Planta (I 984)160 : 559-568

P l ~ n ~ 9 Springer-Verlag 1984

Regulation of the transcription of storage-protein mRNA in nuclei isolated from developing pea (Pisum satirum L.) cotyledons I. Marta Evans*, John A. Gatehouse, Ronald R.D. Croy and Donald Boulter Department of Botany, University of Durham, Durham DH1 3LE, UK

Abstract. Two types of storage protein, vicilin and legumin, occur in the developing pea seed. Storageprotein gene expression has been studied during cotyledon development by assaying specific transcripts produced by nuclei isolated at different stages, and from pea leaves. The proportion of vicilin to legumin transcripts changed during development: vicilin transcripts predominated at 9 and 11 days after flower opening (d.a.f.) and were similar in amount to legumin at 14 d.a.f., whereas at 18 d.a.f., legumin transcripts predominated and little vicilin transcription was observed. The rate of storage-protein transcription correlated with previously determined (Gatehouse et al. 1982) m R N A levels during seed development; these transcripts were not detected in similar assays using leaf nuclei. Transcription by cotyledonary nuclei for short times indicated that post-transcriptional processing may be a factor in regulating m R N A levels, at least in the earlier part of seed development.

Key words: DNA hybridisation - Pisum (storage protein mRNA) - RNA (transcription) - Storage protein - Transcription.

Introduction

The pea haploid genome contains in the order of 4.8.109 base pairs DNA (Thompson 1975-1976). It is estimated that up to 15% is constituted by low copy number D N A (Murray et al. 1978) which potentially codes for m R N A sequences. In the developing cotyledon, m R N A sequences form a complex group containing about 20000 spe* To whom correspondence should be addressed Abbreviations: d.a.f. = days after flower opening; RNase = ribo-

nuclease; UTP = uridine 5'-triphosphate

cies at the early stage of development (Morton et al. 1983). The most abundant at mid-maturation stage, about six discrete species, code for the major storage proteins, vicilin and legumin which constitute ~ 80% of the protein mass at seed maturity. These m R N A sequences are polyadenylated and are in the range 17-19S (1800-2200 bases) (Croy et al. 1982). The major vicilin initial translation products are polypeptides of M, 50000 and 47000 (Croy et al. 1980b) whereas the legumin initial translation products are 60000-Mr precursor polypeptides (Croy et al. 1980a). It has been previously shown that levels of specific mRNAs coding for storage-protein precursor polypeptides change during seed development in pea (Gatehouse et al. 1982) in a manner that correlated with the observed synthesis of storage proteins. Under standardised growth conditions, seed development was synchronised, and could be measured in days after flower opening (d.a.f.) with cotyledon differentiation commencing at 7 d.a.f., and storage-protein synthesis commencing 8-9 d.a.f. and ceasing at 21 d.a.f. Vicilin and legumin mRNAs, which were present in very low concentrations during earlier stages of seed development, became major components of total m R N A after approximately two thirds of the interval from flower opening to cessation of synthetic activity in the seed (14 d.a.f.). Legumin m R N A remained at relatively high levels until the end of this period (approx. 21 d.a.f.) whereas the m R N A encoding vicilin 50000-M~ polypeptides declined to lower levels before storage-protein deposition ceased (i.e. by 19 d.a.f.). A second vicilin mRNA, encoding the 47000-M~ polypeptide, became a detectable component in total m R N A 2-3 d earlier in seed development than the other mRNAs and also declined earlier (by 16 d.a.f.). These results demonstrate developmental regulation of m R N A concentration

