and b, convicilin subunits purified by elution from Weber-Osborn gels (Weber and Osborn 1969), when the subunits shown in a were separated by electrophoresis on 5% Weber-Osborn gels
Fig. 3. An analysis by PAGE and fluorography of a, the total products synthesized in a rabbit reticulocyte cell-free protein synthesizing system by poly(A) § RNA purified from developing seeds of cv. Birte; b, the products from those in a which were immunoprecipitated by anti-convicilin; and c, the products from those in a which were immunoprecipitated by anti-vicilin
Fig. 4. An analysis by PAGE and fluorography of: the total products of RNA selected by four different plasmids (a, d, g, j); the products of selected RNA which were immunoprecipitated by anti-legumin (b, e, h, k); the products of selected RNA which were immunoprecipitated by anti-vicilin (c, f, i,/); the products of RNA selected by pCD 59 which were immunoprecipitated by anti-convicilin(rn); and the products of the poly(A) +RNA preparation used for cDNA synthesis (n)
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
Fig. 5. A 'Northern' blot analysis of poly(A)+RNA. Filters were hybridized with pCD 4 (a), pCD 48 (b), pCD 59 (c) or pAT 153 (at). Numbers refer to the nucleotide lengths of the markers shown in (e) 0 I
:
0.5 i
,
HTP -,
,
,
I
1 ,
,
~
,
~
R CBN ,
9
BT , ,
,
PCBN ,i .
C ,,
though the two vicilin eDNA clones (pCD 4, pCD48) hybridize to RNAs of similar size (Fig. 5a, b), the convicilin cDNA clone (pCD 59) was found to hybridize to R N A which was appreciably longer than that hybridizing to the other two plasmids (Fig. 5c). The estimated sizes of the two m R N A classes were 1,725 _+35 nucleotides (vicilin mRNAs) and 2,210 _+30 nucleotides (convicilin mRNA). Analysis of mixtures of markers and RNA samples showed that there was no significant error arising from the differences in mass of the R N A samples compared with the markers. Treatment of the RNA samples with deoxyribonuclease did not affect the hybridization patterns. Figure 6 shows a comparison of the restriction maps obtained for the cDNA inserts from pCD 48 and pCD 59 (inserts of 1200 and 650 base pairs, representing 70% and 30% of their corresponding mRNAs, respectively). Out of sixteen enzymes tested, restriction sites for five (BglII, BstNI, HincII, PstI, TaqI) were present in the inserts from both pCD 48 and pCD 59; sites for an additional two enzymes (HindIII, RsaI) were present in pCD 48 only. No major similarity in the arrangement of common sites within the two eDNA inserts was apparent (Fig. 6).
Kb
Discussion
C C
,,,
451
,
,
p C D 4 8
p C D 5 9
Fig. 6. A restriction enzyme analysis of the eDNA inserts from pCD 48 and pCD 59. B=BglII, C=HincII, H = H i n d I I I , N = BstNI, P = PstI, R = RsaI, T : TaqI
Figure 4 shows the identification by hybrid-selection and immunoprecipitation of cDNA clones for the major storage protein polypeptides evident among total translation products (Fig. 3 a). In the case of each cDNA clone, a positive and clearcut identification of the products of selected RNAs was possible using specific antibodies. Legumin (pCD 40), vicilin (pCD 4, pCD 48) and convicilin (pCD 59) clones were identified in this manner (Fig. 4). The polypeptides of M r 70,000 synthesized by R N A selected by pCD 59 were immunoprecipitated by both anti-convicilin and anti-vicilin; however, a higher proportion of the radioactivity present in total products was consistently immunoprecipitated by anti-convicilin when compared with anti-vicilin. Figure 5 shows a 'Northern' blot analysis of the hybridization of size-fractionated poly(A) + RNA to pCD 4, pCD 48 and pCD 59. AI-
The results presented describe the isolation and identification, using antibody raised against convicilin, of a convicilin cDNA clone, pCD 59. Chandler (1982) has reported the isolation of two cDNA clones which hybridize separately to mRNAs coding for polypeptides of M r 70,000 and 76,000. These polypeptides, however, have not been identified by immunoprecipitation (Chandler 1982), but it is reported (Chandler 1982) that the polypeptide of M r 76,000 is a precursor to one of the pea storage globulins; it is probably equivalent to the polypeptide of M r 76,000 reported by Higgins and Spencer (1981), previously assumed to be convicilin (see Results section). On this basis, it may be assumed that the cDNA clone for the polypeptide of M r 70,000 observed by Chandler (1982) is not equivalent to the cDNA clone described by us. Chrispeels et al. (1982) have previously observed a minor vicilin polypeptide of M r 70,000 which they report to be processed to give quantitatively minor products, mainly of Mr 50,000. This discrepancy in molecular weight estimations is unfortunate, but both we (Casey and Sanger 1980), and Croy et al. (1980) have reported a major component of 7S protein (convicilin) to have subunits of M r approx. 70,000.
452
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
Under the conditions employed for hybrid-selection in this study, the RNA selected by pCD 59 coded only for convicilin polypeptides and there was no evidence for selection of RNAs coding for either of the two vicilin polypeptides (Fig. 4j). Conversely, there was no evidence for selection of RNA coding for convicilin by either pCD 4 or pCD 48 (Fig. 4d, g). This is hardly surprising since, under these conditions, no cross-selection of the two RNAs coding for vicilin polypeptides takes place with either pCD 4 or pCD 48 (Fig. 4d, g), despite the extensive sequence homology reported by Gatehouse et al. (1982) for two corresponding cDNAs. In addition, there was no evidence from 'Northern' blot analyses of any hybridization of pCD 59 to RNA in the size class to which pCD 4 and pCD 48 hybridized (Fig. 5); the converse was also true in these analyses. Although there was no evidence of relatedness of the two proteins, convicilin and vicilin, from the RNA hybridization analyses in this study, it might be expected from the serological cross-reactivity that they share some sequence homology and that these sequences may be responsible for common antigenic determinants on the outer surfaces of the molecules. The degree of sequence homology might not be expected to be great, based on previous observations of dissimilarities in the two proteins (see Introduction section). The isolation and identification of cDNA clones for these two proteins, as reported here, will facilitate DNA sequence comparisons and the possible divergent or convergent evolution of the two proteins might be delineated. We are grateful to Agrigenetics Research Corporation, Golden, Colo., USA, for financial support. We thank Dr. C.L. Hedley for providing us with seeds of genotype BC1/1R, Dr. T.H.N. Ellis for invaluable advice and Wendy Cleary for excellent technical assistance.
