Pl~.Jn~ 9 Springer-Verlag1985
Transcription of a legumin gene from pea (Pisum sativum L.) in vitro I.M. Evans, D. Bown, G.W. Lycett, R.R.D. Croy, D. Boulter and J.A. Gatehouse Department of Botany, Universityof Durham, South Road, Durham DHI 3LE, UK
Abstract. The transcriptional activity of the pea legumin gene Leg A in the HeLa cell lysate in-vitro transcription system has been assayed. Labelled transcripts were examined by hybridisation to restriction fragments of Leg A, and by glyoxalation followed by agarose gel electrophoresis. Although the legumin gene was not transcribed efficiently, transcripts were shown to be produced from the correct region of Leg A, and analysis by S1 nuclease mapping was used to show that the transcription start was the same as in vivo. The transcripts produced in vitro did not extend over the whole coding sequence of the gene; termination of transcripts occurred in a semi-random fashion. Transcription of templates truncated at their 3" ends (i.e. in coding sequence) showed that discrete transcripts were produced from the start to restriction sites at approx. + 520 and + 900 bases. Transcription of templates truncated at their 5' ends showed that sequences upstream of - 9 7 bases relative to the transcription start had no appreciable effect on transcription in vitro.
Key words: Legumin - Pisum (storage protein) Storage-protein gene - Transcription (in vitro).
Legumin is a major storage protein in peas and other important crop plants (Derbyshire et al. 1976). It is synthesised in the developing embryo during seed development but not in other tissues of the plant (Croy et al. 1982). The expression of legumin genes has been shown to be primarily Abbreviations:
A=adenine; C=cytosine; G=guanine; T=
under transcriptional control, both in pea (Chandler et al. 1983; Evans et al. 1984) and in soyabean (Meinke et al. 1981; Goldberg et al. 1981). The legumin genes of pea make up a small gene family, with four to five closely homologous genes encoding the major legumin polypeptides (Croy et al. 1982). Several legumin genes from pea have been cloned (Croy, R.R.D., personal communication), and the complete sequence of a gene (Leg A) transcribed in vivo including the whole protein-coding region (containing three short introns), and the 5" and 3' flanking sequences has been reported recently (Lycett et al. 1984). The activity of putative "short-range" controlpromoter sequences has been assayed in vitro using a transcription system based on a whole cell extract from human tumour (HeLa) cells. This system has been shown to contain all the factors and enzymic activities required for specific transcription of exogenous DNA templates by RNA polymerases I, II and III (Manley et al. 1983). Although s u c h whole-cell extracts do not seem able to transcribe accurately genes normally transcribed by RNA polmerase II in yeast, several other genes from higher eukaryotes and their viruses have been specifically transcribed (Proudfoot et al. 1980; Wasylyk et al. 1980; Handa et al. 1981; Tsujimoto et al. 1981; H6rcher and Seifart 1984), and hence it could be concluded that RNA polymerase II in the HeLa system does not display a strict species/ tissue specificity. The transcripts synthesised were efficiently capped and methylated at their 5' ends (Manley et al. 1980) and m R N A precursors were polyadenylated (Manley 1983), but splicing activities, although occurring (Kole and Weissman 1982), were not reproducibly detected (Weing/irtner and Keller 1981). The present paper reports experiments designed to test the usefulness of the HeLa cell lysate in-
I.M. Evans et al. : Transcription of a legumin gene from pea
vitro transcription system to assay control-promoter sequences for legumin gene transcription. Materials and methods Materials Restriction endonucleases were from Boehringer Corporation (London, UK), Bethesda Research Laboratories (BRL; Cambridge, UK) or New England Biolabs (CP Laboratories, Bishop's Stortford, UK). Deoxyribonuclease I (DPFF) was obtained from Worhington Biochemicals (Millipore U.K., London, UK), bovine alkaline phosphatase, T4 polynucleotide kinase and S1 nuclease were from Boehringer. Deoxy- and dideoxynucleotide triphosphates were from P-L Biochemicals (Northampton, UK). et-[32P]Uridine-5"-triphosphate (UTP; 15.17 TBq/mmol) and 7-[32P] ATP (185 TBq/mmol) were from Amersham International (Amersham, Bucks., UK). The HeLa cell lysate or transcription-system kit were obtained from BRL.
