Identification of tomato golden mosaic virus-specific RNAs in infected plants

VIROLOGY

170,243-250

(1989)

Identification

of Tomato Golden Mosaic Virus-Specific

RNAs in Infected

Plants

GARRY SUNTER, WILLIAM E. GARDINER,’ AND DAVID M. BISARO’ Department

of Molecular

Genetics and The Ohio State Biotechnology Received December

Center, The Ohio State University,

Columbus, Ohio 432 10

1) 1988; accepted February 3, 1989

The bipartite genome of the geminivirus tomato golden mosaic virus (TGMV) contains at least six open reading frames (ORFs) with the potential to code for proteins of greater than 100 amino acids. In order to investigate the expression of these coding regions, RNA preparations from plants infected with TGMV have been examined for the presence of viral transcripts. We have identified six polyadenylated, virus-specific RNAs which correspond in size, polarity and map location to the six ORFs. Primer extension and S, nuclease analysis of an RNA which maps to the viral coat protein gene (ORF ARl) has shown that this transcription unit begins at nucleotide 319 or 320 and ends in the vicinity of nucleotide 1090 of the TGMV A sequence, in agreement with a previous report (I. T. D. Petty, R. H. A. Coutts, and K. W. Buck, 1988,). Gen. Viral 69, 1359-1365). The data presented here confirm the bidirectional transcription strategy implied by the arrangement of ORFs on both strands of double-stranded TGMV DNA intermediates and lay the groundwork for further studies of viral transcription and its control. 0 1989 Academic Press, Inc.

INTRODUCTION

stranded DNAforms, that could encode proteins larger than 10,000 Da (Hamilton et al., 1984). Four of the ORFs lie on component A and two on component B, and all have conserved counterparts in the genomes of other bipartite geminiviruses (Howarth and Goodman, 1986). An ORF arrangement similar to that of the A component is also apparent in the genomes of all monopat-tite geminiviruses sequenced thus far. Such a genome organization, with ORFs on both strands of a circular double-stranded DNA template, strongly suggests a common bidirectional transcription strategy. This has been substantiated in the case of CLV (Townsend et a/., 1985) and MSV (Morris-Krsinich et al., 1985) by the identification in infected plant extracts of virus-specific polyadenylated transcripts which map to both strands of the viral DNA. The data presented in this report demonstrate that extracts prepared from plants infected with TGMV also contain virus-specific, polyadenylated RNAs transcribed from both strands of the viral DNA. These RNAs have been characterized further and mapped to specific locations on either the A or B genome component by Northern blot hybridization using region- and strand-specific probes, and by low resolution S, nuclease analysis using full-length viral DNA probes. The 5’- and 3’-termini of a viral transcript which spans the coat protein gene have been further defined using both primer extension and high resolution S, nuclease mapping techniques, and our results confirm those of Petty eta/. (1988). The information obtained from these experiments is compared with transcription data available for CLV and MSV and is discussed in relation to potential transcriptional regulatory signals.

Tomato golden mosaic virus (TGMV) is a whiteflyborne agent that belongs to the geminivirus group, whose members are characterized by geminate virions and circular single-stranded DNA genomes (for review see Davies et al., 1987; Lazarowitz, 1987). The genome of TGMV is divided between two DNA components, designated A and B (Bisaro eta/., 1982) both of which are required for infectivity (Hamilton et a/., 1983) and both of which are represented in circular, doublestranded DNA forms found in infected cells (Hamilton et al., 1982). The A component encodes all viral functions necessary for the replication and encapsidation of viral DNA, while the B component encodes functions necessary for the movement of the virus throughout the infected plant (Rogers et al., 1986; Sunter et a/., 1987). Other whitefly-transmitted geminiviruses, for example, cassava latent virus (CLV) (Stanley and Gay, 1983) also have bipartite genomes and in general appear quite similar to TGMV. In contrast, a second subgroup of geminiviruses are leafhopper transmitted and have single component genomes. This subgroup is represented by maize streak virus (MSV) (Mullineaux er al., 1984; Howell, 1984; Lazarowitz, 1988) the type-member of the geminivirus group. Sequence analysis of TGMV DNA has revealed at least six open reading frames (ORFs). located on the viral and the complementary strands of double’ Present address: Department of Biological Sciences, Mississippi State University, Starkville, MS 39762. ’ To whom requests for reprints should be addressed. 243