560

I.M. Evans et al. : Transcription of storage-protein mRNA in pea cotyledons

and it was suggested that control was likely to occur at the level of transcription. To study further the expression of genes coding for these m R N A sequences, it is necessary to examine transcription of isolated nuclei from pea cotyledons at different developmental stages, and to attempt to assay specific transcripts from these nuclei. These products will be run-off transcripts due to the activity of endogenous RNA polymerase II and are likely to reflect in-vivo transcription, since the intact nuclei should preserve the native state of chromatin, including regulatory proteins. Transcription of cellular chromosomal genes in eukaryotes is performed by three different RNA polymerases: R N A polymerase I is responsible for the transcription of the ribosomal genes (18S and 25S), R N A polymerase II for genes encoding mRNAs, and R N A polymerase III for transcription of the transfer-RNA and 5S-ribosomal-RNA genes. These different R N A polymerases are distinguished by their differential sensitivity to ~zamanitin with R N A polymerase II being sensitive to the lowest level of 0~-amanitin (Roeder 1976; Guilfoyle et al. 1980). The developmental stages investigated were chosen to compare transcribed sequences at the beginning, during and towards the end of the storage-protein accumulation in pea seeds. Isolated-nuclei transcription was also investigated in leaves where storage proteins and their corresponding mRNAs are not detected (Croy et al. 1982; Gatehouse et al. 1982). Materials and methods Materials. Chemicals used in the work described were obtained from BDH Chemicals, Poole, Dorset, UK, unless otherwise stated, and were of analytical grade or the best available. Trizma base, glyoxal, acridine orange, c~-amanitin, herring sperm DNA and nucleotide triphosphates, were purchased from Sigma Chemical Co., Poole, Dorset, UK; agarose and globin mRNA were from Miles Laboratories, Stoke Poges, Slough, Berks, UK Percoll was from Pbarmacia, Uppsala, Sweden. Restriction enzymes were obtained from BRL, Cambridge, UK; nitrocellulose (type BA 85) from Schleicher and Schuell, Dassel, FRG; actinomycin D from Calbiochem, La Jolla, Cal., USA. Radiochemicals were supplied by Amersham International Amersham, Bucks., UK. Placental ribonuclease (RNase) inhibitor (RNasin) was obtained from Biotech., Madison, Wis., USA. Plant growth. Plants were grown from pea seeds (Suttons Seeds, Reading, Berks., UK), variety Feltham First, as previously described (Evans et al. 1979). Cotyledons were aseptically removed at defined developmental stages of 7, 9, 11, 14 and 18 d.a.f. (Gatehouse et al. 1982); under these conditions seed development was essentially complete at 21 d.a.f. Leaves were harvested from separate plants, grown in a growth cabinet in sterile glass containers on a 1.2% agar media, at about 9 d after seed germination.

Isolation of nuclei. Fresh cotyledons and leaves were used for isolation of nuclei using a modification of the procedure described by Willmitzer and Wagner 1981, which was shown to yield purified nuclei containing RNA polymerase I, II and III activities (Willmitzer et al. 1981). The modification involved incubation of cotyledons at 25~ C for 4 h before homogenization by means of either a hand Quickfit glass-to-glass homogenizer (BC 15/150 and BC 15P) or an ()sterizer blender for 3 x 10 s (J. Oster, M.F.G. Co., Milwaukee, Wis., USA). Final preparations were stored at - 80~ C in 50% glycerol. Nuclei were examined by a fluorescence microscopy (Nikon Diaphot) with a TMD fluorescence attachment (The Projectina Co., Skelmorlie, Ayrshire, UK), using 4, 6-diamidino-2-phenylindolestain (Kuroiwa et al. 1982). They were counted by means of a modified Fuchs-Rosenthal haemocytometer (Hawksley; Gallenkamp & Co., Stockton-on-Tees, Cleveland, UK). The DNA content in nuclei was determined by fluorescence with 3, 5-diaminobenzoic acid (Thomas and Farquhar 1978), using herring sperm DNA as a standard. Transcription in isolated nuclei. Nuclei were washed three times (5 rain; 800 g) in buffer I (60 mM 2-amino-2-(hydroxymethyl)1,3-propanediol (Tris)-HC1, pH 7.4; 5raM MgClz; 5raM 2-mercaptoethanol; 0.1 mM ethylenediaminetetraacetic acid, 0.5 % bovine serum albumin; 15% (w/v) glycerol), resuspended in 300 lal of buffer I and divided into three 100-lal aliquots. To one aliquot, 20 lal of buffer I supplemented with 240 mM ammonium sulphate (buffer II) was added, and to the remaining two, 20 lal of buffer II supplemented with e-amanitin (3.5 lag m1-1) or actinomycin D (700 lag ml-1), respectively; the mixtures were incubated on ice for 15 rain. To each tube, 20 ~1 of buffer II supplemented with 2 mM each of ATP, guanosine 5'-triphosphate (GTP), cytidine 5'-triphosphate (CTP), 0.15 mM uridine 5'-triphosphate (UTP) and 74 kBq (0.44 TBq retool-l) of [5-3H]UTP (analytical scale) or 5.55-29.6 MBq (15.17 TBq mmo1-1 of c~-[~zP]UTP (preparative scale) were added and the mixture (total assay volume 140 gl) incubated at 26~ for a given time. In analytical assays, transcription was stopped by adding 0.5 ml cold 10% trichloroacetic acid (TCA) and precipitated transcripts were washed on GF/C filters (Whatman Labsales, Maidstone, Kent, UK) with 10% TCA, 5% TCA and ethanol. The discs were dried at 80~ C in a vacuum oven and radioactivity incorporated determined by scintillation counting with 2,5-diphenyloxazole (3 g 1-1) plus 1,4-di[2-(5 phenyloxazolyl)-benzene] (0.3 g 1-1) in toluene. Isolation of RNA from transcribed nuclei. When total RNA was to be isolated from nuclei, transcription was routinely performed in the presence of ~-[32P]UTP, with no unlabelled UTP added, with 1.4 ~tl of RNasin (placental RNase inhibitor), for 45 min at 26~ C. The isolation of RNA was based on the method of McKnight and Palmiter (1979) and final samples were either immediately used for hybridisation or stored in sterile H20 at - 8 0 ~ C for short period of time. The RNA was glyoxalated and analysed on 1.5% agarose gels (McMasters and Carmichael 1977). Gels were dried down and autoradiographed (Fuji X-ray film; Fujimex, Swindon, Wilts., UK) with an intensifying screen (du Pont; M.A.S. Northern, Aycliffe, Durham, UK). Immobilisation of plasmid DNAs. Recombinant plasmids containing sequences specific for pea legumin (pDUB3 and pDUB6, previously referred to as pRC 2.11.7 and pAD4.4), pea vicilin M r 47000 [pDUB7 (pAD 3.4)], pea vicilin M r 50000 [pDUB2 (pRC 2.2.1)], pBR 322 and pAT 153, were prepared in this laboratory and are described elsewhere (Croy et al. 1982); the probe for the light-harvesting chlorophyll a/b-bind-