References Bedbrook, J.R., Smith, S.M., Ellis, R.J. (1980) Molecular cloning and sequencing of cDNA encoding the precursor to the small subunit of chloroplast ribulose-l,5-bisphosphate earboxylase. Nature (London) 287, 692-697 Casey, R. (1979 a) Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds. Biochem. J. 177, 509-520 Casey, R. (1979b) Genetic variability in the structure of the a-subunits of legumin from Pisum - a two-dimensional gel electrophoresis study. Heredity 43, 265-272 Casey, R., Sanger, E. (1980) Purification and some properties of a 7S seed storage protein from Pisum (pea). Biochem. Soc. Trans. 8, 658 Chandler, P.M. (1982) The use of single-stranded phage DNAs
in hybrid arrest and release translation. Anal. Bioehem. 127, 9-16 Chrispeels, M.J., Higgins, T.J.V., Spencer, D. (1982) Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons. J. Cell Biol. 93, 306-313 Christophe, D., Brocas, H., Vassart, G. (1982) Improved synthesis of DBM paper. Anal. Biochem. 120, 259-261 Covey, S.N., Lomonossoff, G.P., Hull, R. (1981) Characterisation of cauliflower mosaic virus DNA sequences which encode major polyadenylated transcripts. Nucl. Acids Res. 9, 6735-6747 Croy, R.R.D., Derbyshire, E., Krishna, T.G., Boulter, D. (1979) Legumin of Pisum sativum and Vieiafaba. New Phytol. 83, 29-35 Croy, R.R.D., Gatehouse, J.A., Tyler, M., Boulter, D, (1980) The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.). Biochem. J. 191, 509-516 Danielsson, C.E. (1949) Seed globulins of the Graminae and Leguminosae. Biochem. J. 44, 387-400 Davis, R.W., Thomas, M., Cameron, J., St. John, T.P., Scherer, S., Padgett, R.A. (1980) Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 65, 404-411 Denhardt, D.T. (1966) A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646 Domoney, C., Davies, D.R., Casey, R. (1980) The initiation of legumin synthesis in immature embryos of Pisum sativum L. grown in vivo and in vitro. Planta 149, 454-460 Domoney, C. (1981) Legumin synthesis and accumulation in Pisum sativum L. Ph.D. thesis, University of East Anglia, Norwich, UK Gatehouse, J.A., Croy, R.R.D., Morton, H., Tyler, M., Boulter, D. (1981) Characterisation and subunit structures of the vicilin storage proteins of pea (Pisum sativum L.). Eur. J. Biochem. 118, 627-633 Gatehouse, J.A., Evans, I.M., Brown, D., Croy, R.R.D., Boulter, D. (1982) Control of storage-protein synthesis during seed development in pea (Pisum sativum L.). Biochem. J. 208, 119-127 Goldsbrough, P.B., Ellis, T.H.N., Lomonossoff, G.P. (1982) Sequence variation and methylation of the flax 5S RNA genes. Nucl. Acids Res. 10, 4501-4514 Grunstein, M., Hogness, D.S. (1975) Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72, 3961-3965 Higgins, T.J.V., Spencer, D. (1981) Precursor forms of pea vicilin subunits. Plant Physiol. 67, 205-211 Jen, G., Thach, R.E. (1982) Inhibition of host translation in encephalomyocarditis virus-infected L cells: a novel mechanism. J. Virol. 43, 250-261 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685 Larkins, B.A., Davies, E. (1975) Polyribosomes from peas. V. An attempt to characterize the total free and membranebound polysomal population. Plant Physiol. 55, 749-756 Martin, C., Northcote, D.H. (1982) The action of exogenous gibberellic acid on isocitrate lyase-mRNA in germinating castor bean seeds. Planta 154, 174-183 Matta, N.K., Gatehouse, J.A. (1982) Inheritance and mapping of storage protein genes in Pisum sativum L. Heredity 48, 383-392 McMaster, G.K., Carmichael, G.G. (1977) Analysis of singleand double-stranded nucleic acids on polyacrylamide and
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74, 4835-4838 Millerd, A., Thomson, J.A., Schroeder, H.E. (1978) Cotyledonary storage proteins in Pisum sativum. III. Patterns of accumulation during development. Aust. J. Plant Physiol. 5, 519-534 Osborne, T.B., Campbell, G.F. (1898) Proteids of the pea. J. Am. Chem. Soc. 20, 348-362, 36~375, 393-405 Pelham, H.R.B., Jackson, R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256 Roychoudhury, R., Jay, E., Wu, R. (1976) Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucl. Acids Res. 3, 101 116 Spencer, D., Higgins, T.J.V., Button, S.C., Davey, R.A. (1980) Pulse-labeling studies on protein synthesis in developing pea seeds and evidence of a precursor form of legumin small subunit. Plant Physiol. 66, 510-515
453
Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201-5205 Thomson, J.A., Schroeder, H.E., Dudman, W.F. (1978) Cotyledonary storage proteins in Pisum sativum. I. Molecular heterogeneity. Aust. J. Plant Physiol. 5, 263-279 Weber, K., Osborn, M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406M412 Weber, K., Osborn, M. (1975) Proteins and sodium dodecyl sulfate: molecular weight determination on polyacrylamide gels and related procedures. In: The proteins, vol. 1, edn. 3, pp. 179-223, Neurath, H., Hill, R.L., eds. Academic Press, New York
Received 30 May; accepted 5 July 1983
Planta 9 Springer-Verlag 1983
Cloning and characterization of complementary DNA for convicilin, a major seed storage protein in Pisum sativum L. C. Domoney and R. Casey John Innes Institute, Colney Lane, Norwich NR4 7UH, UK
Abstract. A complementary DNA (cDNA) clone for convicilin, a major storage protein, has been isolated from a cDNA library prepared in the plasmid vector pAT 153, using poly(A)+RNA from developing seeds of Pisum sativurn L. The clone was identified by hybrid-selection with poly(A) + RNA, translation of selected RNAs and immunoprecipitation of the translation products by antibody raised against purified convicilin subunits. The size of the m R N A coding for convicilin polypeptides has been established using this convicilin cDNA clone and has been found to be appreciably longer than the mRNAs coding for the polypeptides of vicilin, a related but separate storage protein. The insert from this convicilin cDNA clone has been compared with the insert from a vicilin cDNA clone by restriction enzyme analysis. Key words: Complementary DNA cloning - Convicilin - Hybrid selection - Pisum (protein) - Polyadenylated RNA - Storage protein.