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244

SUNTER, GARDINER, AND BISARO

MATERIALS

AND METHODS

DNA techniques All restriction endonucleases and other DNA-modifying enzymes were used as recommended by the suppliers. Other techniques were performed according to Maniatis et al. (1982) unless otherwise stated. All nucleotide coordinates used refer to the TGMV sequence of Hamilton eta/, (1984). Inoculation

of plants

Nicotiana benthamiana plants were agroinoculated at the six- to eight-leaf stage. Agrobacterium cells containing tandem copies of the TGMV A and B components were applied to cut stems as previously described (Elmer eta/., 198813). Leaves from plants showing systemic symptoms were harvested 14 to 2 1 days postinoculation. RNA isolation Total cellular RNA was isolated from uninoculated and TGMV-infected N. benthamiana plants according to the procedure of Rochester et a/. (1986). RNA was ethanol precipitated at -20” overnight, recovered by centrifugation, and resuspended in sterile distilled water. The RNA was stored in aliquots at -80”. Small amounts of cellular RNA were incubated at 37” for 15 min in 10 mM Tris, pH 7.4, 10 mM MgCI,, containing 10 pg/ml DNase I (RNase-free, Promega) and 2 units/ ml RNasin (Promega). Reactions were extracted with an equal volume of phenol/chloroform and the RNA was precipitated with ethanol. Total cellular RNA, free from contaminating DNA, was resuspended in sterile distilled water and aliquots were stored at -80”. Poly(A)+ RNA isolation Total cellular RNA samples (1-l 0 mg) at concentrations of l-2 mg/ml were heat-denatured and fractionated on oligo(dT) cellulose columns (Collaborative Research) as described by Bantle et al. (1976). Eluted poly(A)+ RNA was precipitated with ethanol, resuspended in sterile distilled water, and treated with DNase I as described above. Finally, poly(A)+ RNA, free from contaminating DNA, was resuspended in sterile distilled water and stored in aliquots at -80“. Northern

blot hybridization

RNA samples were denatured in glyoxal according to the procedure of McMaster and Carmichael (1977). Up to 10 pg of poly(A)+ RNA was incubated at 50”for 60 min in the presence of 1 1\/1glyoxal, 50% (w/v) dimethyl sulfoxide in TAE buffer (12 mn/r Tris-acetate, pH 7.0,

6 mn/l sodium acetate, 0.3 mM EDTA). Samples were cooled on ice prior to electrophoresis through a 1.5% agarose gel in TAE buffer and transferred to GeneScreen (New England Nuclear) or Nytran (Schleicher and Schuell) membranes in 10X SSC. Membranes were baked and then prehybridized for 4 hr at 42” in 50% formamide, 5X SSC, 5x Denhardt’s solution, 0.1% SDS, 100 pg/ml denatured salmon DNA (200 PI/ cm’). Hybridizations were carried out overnight at 42” in the same solution (50 pl/cm2) containing a radioactively labeled probe. Washes were carried out in 2x SSC, 0.1% SDS at room temperature for 5 min and 30 min, followed by two washes at 65” in 0.1 X SSC, 0.1% SDS. Northern blots were hybridized with a variety of highspecific-activity RNA probes prepared from TGMV subclones in the pGEM plasmids (Promega) discussed below. Plasmid DNA templates were cleaved downstream from the bacteriophage SP6 or T7 polymerase promotors and incubated with all four ribonucleotides (including [a-32P]CTP, >800 CilmM, New England Nuclear) and either SP6 or T7 RNA polymerase to yield region-specific probes of the viral (+) or complementary (-) sense. RNA size markers were obtained in the same manner using unlabeled ribonucleotides. Construction