I.M. Evans et al. : Transcription of storage-protein mRNA in pea cotyledons

561

ing protein complex ofPisum, pFa/b31, was obtained from S.M. Smith, University of Warwick, Coventry, U K ; fl-globin DNA, pflG-1, was obtained from C. Shaw, University of Durham, UK, and the plasmid, pHAI, containing pea ribosomal genes was obtained from R. Cuellar, PBI, Cambridge, UK. Plasmid DNAs were restricted with BamHt (pDUB3, pDUB6, pDUB7, pDUB2 and pFe{/~31), EcoRI (pBR322 and pAT153) or Hind III(pflG-i and pHA1) (according to the suppliers instructions); digests were elec'trophoresed on 0.7% agarose gels and DNA transferred to nitrocellulose sheets by the procedure of Southern (1979). Some plasmid DNAs were also linearised with EcoRI (pDUB6) or Hind III (pHA1), further treated and attached to nitrocellulose filters (Schleicher and Schuell BA85, 0.45 lain) according of Kafatos et al. (1979), except that for the binding of DNA we used a Hybri. Dot TM manifold (Bethesda Research Laboratories, Cambridge, UK). Discs of 1 cm diameter were cut out of nitrocellulose sheets before being airdried and baked at 80 ~ c. Hybridisation to immobilised DATA. Hybridisation of nuclear transcripts to Southern transfers of plasmid DNA, and to nitrocellulose filters bearing similar complementary-DNA (cDNA)containing plasmids was performed according to Gallagher and Ellis (1982). Each transfer contained 2.5 lag or 5 ~tg of a cloned plasmid, and in filter hybridisation each reaction mixture (100 gl) contained a filter bearing 5 lag of a cloned probe and a control filter bearing 5 lag of pAT153 (a derivative of pBR322) or pBR322. The filters were overlaid with 0.5 ml of paraffin oil and hybridised at 41 ~ C for 48-60 h. The efficiency of hybridisation, and the relationship between input 3zp nuclear RNA and 32p hybridised RNA, were measured by hybridising azp cRNA synthesised on pDUB6 and pDUB7 templates (excised and isolated by gel electrophoresis) according to the method of McKnight and Palmiter (] 979).