Introduction The storage proteins of pea (Pisum sativum L.) seeds have been divided into two major groups on the basis of solubility (Osborne and Campbell 1898) and sedimentation coefficient (Danielsson 1949): these are the legumin (11S) and the vicilin (7S) fractions which together may comprise 80% of the total seed protein. Legumin has been shown to be the simpler fraction of the two in being composed predominantly Abbreviations: cDNA=complementary DNA; IgG=immunoglobulin G; PAGE=polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate; Poly(A) +RNA = polyadenylated RNA
of only two classes of subunits of M r approx. 40,000 and 20,000. Heterogeneity and inheritance patterns have been well-documented for these subunits (Casey 1979a, b; Croy etal. 1979; Matta and Gatehouse 1982). The 7S fraction is more complex in being composed of many classes of subunits of M r ranging from approx. 70,000 to 12,000 (Thomson et al. 1978; Croy et al. 1980; Gatehouse et al. 1981). It has been demonstrated that a native protein containing only subunits of M r approx. 70,000 is separable from other 7S protein molecules under non-dissociating conditions (Casey and Sanger 1980; Croy et al. 1980) and this protein has been named convicilin (Croy et al. 1980). It has been shown that convicilin and vicilin (7S protein molecules containing subunits in the M r range 50,000 to 12,000, approx.) exhibit reactions of serological identity in Ouchterlony immunodiffusion tests (Croy et al. 1980). The amino-acid compositions of convicilin and vicilin appear to be quite similar (Casey and Sanger 1980), although the glutamic acid: aspartic acid ratio is higher for convicilin than for vicilin; the former protein also contains a small amount of the sulphur-containing amino acids in contrast to the latter which contains none (Croy et al. 1980). Genetic variants have been described for convicilin subunits and the inheritance of variant subunit forms has been shown to follow a simple Mendelian pattern (Casey and Sanger 1980; Matta and Gatehouse 1982). In addition, convicilin subunits have been shown by these authors to segregate independently from the major acidic subunits of legumin and the convicilin gene(s) has been mapped by genetic analysis to chromosome 2, close to the k locus (Matta and Gatehouse 1982). A lack of useful variation in major vicilin subunits has so far hindered their genetic analysis and, therefore, the physical relationship between the genes coding
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
for these two biologically related proteins within the genome remains to be elucidated. In this paper, we report the isolation and characterization of a cDNA clone for convicilin and present evidence that mRNA coding for convicilin subunits is present in a distinct mRNA size class which differs from that containing mRNAs coding for the major vicilin subunits.
447
Materials and methods
(w/v) SDS at 37~ for 20 h (Weber and Osborn 1975). The gel pieces were removed by centrifugation and re-eluted in a further 10 ml buffer. The eluates were combined and freezedried. The freeze-dried material from each gel was acetone-precipitated, dissolved in 1 ml 0.3 M NaC1, 0.02 M sodium phosphate, pH 7.0 and used to raise antibody in a rabbit, as previously described (Casey 1979a); three injections of approximately 300 ~tg were given at weekly intervals. Antisera to legumin and vicilin were raised in rabbits; IgG fractions were prepared and purified by affinity chromatography on columns of both legumin- and vicilin-Sepharose 4B, all as previously described (Casey 1979 a).
Materials. Radiochemicals were purchased from Amersham International plc, Amersham, Bucks., UK. Avian myeloblastosis virus (AMV) reverse transcriptase was from Dr. J.W. Beard, Life Sciences Incorp., St. Petersburg, Fla., USA. Restriction enzymes (HincII, TaqI, PstI) and $1 nuclease were obtained from Bethesda Research Laboratories, Cambridge, UK. The ribonuclease inhibitor from human placenta (RNasin) was from Biotec, Madison, Wis., USA. Proteinase K was purchased from The Boehringer Corp., Lewes, Sussex,UK. Restriction enzymes BstNI and RsaI were from New England BioLabs, CP Laboratories, Bishop's Stortford, Herts., UK. Sephadex and Protein A-Sepharose were purchased from Pharmacia (Great Britain), Hounslow, Middlesex, UK. Oligo dT and terminal transferase were products of P-L Biochemicals, Northampton, UK. BglII, HindIII and SalG restriction enzymes and the plasmid pBG6 were gifts from Mr. P.E. Dickerson and Dr. T.H.N. Ellis, respectively, of the John Innes Institute.