of TGMV subclones

Defined regions of the TGMV A and B genome were subcloned between the bacteriophage SP6 and T7 RNA polymerase promoters which lie in opposite orientation in the pGEM plasmids. To prepare the subclones, TGMV A or B DNA was cleaved with the appropriate restriction enzymes, made blunt-ended, and ligated into the pGEM3 (B-DNA) or pGEM4 (A-DNA) plasmids cleaved at the unique Smal site in the multilinker. The pGEM-derived plasmids and the inserts they contain are described further below and illustrated in Fig. 1. Full-length component A and B inserts in phage M 13mp8 (Messing and Viera, 1982) were obtained by cloning the TGMV DNAs at their unique fcoRl sites. The recombinants are referred to as TGAE 101 and TGBE 105, respectively. A full-length component A clone at the Xhol site (pTGAS 210) was also constructed in pGEM 3Zf+ (Promega). pMON 309, which contains four tandemly repeated copies of the B component linearized at the C/al site, has been described previously (Rogers et al., 1986). S, nuclease mapping Poly(A)+ RNAs were mapped according to the procedure of Berk and Sharp (1977) as modified by Favaloro et a/. (I 980). Varying amounts of polyadenylated RNA

245

TGMV TRANSCRIPTS

isolated from infected plants were used to protect different full-length TGMV A or B DNAs: 9 pg with single-stranded TGAE 101 (10 pg), pTGAS 210 (1 pg) or TGBE 105 (1 pg) DNA, or 2 gg with Bglll restricted pMON 309 DNA (0.5 pg). All hybridizations were carried out at 42” for 16 to 18 hr. Following incubation with S, nuclease and isopropanol precipitation, resistant RNA/DNA hybrids were electrophoresed through 1.5% alkaline agarose gels and neutralized. Alternatively, RNA/DNA hybrids were hydrolyzed in 0.15 N NaOH/l 0 mM EDTA at 60” for 15 min, neutralized, and precipitated. The DNA fragments were then recovered and analyzed on glyoxal gels (McMaster and Carmichael, 1977). Following electrophoresis, gels were transferred to GeneScreen or Nytran membranes and hybridized with TGMV strand-specific probes. Labeled RNA probes of the same sense as the protecting RNA were used to identify DNA fragments. For high resolution S, mapping of the 5’ and 3’ ends of the AR1 transcript, selected restriction fragments of the A component were isolated from agarose gels and labeled either at the 5’ end using [Y-~‘P]ATP (>3000 CilmM, New England Nuclear) and T4 polynucleotide kinase, or at the 3’ end using [a-32P]dCTP (>800 Ci/ mM, New England Nuclear) and the Klenow fragment of DNA polymerase I. The labeled DNA fragments were digested with an appropriate restriction enzyme to remove one of the labeled termini and probe fragments were reisolated from agarose gels. Polyadenylated RNA from TGMV-infected (5 pg) or uninoculated (5 pg) plants was annealed to 5’ end labeled (ml 50,000 cpm) or 3’ end labeled (-10,000 cpm) DNA probes under the same conditions used for low resolution S, mapping. Protected DNA fragments were resolved on 6% polyacrylamide sequencing gels @anger and Coulson, 1978; Garoff and Ansorge, 1981). Primer extension Total cellular RNA (15-30 pg) and poly(A)+ RNA (2.55.0 pg) from TGMV-infected plants, as well as total cellular RNA (15-30 pg) and poly(A)+ RNA (2.5-5.0 pg) from uninoculated plants, were primed with a 32P-labeled synthetic oligomer (“2 X 1O6cpm) and extended along the RNA template with reverse transcriptase as described by Shah eta/. (1986). A 2 1nucleotide primer complementary to nucleotides 425-405 of component A WATCACGCTTAGGCAAACTTCC-3’) was used to locate the 5’ end of the AR1 transcript. Primer extension products were resolved on 6%~polyacrylamide sequencing gels. Nucleotide sequencing TGMV A DNA was cloned into the bacteriophage M 13mp8 at the EcoRl site (M 13 derivative TGAE108)

and its sequence determined by dideoxynucleotide chain termination (Sanger et al., 1977) using the 21mer primer described above. Sequencing and primer extension reactions were electrophoresed in adjacent lanes on 6% polyacrylamide gels to permit the exact sizing of primer extension products. Following electrophoresis, gels were fixed in 1O”b acetic acid and dried for autoradiography. RESULTS Construction RNA probes