Me~o~ Full details of cloning, isolation, restriction mapping and sequencing of )~ Leg 1, the pea genomic clone containing legumin gene Leg A have been given elsewhere (Lycett et al. 1984).
Template DNA. The legumin gene used, Leg A, was obtained by subcloning sections of the genomic clone )~ Leg 1 into the plasmid vector pUC 8. The area of )~ Leg 1 covered by the subclones is shown in Fig. 1. Whenever complete Leg A was used, this refers to the subclone containing an insert extending from a Bam HI site approx. 1300 base pairs (bp) upstream from the start of transcription to a Barn HI site approx. 270 bp downstream of the observed end of the legumin m R N A (pDUB 24). The Leg A insert was isolated by digestion of the plasmid with the appropriate restriction enzyme; the vector and insert were then separated by electrophoresis on agarose gels. Bands containing D N A were excised, and the D N A was isolated on glass-fibre discs (GF/C; Whatman, Maidstone, Kent, UK) after dissolutiion of the agarose with sodium perchlorate (Chen and Thomas 1981). The D N A was estimated fluorimetrically by the diaminobenzoic-acid assay (Thomas and Farquhar 1978) and by spectrophotometric estimation at 260 nm. Good agreement between the two methods was obtained, and as the latter was more convenient, it was routinely used. Estimation of D N A by band intensities on minigels was found less reliable. The isolated inserts were further digested with restriction enzymes to produce truncated templates. Transcription assay. The HeLa cell-free transcription system was used essentially as described in the BRL protocol, except that transcription was optimised for concentration of D N A template, salt and HeLa cell lysate. Routinely, transcription was performed in 20-gl reaction mixtures containing 10 lal cell lysate, 100 gg/ml D N A template, 140 I~M ethylenediaminetetraacetic acid (EDTA), 1 mM creatine phosphate, 500 gM each of ATP, guanosine-5'-triphosphate (GTP) and cytidine-5'-triphosphate (CTP), 50/aM UTP and 0.37 MBq of a-[32p]UTP for 1 h at 30 ~ C. Incubation was followed by addition of 100 gl of 7.5 M urea containing 0.5% sodium dodecyl sulfate and 10 m M EDTA, repeated extraction with phenol-chloroform and ethanol precipitation of nucleic acids. Recovered transcripts were denatured with glyoxal and analysed by electrophoresis on 1.4% agarose gels (McMaster and Carmichael 1977). After eleetrophoresis, gels were dried down on a gel drier at room temperature, and autoradiographed at - 7 0 ~ using flashed X-ray film (Fuji RX; Fujimex, Swindon, Wilt.
555 UK) and an intensifying screen (du Pont Lightning Plus; M.A.S. Northern, Aycliffe, Durham, UK).
Hybridisation to imrnobilised DNA. Southern blots of restriction fragments of Leg A-containing plasmids were produced as previously described, except that each track on the gel contained 1 ~tg of digested plasmid. The blots were hybridised to 32p_ labelled transcripts synthesised on a Leg A insert. The conditions for hybridisation were as described by Evans et al. (1984). $1 nuclease mapping. Unlabelled transcripts synthesised on a Leg A template (25 gg) were purified by centrifugation on CsC1 gradients to remove any contaminating D N A (Weingfirtner and Keller 1981). Labelled transcripts were undegraded by this procedure when analysed on glyoxal gels. A D N A fragment from Leg A, 5'-end labelled at the Xho I site + bases relative to the transcription start determined in vivo, and extending to the Pst I site at - 9 7 bases relative to the transcription start was prepared as described elsewhere (Lycett et al. 1984). Hybridisation of the labelled D N A fragment to the unlabelled transcripts and subsequent treatment with S1 nuclease were carried out as described previously (Lycett et al. 1984), using methods of Favaloro et al. (1980) and Pedersen et al. (1982). A hybridisation and Sl-nuclease treatment with polyadenylated R N A from developing pea cotyledons, and with no added RNA, were carried out as positive and negative controls. The processed samples were loaded onto a normal sequencing gel alongside four sequencing tracks of the Xho I-Pst I fragment of Leg A prepared as described (Lycett et al. 1984).