Preparation of RNA. Membrane-bound polysomes were prepared from seeds or cotyledons of P. sativum ev. Birte and from cotyledons of P. sativum BC1/1R and P. fulvum JI 224 Sp at selected stages of seed development, using a modification (Domoney 198l) of the method of Larkins and Davies (1975). Polysomes were digested with proteinase K; poly(A)+RNA was selected by two passages through a column of oligo dTcellulose and ethanol-precipitated. The RNA for cDNA synthesis was further purified by sizeselection on sucrose density gradients (10-40% (w/v) sucrose in 100 mM KC1, 10 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1, pHT.5; 18h at an average 110,000g, 2~ C). Gradients were fractionated and the RNA in each fraction identified by translation using messenger-dependent reticulocyte lysate as described below. Fractions which were enriched for convicilin mRNA were pooled, subjected to another cycle of oligo dT-cellulose chromatography and ethanol-precipitated.
Radioactive labelling of proteins by cotyledons in vivo. Cotyledons from seeds at the later stages of development (Domoney et al. 1980) were harvested from plants of Pisum sativum cv. Birte and placed on [14C]-labelled amino acids (Spencer et al. 1980). Extracts of slices from the labelled cotyledons were passed through columns of Sepharose coupled to anti-legumin immunoglobulinG (IgG) (Casey 1979a), Unbound and bound protein fractions were recovered by precipitation (Domoney 1981) and analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS) on 15% (w/v) gels (Laemmli 1970). Gels were stained, dried and autoradiographed.
Cell-free protein synthesis and immunoprecipitation. Polyadenylated RNA samples were translated in messenger-dependent rabbit reticuloeyte lysate (MDL; Pelham and Jackson 1976) using [3H]leucine as the label source. Incubations consisted of 18 gl MDL containing 3.77.104-9.43 910s Bq [3H]leucine, as appropriate, and up to 500 ng RNA in 2 gl water. Samples were incubated for 1 h at 30~ C and 2 lal removed for determination of the extent of incorporation into trichloroacetic-acid (TCA)-insoluble material. A portion of the incubation was diluted with an equal volume of twice-concentrated Laemmli (1970) sample buffer, boiled for 3 min and the total translation products were analysed by PAGE on 15% (w/v) gels (Laemmli 1970), followed by fluorography (Jen and Thach 1982). The remainder of the incubation was diluted with three volumes of 1 M NaC1, 1% (v/v) Triton X-100 and translation products reacted with specific antisera or IgG fractions, essentially as described by Martin and Northcote (1982). After a preincubation with 8 lal pre-immune serum (convicilin) or pre-immune IgG fraction (legumin and vicilin) plus protein A-Sepharose, the supernatant and washings (Martin and Northcote 1982) were incubated for 18 h at 4~ C with 8 gl antiserum (convicilin) or immune IgG (legumin and vicilin) plus protein A-Sepharose. After five washings, the pellet of protein A-Sepharose-IgG, translation products was dissociated in gel sample buffer and the radioactive proteins analysed by PAGE and fluorography.
Purification of convicilin and antibody preparation. A salt extract was prepared from cotyledons of cv. Birte which were harvested from seeds representing the later stages of seed development (Domoney et al. 1980). Legumin was removed from extracts by passage through anti-legumin IgG affinity columns, as previously described (Domoney et al. 1980). A protein fraction enriched in subunits of M r 70,000 was obtained from the unbound protein by zonal isoelectric precipitation. The polypeptides present in this protein preparation were separated by preparative electrophoresis on slab gels (140 minx 160 minx 0.8 ram). Gels were composed of 5% (w/v) acrylamide and 0.14% (w/v) bis-acrylamide in the continuous phosphate-buffer system described by Weber and Osborn (1969). Approximately 2 mg protein (1 mg ml 1 sample buffer) was applied to each gel and electrophoresis was at 50 mA for 20 h. Gels were fixed in 10% (v/v) ethanol, 5% (v/v) acetic acid for 2-3 rain or until a white precipitate became evident in the upper part of the gel. This band, which contained the convicilin subunits of M r 70,000, was excised immediately, cut into small cubes and placed in water for 15 rain. The gel pieces were removed and macerated by forcing through a syringe and protein was eluted by shaking the gel pieces in 20 ml 0.05 M NH4HCO3, 0.05%
Radioactive labelling of protein standards for PAGE. Catalase and bovine serum albumin (BSA) were carboxymethylated in 10 M urea, 0.1 M Tris-HC1, pH 8.6 using 3.77.107 Bq iodo-[23H]acetic acid (BSA) or 1.89.106 Bq iodo-[2-a4C]acetic acid (catalase), to specific activities of 2.1.10 e counts per minute (cpm) rag-1 and 6.4.105 cpm mg -1, respectively. The labelled proteins were dialysed into gel sample buffer and stored at 20~ C. The apparent M r values of the subunits of the labelled proteins were 60,700 (catalase) and 70,500 (BSA). -
448
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
Synthesis and cloning of eDNA. The DNA complementary to poly(A) +RNA was synthesized in a 100-p-1reaction volume containing 50 mM Tris-HC1, pH 8.3; 5 mM MgCl2; 80 mM KC1; 1 mM dithiothreitol (DTT); 10 p-g ml- ~ oligo dT; 500 p-M each deoxynucleotide triphosphate; 1.89.105 Bq [~ 32p] deoxycytidine 5'-triphosphate (dCTP); 500 units ml- ~ ribonuclease inhibitor (RNAsin); 80 p-g ml- i RNA (prepared from cv. Birte), and 10 units p-g-1 AMV reverse transcriptase. Following a 1-h incubation at 42~ C this reaction mix was boiled, after addition of potassium phosphate, pH 6.9 to give 110 mM final concentration, for second-strand synthesis. The eDNA was rendered double-stranded in a 200-p-1reaction volume containing 6.5 mM MgC12, 10 mM DTT, 250 p-M each deoxynucleotide triphosphate, 7.45.105 Bq [e-32P]dCTP, 20 units p-g-1 DNA polymerase 1 (Klenow fraction), and a portion of the first-strand reaction mix (approx. 