of TGMV A- and B-specific

Full-length TGMV A and B DNAs were cloned at their unique EcoRl sites into pGEM 4 and 3, respectively, to allow the preparation of high-specific-activity RNA probes of either the viral or the complementary sense using SP6 or T7 RNA polymerase. The resulting constructs were designated pTGA 6 and pTGB 14. DNA fragments internal toTGMV ORFs were also subcloned into the Smal sites of the pGEM plasmids to permit synthesis of probes specific for defined regions of the virion or complementary strands of the genome. Figure 1 shows the location of ORFs in TGMV A and B DNA and illustrates the regions of each that the subclones cover. The subclone constructs include pTGA 10 (528 bp Xhol-Accl fragment), pTGA 12 (437 bp C/al-&oRI fragment), pTGA 17 (279 bp Accl-Dral fragment), pTGB 17 (720 bp EcoRV-Noel fragment), and pTGB 18 (328 bp C/al-EcoRI fragment). Identification

of TGMV-specific

RNAs

Northern blots of poly(A)+ RNA obtained from TGMVinfected N. benthamiana leaves are shown in Fig. 2. Hybridization with a full-length TGMV A plus-strand probe revealed three virus-specific RNA species (Fig. 2, A+) of approximately 1640, 1040, and 710 nucleotides. These three RNAs are of the complementary sense and thus are transcribed from the viral strand DNA template. In contrast, only one major RNA of 860 nucleotides was detected with a minus-strand A probe (Fig. 2, A-). This RNA is of viral sense and is transcribed from the complementary strand DNA template. TGMV B plus- and minus-strand probes each hybridized with only one RNA species of 1330 and 920 nucleotides, respectively (Fig. 2, B+ and B-). The 1330-nucleotide RNA is complementary sense and the 920-nucleotide RNA is viral sense. The RNAs identified as TGMV-specific were sometimes found in poly(A)- RNA preparations from infected plants, but in much lower amounts than in comparable poly(A)+ fractions. RNAs capable of hybridizing with

246

SUNTER, GARDINER, AND BISARO

Common

A 1)

2588bo

AL2

.-.-

FIG. 1. Maps of double-stranded TGMV A and 6 component DNAs. The maps and nudeotide coordinates are drawn from the sequence of Hamilton er al. (1984). The shaded arrows show the positions of open reading frames and the open box indicates the region common to both DNAs. The arrows inside the circles define the regions subcloned for use in preparing hybridization probes.

TGMV-specific probes were not found in extracts from uninoculated plants (data not shown). Mapping of TGMV-specific RNAs The map positions of the polyadenylated RNAs transcribed from the TGMV A and B component DNAs were located by Northern blot analysis using RNA probes specific for defined regions of the virion or complementary strands of the TGMV genome (see Fig. 1).

A+

A-

B+

A probe internal to ORF AR1 detected only the 860nucteotide RNA (Fig. 3, lane l), which indicates that this transcript traverses all or part of the Xhol-Accl fragment (nucleotides 40 l-929) of the TGMV A component. A probe which spans a region within ORF AL1 hybridized with the 1640- and the 1040-nucleotide RNAs, while a probe specific for a small region within ORF AL3 hybridized with rhe 1640-, 1040-, and 710nucteotide RNAs (Fig. 3, lanes 2 and 3). Thus two of the three A component complementary sense transcripts contain sequences from the C/al-EcoRI fragment (nu-

B-

1

164Ow 1040660-

2

3

4

5

3330 -920

fl o-

FIG. 2. TGMV-specific polyadenylated RNAs in infected Nicotiana benthamiana leaves. Aliquots of poly(A)+ RNA (2 gg) were fractionated on agarose gels and transferred to Genescreen. The immobilized RNA was hybridized to probes specific for either strand of the TGMV A or B component, as indicated.

FIG. 3. Mapping of TGMV RNAs. Either 2 qg (lanes 1,4, and 5) or 6 pg (lanes 2 and 3) of total cellular poly(A)+ RNA from infected plants was hybridized with probes derived from subclones of the A (lanes 1,2, and 3) and B (lanes 4 and 5) genome components. The probes were specific for the following fragments: Lane 1, Xhol-Accl; lane ‘2, EcoRI-C/al; lane 3, Bat-Accl; lane 4, C/al-EcoRI; lane 5, NdelEcoRV.