Fig. 1. Nuclei from 9 d.a.f, pea cotyledons isolated by centrifugation on Percoll gradients and stained with the fluorescent dye, 4,6-diamidino-2-phenylindole. Bar= 25 lam; x 400

Results

Characterisation of nuclei. Nuclei were isolated from pea leaves and cotyledons at four different stages of development, and after purification on two Percoll gradients, appeared to be devoid of major non-nuclear contamination. However, some cell-wall debris were visible and their amount increased in nuclear preparations from cotyledons at later stages of development. Figure i shows nuclei isolated from 9 d.a.f, cotyledons, stained with the fluorescent dye, 4,6-diamidino-2-phenylindole. The yield of leaf nuclei varied between 2.5-9.3-106 nuclei g-1 of fresh weight and that of cotyledonary nuclei between 1.7-14-106; 0.9-2.6,106; 0.5-2.8.105; and 0.4-1.10 s nuclei g- * from 9, 11, 14 and 18 d.a.f., respectively. Leaf nuclei were stored at a concentration of about 4 - 1 0 7 nuclei m1-1, whereas cotyledon nuclei at about 6-10 ~ nuclei ml- 1 (from 9 and 11 d.a.f, cotyledons) and 5"105 nuclei m1-1 (from 14 and 18 d.a,f, cotyledons). Overall transcription assay. Isolated nuclei, both from pea leaves and cotyledons, incorporated [3H]-UTP into RNA when incubated with nucleo-

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Fig. 2. Time course of transcription in isolated nuclei (10 gg DNA, 74 KBq [3H]UTP per assay) at 26 ~ C for the times indicated. Results are the average of duplicate incubations

side triphosphates. Overalt transcription increased with time for about 15 rain (Fig. 2) and then after a short period of decay, remained fairly constant up to 90 min. Transcription was reduced by omission of unlabelled nucleoside triphosphates, addition of actinomycin D (100 gg ml-1) and addition of c~-amanitin (0.5 gg m1-1 and 10 gg m l - 1). Overall, the greatest inhibition of transcription with ~amanitin at 10 gg m1-1 was observed for nuclei isolated at 9 d.a.f. (17%). Actinomycin D, which

562

I.M. Evans et al. : Transcription of storage-protein m R N A in pea cotyledons

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effectively blocks the activity of endogenous nuclear template DNA-bound R N A polymerases (Yu 1974) inhibited transcription by 92%. Similar assays were performed with nuclei isolated from 7 d.a.f., 14 d.a.f, and 18 d.a.f, cotyledons, but because of the much poorer transcription by these nuclei, the results were variable and difficult to quantitate. A 24% inhibition of overall transcription by ~-amanitin at 10 gg m1-1 and 65% inhibition by actinomycin D, were observed in nuclei isolated from pea leaves. For a particular stage of cotyledon development, although total transcription (15 min) increased with the amount of DNA template, the efficiency of transcription (uridine 5'-monophosphate incorporated per gg DNA) was higher at lower concentrations of D N A template. The results in Fig. 3 give the changes in transcriptional efficiency with some stages of cotyledon development (using the same amount of D N A in the assay at each developmental stage) and show that 11 d.a.f. (approx. 2 d after the cessation of cell division) is the stage at which transcription is most efficient. Nuclei isolated from cotyledons at 7 d.a.f., the earliest stage at which physical handling of the cotyledons is possible, were found to transcribe poorly indicating that total transcription is most efficient at about 10-12 d.a.f. The R N A synthesised by isolated pea cotyledon nuclei is heterodispersed in size (Fig. 4). To obtain RNA transcripts of high molecular weight, it was found necessary to include RNasin (a human placental RNase inhibitor) in the transcription mixtures. The presence of this inhibitor also enhanced total transcription (Fig. 4B, lanes 1, 2). Under resolving conditions, ribosomal R N A (rRNA) transcripts at about 18 and 25S were clearly visible in the total transcription products (Fig. 4A).