3 gg single-stranded cDNA, as estimated from the proportion of radioactivity incorporated into DNA when an aliquot was chromatographed on Sephadex G50). Following a 4-h incubation at 15~ C, the reaction mix was extracted with phenol, fractionated by gel filtration on Sephadex G50 and double-stranded DNA (approx. 4 p-g) recovered from the DNA fraction by ethanol-precipitation. The eDNA was then treated with S1 nuclease (100 units p.g-1) in a reaction containing 0.3 M NaC1, 30 mM Na acetate, 45 mM ZnClz, pH 4.5. Following a 1-h incubation at 370 C, this reaction mix was layered on an ll-ml 5-29% (w/v) sucrose gradient in 50 mM Tris-HC1, pH 7.5, 0.25 M NaC1 and centrifuged at an average 75,000 g for 40 h at 4~ C. The DNA was recovered from the leading fractions by ethanol precipitation and the average length of DNA molecules present in different fractions assessed by agarose-gel electrophoresis. Pst I-digested pAT 153 and size-fractionated cDNA were tailed with deoxyguanosine (dG) and deoxycytidine (dC) residues, respectively (approx. 14 G's or C's per 3' end group) in reactions containing 140 mM cacodylate; 30 mM Tris-KOH, pH 7.6; 1 mM DTT; 2 mM dGTP or dCTP; 7.45.105 Bq dGTP or 3.77. i06 Bq dCTP; 1 mM CoC12 and terminal deoxynucleotidyl transferase (5 units pmol- 1 3' end group) (Roychoudhury et al. 1976). Following a 0.5-rain (plasmid) or 8-rain (eDNA) incubation at 37~ C, samples were extracted with phenol, fractionated by gel filtration as previously and equimolar proportions of plasmid and cDNA co-precipitated with ethanol. Annealing was in 10 mM Tris-HC1, pH 8; 100 mM NaC1; 10 mM ethylenediaminetetracetate (EDTA) (5 ng DNA p-1 1) ; at 65~ C for 4 min followed by slow cooling to room temperature over a 20-h period. Annealed DNAs were made to 0.1 M CaC1z before addition of 200 gl competent Escherichia coli HBI01 cells. Following a 1-h incubation on ice and 2 min at 42~ C, 5 ml L-broth (1% tryptone, 1% NaC1, 0.4% glucose and 0.5% Difco Bacto Yeast extract, all w/v) were added and the cells incubated at 37~ for 90 min. Transformants were selected on L-agar (L-broth containing 1.5% Bacto Agar; Difco, Detroit, Michigan, USA) containing tetracycline (10 p-g ml-1) and recombinants identified by failure to grow on L-agar containing ampicillin (100 p-g ml- 1). Characterization of plasrnids containing cDNA inserts. A total of 120 recombinant clones were obtained by transformation when the longest eDNA fraction (DNA of 1.3 kilobase pairs mean length) from sucrose gradients was used. Plasmid DNA was prepared from mini-cultures of these clones by a modification (A.O. Jackson, Department of Plant Pathology, Purdue University, West Lafayette, Ind., USA, personal communication) of the method of Davis et al. (1980). Digestion by Pst I of plasmid preparations followed by agarose-gel electrophoresis allowed the determination of insert size for each clone. Colony hybridizations were performed essentially as de-
scribed by Grunstein and Hogness (1975). Individual nitrocellulose fiters were screened by hybridization with poly(A)+RNA which had been hydrolyzed and labelled with T4 polynucleotide kinase and [?~-32p]ATP(Bedbrook et al. 1980). The RNA preparations used were from different genotypes and were enriched for one or more RNA species as judged from their translation products. Hybridization was in 3 x SSC (SSC is 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 4 x Denhardt's mixture (Denhardt 1966) at 65~ for 16 h. Filters were washed in 2xSSC, 0.1% (w/v) SDS at 65~ for 10h and exposed to X-ray film at - 7 0 ~ C. On the basis of insert size and hybridization intensity, a number of clones were selected for identification by hybridrelase translation and immunoprecipitation. Plasmid DNA, prepared from litre cultures of selected clones, was digested with Pst I and, following phenol extraction and ethanol precipitation, bound to diazobenzyloxymethyl (DBM) paper (10 p.g per 1 cm z disc), as described by Christophe et al. (1982). Hybridization of poly(A)+RNA to plasmid DNA bound to DBM paper was at 42~ for 18 h in 50% (v/v) formamide (deionized); 0.4 M NaC1; 20 mM 1,4-piperazinediethanesulfonic acid (Pipes), pH 6.4; 0.2% (w/v) SDS; 1 mM EDTA (200 gl per disc) containing 25-100 p-gml 1 RNA (prepared from cv. Birte). Papers were washed five times with 1 ml 50% (v/v) formamide; 0.15 M NaC1; 10raM Tris, pH 7.4; 1 mM EDTA; 0.1% (w/v) SDS; at 42~ C for 15 rain. Bound RNA was eluted with two successive 200-p-1 aliquots of 90% (v/v) formamide; 20mM Pipes, pH6.4; 1 mM EDTA; 15p-gm1-1 calf-liver tRNA; at 68~ C for 10 min. The eluates were diluted 1:1 with water and RNA recovered by ethanol-precipitation. Hybridselected RNAs were dissolved directly in 22 p-1MDL containing [3H]leucine (1.89.105 Bq dried) as label-source. Following a 1-h incubation at 30~ C, 3 p-1of the translation mix was retained for analysis of products of selected RNAs, while aliquots of the remainder were used for immunoprecipitationwith different antibody preparations. Total products of selected RNAs and immunoprecipitated products were analysed by PAGE and fluorography. The RNAs complementary to various clones were further characterized by hybridization of 'Northern' transfers to nicktranslated plasmid DNAs. One p-g poly(A)+RNA and 0.5 ng of each of the DNA standard mixtures (see below) were denatured in glyoxal-formamide (Covey et al. 1981) and fractionated by electrophoresis on agarose gels. Reference samples for the determination of RNA sizes consisted of a TaqI partial restriction digest of pAT 153 and a PstI-SalG digest ofpBG 6 (Goldsbrough et al. 1982). DNA markers and RNA were transferred to nitrocellulose in 20 x SSC (Thomas 1980), hybridized to nicktranslated plasmid and the filters washed as above. Apparent RNA sizes were estimated from the autoradiographs by reference to the DNA standards (McMaster and Carmichael 1977). The cDNA inserts were further characterized by restriction analysis using various restriction enzymes which either were prepared in the laboratory or were obtained commercially.