247

TGMV TRANSCRIPTS 1

2

3

4 4

990-

-650

790620‘;, .“ab :_

:

FIG. 4. Low resolution S, mapping of TGMV transcripts. Total cellular poly(A)+ RNA from infected plants was annealed with full-length TGMV DNA, and nuclease-resistant DNA fragments were subjected to Southern blot analysis. The senses of the probes used to detect protected DNA fragments were as follows: Lane 1, A(+); lane 2, A(-); lane 3, B(+); lane 4 B(-).

cleotides 1815-2252) while all extend into or past the AC&&al fragment (nucleotides 929-l 208). These data indicate that the 1640-nucleotide transcript spans all three leftward coding sequences (ORFs AL1 ,2, and 3) while the 1040- and 7 10-nucleotide transcripts traverse ORFs AL2 and AL3. The data are also consistent with the idea that the three transcripts share a common 3’-terminus. When probes internal to the two ORFs on the TGMV B component were used, the 920-nucleotide RNA hybridized with the BRl probe while the 1330-nucleotide RNA hybridized with the BLl probe (Fig. 3, lanes 4 and 5, respectively). Thus the former is transcribed across all or part of the TGMV B fragment between nucleotides 323 and 651 (C/al-EcoRI) and the latter is transcribed across all or part of the TGMV B fragment from nucleotides 1740-2460 (EcoRV-Noel). Low resolution

the three complementary sense RNAs previously identified by Northern blot analysis. Only one complementary sense TGMV B DNA fragment (850 nucleotides) was protected by poly(A)+ RNA when double-stranded TGMV B DNA, which was band-isolated from Bglll-digested pMON 309 DNA, was the probe (Fig. 4, lane 3). When DNA obtained from an M 13 fcoRl clone of the 8 component (TGBE 105) was used, only a single 1300nucleotide fragment of protected DNA was observed (Fig. 4, lane 4). The protected DNA fragments correspond with the RNA species identified by Northern blot analysis (Figs. 2 and 3) and the 30-to 1OO-nucleotide size differences between them are likely due to the 3’ poly(A) tracts in the RNAs. Therefore, large-scale RNA processing does not appear to play a role in the generation of these TGMV transcripts, although removal by splicing of a small number of nucleotides k50) near their 5’- or 3’termini would have escaped detection at the level of resolution employed here. Fine mapping of the AR1 transcripts The locations of the termini of an RNA that spans ORF AR1 and encodes the viral coat protein were more precisely determined by high resolution S, analysis and primer extension (Favaloro et a/., 1980; Shah et al., 1986). Poly(A)+ RNA protected one major 6 10-nucleotide DNA fragment from S, nuclease digestion when a 772nucleotide Paul-Accl fragment (nucleotides 157-929) was used as probe (Fig. 5a, lane 2). This places the 5’ end of the AR1 transcript at nucleotide 319, only 8

a

b

S, nuclease mapping

In order to determine more accurately the sizes of the viral transcripts and to investigate whether any are processed from larger precursors, polyadenylated RNA isolated from infected cells was hybridized with single- or double-stranded probes containing a fulllength TGMV A or B sequence. The resulting hybrids were then analyzed by the S, nuclease mapping procedure. Polyadenylated RNA protected a single 790-nucleotide fragment when complementary sense DNA from an Ml 3 fcoRI clone of TGMV A (TGAE 101) was used as probe (Fig. 4, lane 1). In contrast, protection of viral sense TGMV A DNA derived from an Xhol clone (pTGAS 210) yielded three fragments (1590, 990, and 620 nucleotides; Fig. 4, lane 2) which correspond to

FIG. 5. S, nuclease mapping of the 5’ (a) and 3’ (b) termini of the TGMV AR1 transcript. Total cellular poly(A)+ RNA was annealed to labeled DNA fragments described in the text, and nuclease-resistant DNAs were run on 6% sequencing gels and subjected to autoradiography. Size markers from Haelll-digested 6,X 174 DNA and from sequencing ladders are indicated in numbers of nucleotides. The 4X markers appear to the left and the sequencing ladder markers to the right of each panel. Lanes 1 and 3 in (a) contain RNA from uninoculated plants, and the remainder contain RNA from TGMV-infected plants.