Hybridisation. The dot-hybridisation protocol of

Fig. 4A, B. Size distribution of R N A synthesised by nuclei isolated from pea cotyledons. Nuclei were incubated with c~-[32P]UTP and transcribed R N A was isolated, glyoxalated and resolved on 1.5% agarose gels. Gels were autoradiographed. Arrows indicate the position of 25S and 18S r R N A markers. A Nuclei incubated 50 rain in the presence of RNasin; 5.104 cpm loaded per track to show discrete transcripts. B Effect of RNasin ribonuclease inhibitor. Nuclei were incubated for 45 min in the presence (1) and absence (2) of RNasin. 105 cpm loaded per track

Kafatos et al. (1979) was inadequate for detection of m R N A transcripts in our system, a consequence of significant levels of non-specific hybridisation and hence a too low "signal to noise" ratio. The 3Zp-labelled RNA was therefore hybridised to plasmid D N A which had been bound to nitrocellulose filters under conditions of D N A excess using the methods of Gallagher and Ellis (1982). Figure 5 shows that a semi-quantitative relationship exists under these conditions between the input amounts of 32p-labelled cRNAs used, and their level of hybridisation to the pDUB6 and pDUB7 DNA templates. When these experiments were repeated and the results quantitated over a wider

I.M. Evans et al. : Transcription of storage-protein m R N A in pea cotyledons

563

Fig. 5. Hybridisation of synthesised cRNAs to pDUB7 and pDUB6 c D N A templates, respectively. ~ZP-labelled cRNAs were prepared separately from pDUB7 and pDUB6 DNA, mixed in the indicated proportions and hybridised to 5 gg each of pDUB7 and pDUB6 D N A that had been restricted with BamH1, electrophoresed on 0.7% agarose gel and transferred to nitrocellulose. 1 ~ 5000 cpm. Exposure time for autoradiograph 24 h

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Fig. 6A, B. Quantitative hybridisation of R N A synthesised by pea cotyledon nuclei to p H A I D N A (A), and of c R N A synthesised from pDUB6 template to pDUB6 D N A (B). Increasing amounts of 32P-labelled R N A were hybridised to filters bearing 5 pg each of DNA. Counts bound to control filters bearing 5 gg of pAT153 D N A were subtracted. Straight lines are leastsquares fits over the range of points covered

range of input amounts of labelled RNA, the relationship was shown to be curvilinear. The results given in Fig. 6A show this for hybridisation of rDNA plasmid pHAI (5 gg) to labelled transcripts of the nuclei isolated from developing cotyledons at 11 d.a.f., whereas Fig. 6B shows similar hybridisation, but of labelled cRNA synthesised from pDUB6 template to the template DNA. This type of relationship results in a lower hybridisation efficiency at a higher concentration of input RNA, which may be the result of a higher proportion of non-specific hybridisation at low input RNA. It was, therefore, important to use similar input cpm of labelled R N A to compare the extent of hybridisation for several developmental stages of Pisum. We have also tried to use similar numbers of nuclei for transcriptions from cotyledons at different developmental stages. A comparison of the hybridisation of accumulated R N A transcripts (45 min transcription time), synthesised in nuclei isolated from pea leaves and from pea seed cotyledons at four developmental stages (9, 11, 14 and 18 d.a.f.), to cDNAs encoding seed storage proteins is shown in Fig. 7 (B-F). The m R N A transcripts of the polypeptides of vicilin (47000 M r Track 3; 50000 M r, Track 4) were detected at all four stages of cotyledon development.

564

I.M. Evans et al. : Transcription of storage-protein m R N A in pea cotyledons

Fig. 7A-F. Hybridisation to specific DNA probes of RNA synthesised by isolated nuclei from pea leaves (B) and from cotyledons at 9 d.a.f. (C); 11 d.a.f. (D) 14 d.a.f. (E) and 18 d.a.f. (F). Plasmid DNAs (A) containing inserts for pea legumin (lane 1 = pDUB3, lane 2 = pDUB6), vicilin M r 47000 (lane 3= pDUB7), vicilin M r 50000 (lane 4= pDUB2), pea light-harvesting chlorophyll polypeptide (lane 5 = p Fa/b 31), fl-globin DNA (lane 6=pflG-1), and two vectors, pBR 322 (lane 7) and pAT 153 (lane 8) were digested with restriction enzymes, separated on 0.7% agarose gels, transferred to nitrocellulose and hybridised to 32p-labelled transcripts (4.6.107 cpm); pflG-1 was hybridised to 32p-5'-end-labelled globin m R N A (5-104 cpm). A UV picture of restricted DNAs; each lane was loaded with 2.5 gg DNA. B-F Autoradiographs of RNAs hybridised to blots similar to A; D Lane 9, 10, 5 ~g pHAI DNA hybridised to 32p-labelled R N A (2. I06 cpm); exposure time for autoradiographs 0.5 h. E 8.910a cpm of azP-labelled RNA; 3.410 s cpm of globin mRNA; F 2.5-10 v cpm of 32p-labelled RNA; 3.7" 104 cpm of globin m R N A ; exposure time for autoradiographs, two weeks