Results T a b l e 1 s h o w s the a m o u n t s o f c o n v i c i l i n , e s t i m a t e d as a p e r c e n t a g e o f t o t a l p r o t e i n , i n five d i f f e r e n t p e a g e n o t y p e s . A p a r t f r o m JI 224 Sp. (a P. f u l v u m line), t h e r e w a s l i m i t e d v a r i a t i o n i n the p e r c e n t a g e c o n v i c i l i n ( T a b l e 1). I n this s t u d y , c o n v i c i l i n w a s p u r i f i e d f r o m c o t y l e d o n s o f cv. Birte, w h i c h , although potentially yielding smaller amounts of c o n v i c i l i n p e r u n i t p r o t e i n t h a n JI 224 Sp., was
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
449
Table 1. The amounts of convicilin in five genotypes of pea, estimated by densitometry, as a percentage of the total Coomassie-Blue staining material when 25 I~g total protein were analyzed by PAGE. Each value (+SE) represents the mean of three determinations Genotype
JI 224 Sp cv. Dark skinned perfection cv. Birte BCI/IW BCI/1R
Convicilin
(%)
13.4_+0.6 8,3 + 0.4 6,2_+0.6 5.8_+0.4 5.9_+0.4
chosen since it had been the subject of earlier studies (Domoney et al. 1980; Domoney 1981). Stages of seed development are best defined in terms of the pattern of current protein synthesis and the cotyledons used in this study were defined by pulse-labelling with 14C-amino acids in vivo (Spencer et al. 1980). When extracts of these cotyledons were passed through anti-legumin IgG-Sepharose columns (Domoney 1981), unbound 14Clabelled material was shown by PAGE and autoradiography to consist almost exclusively of subunits of M r approx. 70,000 (Fig. I a). Stained gels of similar material (Fig. 1 b) revealed vicilin subunits in addition to convicilin. Analysis of bound ~4C-labelled material from these columns showed that legumin was also being actively synthesized by these cotyledons as evidenced by the presence of labelled precursor polypeptides of M r approx. 60,000. It has been previously shown that the bulk of convicilin and legumin synthesis occurs relatively late in seed development compared with the major period of vicilin synthesis (Millerd et al. 1978; Spencer etal. 1980; Croy etal. 1980). The lack of coincidence observed between the in-vivo ~4Clabelled and the stained bands of M r 70,000 is in agreement with the observations of others (Higgins and Spencer 1981) that post-translational modification of these polypeptides takes place (assuming that the polypeptides of M r 76,000/75,000 described by these authors are equivalent to those referred to as M r 70,000 in this study). These putative precursors (Fig. i a) must be relatively longlived, since pulse-labelling was carried out for a period of 2 h. The relative purity of convicilin prepared by elution from Weber-Osborn gels (Weber and Osborn 1969) as assessed by PAGE (Laemmli 1970) is shown in Fig. 2. A sample of the vicilin-convicilin preparation used for preparative gel electrophoresis is shown in Fig. 2a. It can be seen that the subunits of M r approx. 70,000 purified by this
Fig. 1. An analysis by PAGE and autoradiography of unbound protein from anti-legumin IgG columns of extracts from cv. Birte cotyledons 14C-labelled in vivo. a, autoradiograph of the gel track shown in b, b, stain: the stained polypeptide in the region M r 70,000 did not correspond with either of the polypep~ tides in the same region of the autoradiograph
method were free from all other subunits (Fig. 2 b); it was estimated that approx. 20% of the protein applied to each gel was recovered as convicilin. Antiserum raised against purified convicilin subunits was used to identify convicilin polypeptides among the translation products of poly(A)§ When total translation products (Fig. 3 a) were challenged with anti-convicilin, polypeptides of Mr approx. 70,000, 50,000 and 47,000 were selectively immunoprecipitated (Fig. 3 b), in agreement with the serological cross-reactivity of convicilin and vicilin previously reported (Croy et al. 1980). The cell-free translation products immunoprecipitated by anti-vicilin are shown in Fig. 3 c; longer film exposures of such immunoprecipitates would also show polypeptides of M r approx. 70,000. A direct comparison of the proportion of cross-reactivity exhibited by each antibody preparation is not possible, however, since antibody was raised against the whole protein in one instance (vicilin) and against denatured subunits in the other (convicilin). It might be expected that, in the latter case, affinity for primary translation products would have been greater.
