_Archives
Vi rology
Arch Virol (1992) 123:223-228
© Springer-Verlag 1992 Printed in Austria
In vitro recognition of an orf virus early promoter in a vaccinia virus extract Brief R e p o r t J. C. Vos 1, A. A. Mercer 2, S. B. Fleming2, and A. J. R o b i n s o n 2
1European Molecular Biology Laboratories, Heidelberg, Federal Republic of Germany 2Medical Research Council Virus Research Unit and Centre for Gene Research, University of Otago, Dunedin, New Zealand Accepted July 26, 1991
Summary. DNA fragments containing varying lengths of the 5' end of an orf virus early gene (ORF 3) and its associated promoter were introduced into sodium deoxycholate-solubilized vaccinia virus extracts capable of initiating transcription in vitro from vaccinia virus early promoters. After separation of the radiolabelled products of the reactions on a 5% polyacrylamide/7 M urea gel, discrete transcripts were detected the sizes of which were consistent with initiation of transcription from the off virus early promoter. This is the first demonstration in a functional assay of the conservation of early transcriptional promoters between an orthopoxvirus and a parapoxvirus.
Poxviruses are a family of complex, DNA-containing viruses that replicate in the cytoplasm of the infected cell. Vaccinia virus, a member of the orthopoxvirus genus, is the prototype of the family and it has been found that viral mRNA is transcribed, capped, methylated and polyadenylated by enzymes coded for by the virus rather than the host cell [for a review see 15]. Sequence analysis of the viral genome, mapping of transcriptional start points (tsp), and mutational analyses have revealed that vaccinia virus transcriptional promoters are A + Trich and that the particular nucleotide sequence determines whether or not they are active early (in the presence of inhibitors of protein synthesis or DNA replication i.e. before the initiation of viral DNA replication) [3, 4, 13, 19] or late (after the initiation of DNA replication) [5, 10, 18] in the replication cycle. The structures of these promoters are unlike those of the host cell and are not recognized by the cellular RNA polymerases.
224
J.C. Vos et al.
Recognition and binding of the RNA polymerase complex to the early promoter sequence are mediated by a protein termed vaccinia early transcription factor (VETF) [2, 21] which consists of two subunits produced late in infection [1, 8] and is packaged in the virion. In fact all of the enzymatic machinery necessary for early transcription is packaged in the virion and preparations of viral cores or permeabilized virions will make viral RNA from their resident viral genomes under suitable reaction conditions [11, 16]. In a later development it was found that virions treated with sodium deoxycholate will synthesize viral RNA from added DNA templates [9, 17]. Early but not late genes are transcribed and mRNAs produced are capped and sometimes polyadenylated. Early transcription initiates in vitro at or close to the initiation site found in vivo [9, 12, 17]. Orf virus is a member of the poxvirus family [14] and it has a genome which is 64% G + C, unlike vaccinia virus which has a genome with a G + C content of 36% [20]. Sequencing of a 6 kb region of the orf virus genome has revealed a number of open reading frames (ORFs) that are preceded by A + T-rich sequences with similarities to vaccinia virus early promoters [7]. S1 and primer extension analysis of one of these ORFs (ORF 3) showed that the tsp for the gene were on an A or a G, 9-10 nucleotides downstream from the 3' end of the sequence 5'-CAAAGTGAAAAAGGAC in a region of the genome which is otherwise over 80% G + C [6]. Davison and Moss [4] have shown that the vaccinia virus early promoter consists of a 16 base-pair critical region followed by a non-critical 11 base-pair spacer region which is in turn followed by a seven base-pair region within which initiation occurs, usually on a purine. The sequence of the critical region of a vaccinia virus strong early promoter, based on