Optimisation of the HeLa cell lysate transcription system for the whole legumin gene, Leg A, established conditions where the DNA-template concentration was 100 gg/ml and the lysate made up 50% of the total reaction volume. The transcripts produced were examined by glyoxalation, electrophoresis on agarose gels and autoradiography, and were seen to contain no molecules of discrete size; instead a disperse smear of transcripts, ranging in size from approx. 100-2000 bases was observed. (A discrete R N A species occasionally seen at approx. 2100 bases was not due to an exogenous template or the activity of R N A polymerase II, and was possibly a result of end-labelling ribosonal RNA.) Although discrete transcripts were not produced by Leg A, transctiption was being carried out by R N A polymerase II, since it was almost completely inhibited by a-amanitin at 0.5 gg/ml. Addition of wheat-germ R N A polymerase II at 2 u/ml did not stimulate transcription, indicating that the polymerase level was not limiting. The addition of RNAsin (human placental ribonuclease inhibitor) had a detrimental effect on transcription. The extent of the transcribed stretch of the legumin gene, Leg A, was estimated by hybridising the labelled transcripts to a Southern blot of restriction fragments from the gene. The restriction
I.M. Evans et al. : Transcription of a legumin gene from pea
B ] ....
I 'l'l"' ....
NWO AV L I / /
C ' ....
it I I
DP X , ,l',
B H ,,~,
I~ 'CAAT' .
10b Fig. 1. Restriction map of Leg A-gene region. 1: Restriction sites and a scale in kilobases; subclones pDUB 24 and pDUB 21 are covered by the regions B-B and H-H, respectively. 2: Coding regions of the sequenced gene are designated by heavy lines. 3: Expanded 5" end of the gene; / = s t a r t of coding sequence; H = transcription start point (beginning of a " C A T C " sequence). Symbols for restriction-enzyme cleavage sites are: A=AccI, B = B a m H I , C=HincII, D = N d e I , G=BglII, H = Hind III, L = BglI, N = BstNI, O = XhoII, P = Pstl, V= AvaI, W = AvaII, X = XhoI
9map of Leg A is shown in Fig. 1. The observed pattern of hybridisation, shown in Fig. 2, indicated that the majority of transcripts were starting in the region of the Pst I and Hind III sites, at - 9 7 and + 40 relative to the known transcription start of Leg A (Lycett et al. 1984). The transcripts hybridised strongly to all restriction fragments including the earlier part of the legumin coding sequence, but hybridised less strongly to fragments towards the 3' end of the coding sequence. Nevertheless, some transcripts extended at least as far as the Nde I site ( + 1665 relative to the transcription start). When Leg A was restricted with enzymes that cut within the first one-third of the coding sequence, and then used as a template in the HeLa system, discrete transcripts were observed. With templates terminated at the 3" end by Hinc II (site at +517 relative to transcription start), a single transcript of estimated size 560 bases (Fig. 3) was the major product. With templates terminated at the Acc I site (+ 901) three transcripts, of estimated sizes 920, 830 and 760 bases were obtained. The transcripts produced by Acc I-truncated template were less prominent components of total transcripts than the transcript produced by Hinc IItruncated template. The effects of truncating the template at its 5' end were also studied. When fragments of Leg A which started at the Hind III site ( + 40), and which terminated at the Hinc II or Acc I sites were used as templates, no specific transcripts were observed (results not shown). However, template starting at the Pst I site ( - 9 7 ) and terminating at the Hinc II or Acc I sites gave similar specific transcripts
Fig. 2. Southern blot analysis of the restriction digests of Leg A region (B-B in Fig. 1). 1 pg of plasmid containing Leg A was digested with BamHI and the specified restriction enzymes, electrophoresed and the gel blot hybridised to 32p-RNA synthesised in the HeLa whole-cell lysate by the Leg A isolated insert (2.7.10 s cpm). Track 1 = H i n c I I ; track 2 = H i n d III; track 3= AccI ; track 4: Pst ]; track 5 = BglI; track 6 = BamHI only; track7=vector pUC 8 + B a m H I only; track 8= Aval; track 9 = NdeI
to those produced by the whole Leg A terminated at the appropriate sites. A further comparison was made between the transcripts produced by the Pst I-Hinc II ( - 9 7 - + 5 1 7 ) and Ava II-Hinc II ( - 2 3 5 - + 5 1 7 ) fragments. Both fragments gave the same specific transcript, with the shorter fragment being the more efficient template. The start of transcription for the transcripts produced in the HeLa system was estimated by Sl-nuclease mapping, comparing the fragment protected by the transcript to that protected by polyadenylated R N A from developing pea seeds (Lycett et al. 1984). Results (Fig. 4) from the invitro transcripts showed a much lower degree of protection compared with mRNA, but it was possible to localise the in-vitro transcription start that encompassed the CATC (cytosine-adenine-thymine-cytosine) sequence identified as the transcription start in vivo. A further weaker transcription start was indicated approx. 10 bp downstream, as observed for transcription in vivo.