450
C. Domoney and R. Casey : Complementary DNA clone for Pisum convicilin
Fig. 2. An analysis by PAGE of a, the convicilin-enrichedprotein fraction obtained by zonal isoelectric precipitation column chromatography of unbound protein from anti-legumin IgG affinity columns; and b, convicilin subunits purified by elution from Weber-Osborn gels (Weber and Osborn 1969), when the subunits shown in a were separated by electrophoresis on 5% Weber-Osborn gels
Fig. 3. An analysis by PAGE and fluorography of a, the total products synthesized in a rabbit reticulocyte cell-free protein synthesizing system by poly(A) § RNA purified from developing seeds of cv. Birte; b, the products from those in a which were immunoprecipitated by anti-convicilin; and c, the products from those in a which were immunoprecipitated by anti-vicilin
Fig. 4. An analysis by PAGE and fluorography of: the total products of RNA selected by four different plasmids (a, d, g, j); the products of selected RNA which were immunoprecipitated by anti-legumin (b, e, h, k); the products of selected RNA which were immunoprecipitated by anti-vicilin (c, f, i,/); the products of RNA selected by pCD 59 which were immunoprecipitated by anti-convicilin(rn); and the products of the poly(A) +RNA preparation used for cDNA synthesis (n)
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
Fig. 5. A 'Northern' blot analysis of poly(A)+RNA. Filters were hybridized with pCD 4 (a), pCD 48 (b), pCD 59 (c) or pAT 153 (at). Numbers refer to the nucleotide lengths of the markers shown in (e) 0 I
:
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,
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,
,
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,
~
,
~
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9
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,
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though the two vicilin eDNA clones (pCD 4, pCD48) hybridize to RNAs of similar size (Fig. 5a, b), the convicilin cDNA clone (pCD 59) was found to hybridize to R N A which was appreciably longer than that hybridizing to the other two plasmids (Fig. 5c). The estimated sizes of the two m R N A classes were 1,725 _+35 nucleotides (vicilin mRNAs) and 2,210 _+30 nucleotides (convicilin mRNA). Analysis of mixtures of markers and RNA samples showed that there was no significant error arising from the differences in mass of the R N A samples compared with the markers. Treatment of the RNA samples with deoxyribonuclease did not affect the hybridization patterns. Figure 6 shows a comparison of the restriction maps obtained for the cDNA inserts from pCD 48 and pCD 59 (inserts of 1200 and 650 base pairs, representing 70% and 30% of their corresponding mRNAs, respectively). Out of sixteen enzymes tested, restriction sites for five (BglII, BstNI, HincII, PstI, TaqI) were present in the inserts from both pCD 48 and pCD 59; sites for an additional two enzymes (HindIII, RsaI) were present in pCD 48 only. No major similarity in the arrangement of common sites within the two eDNA inserts was apparent (Fig. 6).
Kb
Discussion
C C
,,,
451
,
,
p C D 4 8
p C D 5 9
Fig. 6. A restriction enzyme analysis of the eDNA inserts from pCD 48 and pCD 59. B=BglII, C=HincII, H = H i n d I I I , N = BstNI, P = PstI, R = RsaI, T : TaqI
Figure 4 shows the identification by hybrid-selection and immunoprecipitation of cDNA clones for the major storage protein polypeptides evident among total translation products (Fig. 3 a). In the case of each cDNA clone, a positive and clearcut identification of the products of selected RNAs was possible using specific antibodies. Legumin (pCD 40), vicilin (pCD 4, pCD 48) and convicilin (pCD 59) clones were identified in this manner (Fig. 4). The polypeptides of M r 70,000 synthesized by R N A selected by pCD 59 were immunoprecipitated by both anti-convicilin and anti-vicilin; however, a higher proportion of the radioactivity present in total products was consistently immunoprecipitated by anti-convicilin when compared with anti-vicilin. Figure 5 shows a 'Northern' blot analysis of the hybridization of size-fractionated poly(A) + RNA to pCD 4, pCD 48 and pCD 59. AI-
The results presented describe the isolation and identification, using antibody raised against convicilin, of a convicilin cDNA clone, pCD 59. Chandler (1982) has reported the isolation of two cDNA clones which hybridize separately to mRNAs coding for polypeptides of M r 70,000 and 76,000. These polypeptides, however, have not been identified by immunoprecipitation (Chandler 1982), but it is reported (Chandler 1982) that the polypeptide of M r 76,000 is a precursor to one of the pea storage globulins; it is probably equivalent to the polypeptide of M r 76,000 reported by Higgins and Spencer (1981), previously assumed to be convicilin (see Results section). On this basis, it may be assumed that the cDNA clone for the polypeptide of M r 70,000 observed by Chandler (1982) is not equivalent to the cDNA clone described by us. Chrispeels et al. (1982) have previously observed a minor vicilin polypeptide of M r 70,000 which they report to be processed to give quantitatively minor products, mainly of Mr 50,000. This discrepancy in molecular weight estimations is unfortunate, but both we (Casey and Sanger 1980), and Croy et al. (1980) have reported a major component of 7S protein (convicilin) to have subunits of M r approx. 70,000.
452
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin
Under the conditions employed for hybrid-selection in this study, the RNA selected by pCD 59 coded only for convicilin polypeptides and there was no evidence for selection of RNAs coding for either of the two vicilin polypeptides (Fig. 4j). Conversely, there was no evidence for selection of RNA coding for convicilin by either pCD 4 or pCD 48 (Fig. 4d, g). This is hardly surprising since, under these conditions, no cross-selection of the two RNAs coding for vicilin polypeptides takes place with either pCD 4 or pCD 48 (Fig. 4d, g), despite the extensive sequence homology reported by Gatehouse et al. (1982) for two corresponding cDNAs. In addition, there was no evidence from 'Northern' blot analyses of any hybridization of pCD 59 to RNA in the size class to which pCD 4 and pCD 48 hybridized (Fig. 5); the converse was also true in these analyses. Although there was no evidence of relatedness of the two proteins, convicilin and vicilin, from the RNA hybridization analyses in this study, it might be expected from the serological cross-reactivity that they share some sequence homology and that these sequences may be responsible for common antigenic determinants on the outer surfaces of the molecules. The degree of sequence homology might not be expected to be great, based on previous observations of dissimilarities in the two proteins (see Introduction section). The isolation and identification of cDNA clones for these two proteins, as reported here, will facilitate DNA sequence comparisons and the possible divergent or convergent evolution of the two proteins might be delineated. We are grateful to Agrigenetics Research Corporation, Golden, Colo., USA, for financial support. We thank Dr. C.L. Hedley for providing us with seeds of genotype BC1/1R, Dr. T.H.N. Ellis for invaluable advice and Wendy Cleary for excellent technical assistance.