I.M. Evans et al. : Transcription of storage-protein mRNA in pea cotyledons

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vicilin M r 50 000 > legumin) are in reasonable agreement, allowing for the delay factor discussed previously, with the levels of transcripts after 45 min transcription, but do not agree with those after 3 min transcription. Interestingly the relative amounts of transcripts at 3 min relate more closely to the gene copy numbers of the legumin and vicilin genes (Croy et al. 1982; Gatehouse et al. 1983) in that legumin is encoded by three to four genes, vicilin 50 000 M r by three to six genes and vicilin 47000 M r by two to three genes. This indicates that the rates of transcription of all these genes are similar, and that post-transcriptional processing of the transcripts in the nucleus could explain the difference between the 9 d.a.f, legumin transcripts of the 3-rain and 45-min assays. Previous authors have suggested that this mechanism is of great importance in the control of gene expression in differentiated tissues of both plants (Kamalay and Goldberg 1980) and animals (e.g. Bathurst et al. 1980). However, since the assays did not distinguish between unprocessed and processed RNA, the observed difference in the legumin results when compared with vicilin, could also be explained by other factors, such as the persistence of transcription. Nevertheless, the presence of RNase inhibitor makes it unlikely that the differential breakdown of vicilin and legumin transcripts is the cause of this difference. In conclusion, it has been demonstrated that transcription of specific genes in vitro by nuclei isolated from pea leaves and cotyledons correlates with the observed levels of corresponding m R N A s and thus provides evidence for transcriptional control of storage-protein gene expression. In a related study, the structural genes which encode storageprotein m R N A s in soybean embryos were found to be primarily regulated at the transcriptional level (Goldberg etal. 1981; Fisher and Goldberg

567

1982) as was the soybean lectin gene, which is also developmentally regulated (Goldberg et al. 1983). On the basis of our results in vitro, we would advance the following hypotheses: 1) that storageprotein genes are transcriptionally active during seed development but are not active in other tissues of the plant and 2) that, at least, at earlier stages of cotyledon development there is the possibility of cytoplasmic m R N A levels being regulated by post-transcriptional processing of heterogeneous nuclear RNA. We wish to thank Dr. Phil Gates for advice on fluorescence microscopy, Dr. S.M. Smith, University of Warwick, Coventry, for pFa/b 31 clone, Dr. R. Cuellar, Plant Breeding Institute, Cambridge, for pHAI clone and Mr. Russell Swinhoe and Mr. D. Bown for technical assistance.