I.M. Evans et al. : Transcription of a legumin gene from pea
Fig. 3. Analysis of transcripts of Leg A gene truncated with specified restriction enzymes. Purified transcripts were glyoxalated, run on 1.4% agarose gels and the dried gels were autoradiographed. The following D N A templates were used for transcription: track 1 = L e g A insert cut with AccI used in routine transcription assay at 100 gg/ml; track 2 = a s in track 1 +0.5 gg/ml a-amanitin; track 3 = as in track 1 but at 150 gg/ml; track 4: Leg A insert + AccI; track 5: Leg A insert + HincII; track 6 = Leg A insert + AccI + PstI; track 7: Leg A insert + Pstl ; track 8: Leg A insert + HincII + PstI; track 9: Leg A whole plasmid + BamHI + HinclI + PstI; track 10: Leg A whole p l a s m i d + B a m H I + H i n c I I + A v a I I ; track l l = S V 4 0 + P s t I at 50 gg/ml; track 1 2 = 5 ' - e n d labelled ?~ phage D N A + EcoRI + HindIII used as M r markers
The results presented above show that the pea legumin gene, Leg A, is only transcribed poorly by the human cell transcription system. This is in contrast to the maize zein gene, which is transcribed effectively in the same system (Langridge and Feix 1983). However, the promoter region in the legurain gene appears to be active at a low level, since transcripts produced by R N A polymerse II can be shown to originate from the region immediately 5' to the start of the coding sequence of the gene. The legumin gene, Leg A, has sequences in this region which are homologous to the established short-range control-promoter sequences shown in
other eukaryotic genes (Messing et al. 1983); it has both a " T A T A ' " box at - 3 3 bases (relative to the transcription start) and a " C A A T " box at - 9 0 (Lycett et al. 1984). These features have been shown to be important in other eukaryotic genes for transcriptional activity, and the TATA box in particular appears to be necessary for activity in the HeLa system (Concino et al. 1984). The legumin gene would therefore be expected to be actively transcrilbed in the HeLa system. The low level of transcription of this gene cannot be simply explained, since the factors which determine the transcriptional effectiveness of different genes in the HeLa system are not well understood. Transcription of zein genes in the HeLa system
I.M. Evans et al. : Transcription of a legumin gene from pea
gave rise to transcripts representing the whole of the coding sequence of the gene, up to 1800 bases in length. However, we were unable to demonstrate transcripts of Leg A that covered the whole coding sequence, and the results of hybridising the transcripts produced to restriction fragments of the gene indicate that this is the consequence of premature non-specific termination of the transcripts. Attempts to transcribe templates truncated at their 3' ends showed that termination occurred for most transcripts after the Hinc II site (+517), and for many transcripts after the Acc I site (+901), but few transcripts extended much further. Leg A is A-T rich overall, but its sequence after the Acc I site contains several guanine-cytosine (G-C)-rich regions, and the combination of the two features may be giving rise to non-specific termination, analogously to termination of prokaryotic transcripts at A-T regions after G-C regions. The length of the transcript to the Hinc II site (560 bases) is in reasonable agreement with the predicted length. In the case of transcripts to the Acc I site, it is probably the longest one (920 bases) that represents the correct run-off transcript, the other two either being early termination products or even spliced products (estimated lengths 814 and 727 bases); high salt concentrations or suboptimal lysate abolished the longest transcript, possibly by favouring termination. The discrepancy between estimated and observed transcript lengths is likely to be due to inaccuracies in the gel system since the Sl-mapping experiment clearly indicated a correct transcription start; a second initiation point is unlikely. The short-range control-promoter sequences of Leg A seem to function similarly in the HeLa system to the in-vivo situation, since the transcription start deduced by $1 mapping is the same in both cases. This is probably an efffect of the TATA box, which is thought to be important in determining transcription starts (Concino et al. 1984; Brady et al. 1984; Messing et al. 1983). Removal of the