248

SUNTER, GARDINER, AND BISARO v

c

310 -

I

A-

340 -

x

‘fJ

AG

TC

123

FIG. 6. Primer extension analysis of the AR1 transcript. Primer extension reactions were run alongside a sequencing ladder (AGTC) on a 6% denaturing polyactylamide gel and subjected to autoradiography. The viral (V) and complementary(C) sequence around the largest major extension product (*) is given. The AUG initiation codon at nucleotide 327 is boxed. The following RNAs were extended from the primer described in the text: Lane 1, total cellular RNA from infected plants; lane 2, poly(A)+ RNA from infected plants; lane 3, poly(A)+ RNAfrom uninoculated plants.

nucleotides upstream of the AUG initiation codon at nucleotide 327 (Kallender et al., 1988). In a second experiment, several fragments from 79 to 85 nucleotides were protected when a 736-bp /&RI-Xhol fragment (nucleotides 2252-401) was the probe (Fig. 5a, lane 4). These protected fragments place the 5’-terminus between nucleotides 3 16 and 32 1. The observation of several protected DNAfragments in these experiments may reflect heterogeneity in transcriptional start site choice or may be the result of imprecise digestion of DNA/RNA hybrids. None of the fragments was detected when RNA from uninoculated plants was used to protect the probe DNA (Fig. 5a, lanes 1 and 3). Primer extension analysis was performed using a labeled synthetic oligomer complementary to a region downstream of the initiation codon of ORF ARI, and extension products were sized against a sequence ladder generated from an Ml 3 clone of TGMV A DNA primed with the same oligonucleotide. The extension reactions, which contained total or poly(A)+ RNA from plants infected with TGMV (Fig. 6, lanes 1 and 2, respectively), gave two major products and three minor products which were not detected in comparable extensions of RNA from uninoculated plants (Fig. 6, lane 3). The major products represent RNAs with 5’ start sites which map to nucleotides 319 and 320, in good agreement with the S, nuclease protection data discussed above. The minor extension products observed may reflect multiple transcription start sites, or result from premature termination of reverse transcription due to RNA secondary structure, cleavage of the template by RNase, or a combination of both.

The 3’-terminus of the AR1 transcript was also located by S, analysis using a 956-bp Xhol to BarnHI probe fragment (nucleotides 401-l 357) and a 428-bp Accl to BarnHI probe fragment (nucleotides 9291357). After hybridization with polyadenylated RNA from infected plants, one major protected DNA species (690 and 162 nucleotides, respectively) was observed with each probe (Fig. 5b, lanes 1 and 2). These results place the 3’-terminus of theAR1 transcript near nucleotide 1091. The smaller, somewhat diffuse band observed with the 428-bp probe is probably the result of digestion of DNA/RNA hybrids in the A-T-rich region surrounding nucleotide 1080.

DISCUSSION We have found that extracts obtained from plants infected with TGMV contain at least six virus-specific, polyadenylated RNAs. Three of the RNAs (1640, 1040, and 710 nucleotides) are transcribed from the viral DNA strand of the A component; these appear to overlap extensively and may be 3’ co-terminal. Only one RNA (860 nucleotides) is transcribed from the complementary strand of the A component. We have mapped this RNA, which traverses the coat protein gene, between nucleotides 319/320 and 1091, in agreement with the data of Petty et al. (1988). Likewise, only one transcript has been mapped to the viral and complementary strands of the B component (1330 and 920 nucleotides, respectively). The identification of RNAs which map to both strands of the TGMV DNAs is consistent with the bidirectional transcription strategy observed previously for both CLV (Townsend et al., 1985) and MSV (MorrisKrsinich et al., 1985) and proposed for other geminiviruses. All the TGMV transcripts detected appear to have counterparts in CLV with the exception of the 1040-nucleotide RNA, one of three transcribed from the viral strand of TGMV A DNA. Only two RNAs (1700 and 700 nucleotides) derived from the viral strand of the analogous CLV DNA 1 have been found in extracts from N. benthamiana plants infected with this related virus. On the other hand, several minor RNA species (1350, 2000, and 2200 nucleotides) which hybridize with CLV DNA 2 probes have been found in addition to two major 11 OO- and 900nucleotide species (Townsend et a/., 1985). Similar minor RNAs derived from TGMV B have not been detected. The significance of these differences in size and number between TGMV and CLV transcripts is not known at present, and the absence of information concerning the map locations of the CLV RNAs (with the exception of the coat protein transcript) complicates direct comparison. The six TGMV-specific RNAs are of sufficient size and polarity to code for the proteins specified by the