References Bathurst, I.C., Craig, R.K., Herries, D.G., Campbell, P.N. (1980) Differential distribution of poly(A)-containing RNA sequences between the nucleus and post-nuclear supernatant of the lactating guinea-pig mammary gland. Eur. J. Biochem. 109, 183 191 Croy, R.R.D., Gatehouse, J.A., Evans, I.M., Boulter, D. (1980a) Characterisation of the storage protein subunits synthesised in vitro by polyribosomes and RNA from developing pea (Pisurn sativum L.). I. Legumin. Planta 148, 49-56 Croy, R.R.D., Gatehouse, J.A., Evans, I.M., Boulter, D. (1980b) Characterisation of the storage protein subunits synthesized in vitro by polyribosomes and RNA from developing pea (Pisum sativum L.). II. Vicilin. Planta 148, 57-63 Croy, R.R.D., Lycett, G.W., Gatehouse, J.A., Yarwood, J.N., Boulter, D. (1982) Cloning and analysis of cDNAs encoding plant storage protein precursors. Nature (London) 295, 76-79 Cullis, C.A. (1976) Chromatin bound DNA-dependent RNA polymerase in developing pea cotyledons. Planta 131, 293-298 Evans, I.M., Croy, R.R.D., Hutchinson, P., Boulter, D., Payne, P.I. Gordon, M.E. (1979) Cell free synthesis of some storage protein subunits by polyribosomes and RNA from developing seeds of pea (Pisum sativum L.). Planta 144, 455462 Fischer, R.L., Goldberg, R.B. (1982) Structure and flanking regions of soybean seed protein genes. Cell 29, 651-660 Gallagher, T.F., Ellis, R.J. (1982) Light-stimulated transcription of genes for the two chloroplast polypeptides in isolated pea leaf nuclei. Eur. Mol. Biol. Organ. J. 1, 1493-1498 Gatehouse, J.A., Evans, I.M., Bown, 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 Gatehouse, J.A., Lycett, G.W., Delauney, A.J., Croy, R.R.D., Boulter, D. (1983) Sequence specificity of the post-translational proteolytic cleavage of vicilin, a seed storage protein of pea (Pisum sativum L.). Biochem. J. 212, 427-432 Goldberg, R.B., Hoschek, G., Ditta, G.S., Breidenbach, R.W. (1981) Developmental regulation of cloned superabundant embryo mRNAs in soybean. Dev. Biol. 83, 218-231 Goldberg, R.B., Hoschek, G., Vodkin, L.O. (1983) An insertion sequence blocks the expression of a soybean lectin gene. Cell 33, 465-475 Guilfoyle, T., Olszewski, N., Zurfluh, L. (1980) RNA polymer-

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ase during developmental transitions in soybean. In: Genome organization and expression in plants, pp. 93-104, Leaver, C.J., ed. Plenum, New York London Kafatos, F.C., Jones, C.W., Efstratiadis, A. (1979) Determination of nucleic acid sequence homologies and relative concentrations by dot hybridization procedure. Nucleic Acids Res. 7, 1541-1552 Kamalay, J.C., Goldberg, R.B. (1980) Regulation of structural gene expression in tobacco. Cell 19, 935-946 Kuroiwa, T., Kawano, S., Nishibayashi, S. (1982) Epifluorescent microscopic evidence for maternal inheritance of chloroplast DNA. Nature (London) 298, 481 483 McKnight, G.S., Plamiter, R.D. (1979) Transcriptional regulation of the ovalbumin and conalbumin genes by steroid hormones in chick oviduct. J. Biol. Chem. 254, 9050-9058 McMasters, G.K., Carmichael, G.G. (1977) Analysis of singleand double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74, 48354838 Millerd, A., Spencer, D. (1974) Changes in RNA-synthesising activity and template activity in nuclei from cotyledons of developing pea seeds. Aust. J. Plant Physiol. 1,331-341 Morton, H., Evans, I.M., Gatehouse, J.A., Boulter, D. (1983) Sequence complexity of messenger RNA in cotyledons of developing pea (Pisum sativum) seeds. Phytochemistry 22, 807-812

Murray, M.G., Cuellar, R.E., Thompson, W.F. (1978) DNA sequence organisation in the pea genome. Biochemistry 17, 5781-5790 Roeder, R.G. (1976) Eukaryotic nuclear RNA polymerases. In: RNA polymerase, pp. 285-329, Losick, R., Chamberlin, M., eds. Cold Spring Harbor Laboratory, New York Southern E.M. (1979) Gel electrophoresis of restriction fragments. Methods Enzymol. 68, 152-176 Thomas, P.S., Farquhar, M.N. (1978) Specific measurement of DNA in nuclei and nucleic acids using diaminobenzoic acid. Anal. Biochem. 89, 35-44 Thompson, W.F. (1975-1976) Sequence organization in pea DNA. Carnegie Inst. Washington Yearb. 75, 356-362 Willmitzer, L., Wagner, K.G. (1981) Isolation of nuclei from tissue-cultured plant cells. Exp. Cell Res. 135, 69-77 Willmitzer, L., Otten, L., Simons, G., Schmalenbach, W., Schr6der, J., Schr6der, G., Van Montagu, M., De Vos, G., Schell, J. (1981) Nuclear and polysomal transcripts of TDNA in octopine crown gall suspension and callus cultures. Mol. Gen. Genet. 182, 255-262 Yu, F.-L. (1974) Two functional states of the RNA polymerases in the rat hepatic nuclear and nucleolar fractions. Nature (London) 251,344-346 Received 15 November 1983; accepted 25 January 1984