Fig. 4. Sl-nuclease mapping of the transcription start point " i n vitro". Unlabelled transcripts from Leg A gene (track 3 = transcripts from 5 gg insert; traek4=from 0.8 I~g insert tuncated with AccI) were hybridised to D N A fragments 5" end-labelled at the X h o l site near to the initiation codon and extending to the PstI site directly upstream. Polyadenylated R N A from pea cotyledons was hybridised in a parallel experiment (track 1 = 0 . 3 gg; track5=1.2 ~tg). A sequencing ladder prepared from the same end-labelled D N A fragment was run alongside (tracks G,A,T,C). Track 2 is a control (no RNA). The gel had to be grossly over exposed to show the protection of D N A template by the transcripts
I.M. Evans et al. : Transcription of a legumin gene from pea
region containing the CAAT and TATA boxes abolishes transcription of Leg A in vitro further confirming the functional importance of these sequences. Mutations to the T A T A box are known to affect the expression of other eukaryotic genes in the HeLa system, and this gene would be expected to be affected in a similar fashion. The effect of sequences further upstream from the CAAT box on transcription of Leg A was tested by assessing the expression of 5' truncated templates. These experiments indicated that the putative " A G G A " box (Messing et al. 1983) was not active in the HeLa system, since this sequence contained the Pst I site, and truncation at this site did not affect transcription. Further, the similar results obtained from templates truncated at the Pst I and Ava II sites indicated that the "enhancer" sequence showing homology to the core element of the adenovirus enhancer sequence (Hearing and Shenk 1983) was not having any effect either, since this " e n h a n c e r " is situated between the Pst I and Ava II sites (at -148). Although in the case o f SV40 viral late genes and histone genes, sequences other than the TATA box, situated at least 110 bases upstream from the transcription start, have been shown to be required for efficient in-vitro transcription (Grosschedl and Birnstiel 1982; Brady et al. 1984), in the case of adenovirus-2 late genes and the ovalbumin gene, sequences upstream from - 6 6 and - 6 1 bases, respectively, from the transcription start have no effect (Hu and Manley 1981; Tsai et al. 1981). Consequently the present results cannot be taken as evidence against the putative roles of sequences upstream from the Pst I site ( - 9 7 ) of the 5' flanking sequence of Leg A in vivo. Although the control SV40 viral template was efficiently transcribed in our assays, a construct containing the SV40 promoter-enhancer region located 5' to the legumin gene was not, and transcripts originating at t h e legumin transcription start were not obtained, suggesting that Leg A is not an effective template in this system even in the presence of enhancers. The failure to detect any effect of sequences 5" to the Pst I site ( - 97) of Leg A on transcription in vitro possibly reflects the absence of speciesspecific control factors in the HeLa system. The legumin gene may well require such factors for its effective transcription, since no transcripts were detected from cloned Leg A fragments microinjected into Xenopus oocytes (results not presented); this system would be expected to contain all the non-specific factors necessary for active transcription. This behaviour is in contrast to the maize zein gene, which is effectively transcribed in oo-
cytes as well as in the HeLa system (Langridge and Feix 1983). The authors would like to thank Professor Hugh Woodland for carrying out the experiments using Xenopus oocytes, and Russell Swinhoe, Margaret Richards and Paul Preston for technical assistance. This work was supported by a grant from the Agriculture and Food Research Council.
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Received 5 February; accepted 15 March 1985