249

TGMV TRANSCRIPTS

ORFs to which they map. It should be pointed out, however, that in the absence of information on the translation of these RNAs it is not possible to state with confidence the protein(s) for which a given transcript might serve as messenger. Low resolution S, nuclease mapping of the TGMV transcripts has not provided evidence for the involvement of RNA processing in the generation of the RNAs detected in these experiments. On the other hand, the occurrence of RNA processing cannot be ruled out since the sensitivity of the gel system employed would not be sufficient to detect the removal of small (~50 nucleotide) introns near the 5’- or 3’-terminus. This does not occur at the S’end of the AR1 transcript, however, because the results of high resolution nuclease protection and primer extension experiments indicate the same transcription star-t site. As mentioned previously, the three complementary sense A component transcripts appear to be 3’ co-terminal, and if this proves to be so, it is likely that they share a single polyadenylation signal (AATAAA) (Proudfoot and Brownlee, 1976) located at nucleotide 1088. The 3’terminus of the AR1 virus sense transcript probably employs a polyadenylation signal found at nucleotide 1069 (Hamilton et al., 1984). This, as well as the observation that ORFs AR1 and AL3 share two of three nucleotides in their termination codons, indicates that there is a short region of overlapping transcription from opposite directions at the 3’ end of the virion and complementary sense transcripts. Overlapping transcription in opposite directions also has been suggested to occur in CLV (Townsend et al., 1985), but evidence for this phenomenon is lacking in the case of MSV (MorrisKrsinich et a/., 1985). The significance of such an overlap is unknown, but it is conceivable that it might play a role in the regulation of transcription. This is particularly interesting in light of the recent finding that the AL1 ORF encodes the only viral gene product necessary for viral DNA replication (Elmer et al., 1988a), while the AR1 ORF encodes the coat protein of the virus which is not required for infectivity (Gardiner et a/., 1988). This raises the possibility of the involvement of transcription overlap in an early to late transcription switch, with events separated by viral DNA replication. The 860-nucleotide transcript which maps to ORF AR1 , the coat protein gene, has its 5’terminus at nucleotides 31 g/320. These are both adenine residues, and adenine appears to be the predominant nucleotide used for eukaryotic transcription initiation (Breathnach and Chambon, 1981; Joshi, 1987). The transcription initiation site is 26 nucleotides downstream of the consensus eukaryotic RNA polymerase II promoter sequence TATAT/AA (Breathnach and Chambon, 1981), and 89 nucleotides downstream of a consensus

CCAAT sequence (GGCCAATC) (Benoist et al., 1980). As the translation initiation codon lies at nucleotide 327 (Kallender et a/., 1988), the transcript has an untranslated leader sequence of only 7 or 8 nucleotides which, although very short, is within the size limits reported by Kozak (1984). This is in marked contrast to the relatively long (- 160 nucleotides) untranslated leader of the lOOO-nucleotide CLV coat protein transcript, which also contains four apparently nonfunctional AUGs before the initiation codon (Townsend et a/., 1985). The significance of this difference, and of the additional AUGs, is presently unknown. The identification and preliminary characterization of TGMV RNAs reported here represent a first step toward the elucidation of viral gene expression and its control. We are currently mapping, by high resolution S, and primer extension analysis, the 5’- and 3’-termini of the remaining RNAs we have identified in order to accurately define the limits of all viral transcription units. These studies will be necessary for subsequent investigations of viral promotor sequences and their possible interactions with host and viral proteins.

ACKNOWLEDGMENTS We thank Dr. S. J. Pilistine for critical reading of the manuscript. Thiswork was supported by USDA Grant 87-CRCR-l-2541.

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