References Bedbrook, J.R., Smith, S.M., Ellis, R.J. (1980) Molecular cloning and sequencing of cDNA encoding the precursor to the small subunit of chloroplast ribulose-l,5-bisphosphate earboxylase. Nature (London) 287, 692-697 Casey, R. (1979 a) Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds. Biochem. J. 177, 509-520 Casey, R. (1979b) Genetic variability in the structure of the a-subunits of legumin from Pisum - a two-dimensional gel electrophoresis study. Heredity 43, 265-272 Casey, R., Sanger, E. (1980) Purification and some properties of a 7S seed storage protein from Pisum (pea). Biochem. Soc. Trans. 8, 658 Chandler, P.M. (1982) The use of single-stranded phage DNAs
in hybrid arrest and release translation. Anal. Bioehem. 127, 9-16 Chrispeels, M.J., Higgins, T.J.V., Spencer, D. (1982) Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons. J. Cell Biol. 93, 306-313 Christophe, D., Brocas, H., Vassart, G. (1982) Improved synthesis of DBM paper. Anal. Biochem. 120, 259-261 Covey, S.N., Lomonossoff, G.P., Hull, R. (1981) Characterisation of cauliflower mosaic virus DNA sequences which encode major polyadenylated transcripts. Nucl. Acids Res. 9, 6735-6747 Croy, R.R.D., Derbyshire, E., Krishna, T.G., Boulter, D. (1979) Legumin of Pisum sativum and Vieiafaba. New Phytol. 83, 29-35 Croy, R.R.D., Gatehouse, J.A., Tyler, M., Boulter, D, (1980) The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.). Biochem. J. 191, 509-516 Danielsson, C.E. (1949) Seed globulins of the Graminae and Leguminosae. Biochem. J. 44, 387-400 Davis, R.W., Thomas, M., Cameron, J., St. John, T.P., Scherer, S., Padgett, R.A. (1980) Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 65, 404-411 Denhardt, D.T. (1966) A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646 Domoney, C., Davies, D.R., Casey, R. (1980) The initiation of legumin synthesis in immature embryos of Pisum sativum L. grown in vivo and in vitro. Planta 149, 454-460 Domoney, C. (1981) Legumin synthesis and accumulation in Pisum sativum L. Ph.D. thesis, University of East Anglia, Norwich, UK Gatehouse, J.A., Croy, R.R.D., Morton, H., Tyler, M., Boulter, D. (1981) Characterisation and subunit structures of the vicilin storage proteins of pea (Pisum sativum L.). Eur. J. Biochem. 118, 627-633 Gatehouse, J.A., Evans, I.M., Brown, D., Croy, R.R.D., Boulter, D. (1982) Control of storage-protein synthesis during seed development in pea (Pisum sativum L.). Biochem. J. 208, 119-127 Goldsbrough, P.B., Ellis, T.H.N., Lomonossoff, G.P. (1982) Sequence variation and methylation of the flax 5S RNA genes. Nucl. Acids Res. 10, 4501-4514 Grunstein, M., Hogness, D.S. (1975) Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72, 3961-3965 Higgins, T.J.V., Spencer, D. (1981) Precursor forms of pea vicilin subunits. Plant Physiol. 67, 205-211 Jen, G., Thach, R.E. (1982) Inhibition of host translation in encephalomyocarditis virus-infected L cells: a novel mechanism. J. Virol. 43, 250-261 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685 Larkins, B.A., Davies, E. (1975) Polyribosomes from peas. V. An attempt to characterize the total free and membranebound polysomal population. Plant Physiol. 55, 749-756 Martin, C., Northcote, D.H. (1982) The action of exogenous gibberellic acid on isocitrate lyase-mRNA in germinating castor bean seeds. Planta 154, 174-183 Matta, N.K., Gatehouse, J.A. (1982) Inheritance and mapping of storage protein genes in Pisum sativum L. Heredity 48, 383-392 McMaster, G.K., Carmichael, G.G. (1977) Analysis of singleand double-stranded nucleic acids on polyacrylamide and
C. Domoney and R. Casey: Complementary DNA clone for Pisum convicilin agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74, 4835-4838 Millerd, A., Thomson, J.A., Schroeder, H.E. (1978) Cotyledonary storage proteins in Pisum sativum. III. Patterns of accumulation during development. Aust. J. Plant Physiol. 5, 519-534 Osborne, T.B., Campbell, G.F. (1898) Proteids of the pea. J. Am. Chem. Soc. 20, 348-362, 36~375, 393-405 Pelham, H.R.B., Jackson, R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256 Roychoudhury, R., Jay, E., Wu, R. (1976) Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucl. Acids Res. 3, 101 116 Spencer, D., Higgins, T.J.V., Button, S.C., Davey, R.A. (1980) Pulse-labeling studies on protein synthesis in developing pea seeds and evidence of a precursor form of legumin small subunit. Plant Physiol. 66, 510-515
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Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201-5205 Thomson, J.A., Schroeder, H.E., Dudman, W.F. (1978) Cotyledonary storage proteins in Pisum sativum. I. Molecular heterogeneity. Aust. J. Plant Physiol. 5, 263-279 Weber, K., Osborn, M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406M412 Weber, K., Osborn, M. (1975) Proteins and sodium dodecyl sulfate: molecular weight determination on polyacrylamide gels and related procedures. In: The proteins, vol. 1, edn. 3, pp. 179-223, Neurath, H., Hill, R.L., eds. Academic Press, New York
Received 30 May; accepted 5 July 1983