mutational analysis of the 7.5kDa promoter, was 5'-AAAAgTaGAAAataTA where the upper case letters represent nucleotides which could not be substituted without reducing, usually drastically, the promoter activity and the lower case letters represent nucleotides that could be substituted without affecting, and sometimes enhancing, activity. The ORF 3 promoter is a strong promoter in off virus but a comparison of its sequence with that of the vaccinia virus strong promoter suggests that it might not be a strong vaccinia virus promoter and that there might be some differences in the regulation of transcription between the two viruses. We have yet to compare directly the activity of off virus promoters in vaccinia virus, but show here that a soluble extract ofvaccinia virus virions will initiate transcription in vitro at the ORF 3 promoter. Vaccinia virus WR virions and soluble extracts for in vitro transcription were prepared as described by Rohrmann and Moss [ 17]. To prepare templates, a 756 bp SmaI-HpaI fragment from the BamHI E fragment of off virus DNA, which contained the ORF 3 promoter [7], was cloned into the Sinai site of plasmid pTZ18R (Pharmacia, Uppsala, Sweden). A number of clones were isolated and one with the orientation shown in Fig. 1 A was used to prepare the templates. After digestion with PvuII-ApaI, PvuII-RsaI, PvuII-NotI or PuvII alone, fragments of 397, 502, 662, and 694 bp were purified from agarose gels
In vitro recognition of an off virus early promoter in vaccinia virus
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P-P I p-r p-a p-n Fig. 1. Origin of fragments used in the in vitro run-off assays. A The bar represents the relevant section of the plasmid pTZ18R containing a 756 bp SrnaI-HpaI fragments from the orf virus BamHI E fragment. The hatched portion represents pTZ18R DNA, the stippled portion, orf virus ORF 3 from the putative promoter region (P, solid arrow), and the open region, the flanking regions of orf virus DNA. Restriction endonuclease cutting sites are indicated above the bar with the relevant nucleotide from either pTZ18R (sequence available from Pharmacia, Uppsala, Sweden) or orf virus DNA [7]. PvuII (p), Sinai (s), ApaI (a), RsaI (r), NotI (n), and HpaI (h). Represented below the bar are the fragments used in the assays with their sizes in bp. B The nucleotide sequence of the off virus putative promoter region is shown. Underlined is the A + T-rich sequence that shows strong homology with the critical region of vaccinia virus early promoters [4]. The tsp of ORF 3 transcripts in off virus infected cells as determined by Sl ($1) and primer extension analysis (pe) [6]. pp, p-n, p-r, p-a The tsp calculated for transcripts initiated in vitro on the fragments shown in (B) by vaccinia virus RNA polymerase
using glass powder (Fig. 1 A). Reaction mixtures in 30 gl final volumes contained 3 gl extract (5 ng/ml), 50ng fragment, 5 gCi of [32p]GTP, 1 m M ATP, CTP, and UTP, 0.05 m M GTP, 50 m M KCt, 5 m M MgC12, 0.1 m M EDTA, 25 m M Hepes (pH 7.9) and 7.5% glycerol and were incubated for 45 min at 30 °C. The components of the reaction were treated with proteinase K (1 mg/ml final concentration), ethanol precipitated and then separated in a 5% polyacrylamide gel containing 7 M urea in 0.1 M Tris-borate (pH 8.0), 1 m M EDTA. Radiolabelled fragments of an HpaII digest of the plasmid pBR 322 were used as size markers. After autoradiography of the gel (Fig. 2), discrete products corresponding to 190 (PvuII-ApaI), 305 (PvuII-RsaI), 475 (PvuI-NotI), and 510 (PvuI alone) nucleotides, were found. The two shorter run-off transcripts correspond to tsp
226
J.C. Vos et al.
Fig. 2. Autoradiograph of the products of in vitro transcription after electrophoresisin a 5% polyacrylamide-7M urea gel. 1 and 2 Molecular weight markers; 3 products from the PvuII-ApaI fragment; 4 the PvuII-RsaI fragment; 5 the PvuII-NotI fragment; 6 the PvuII-PvuII fragment
at 17 and 12nts downstream from the putative promoter sequence for ORF 3 (Fig. 1 B). The longer transcripts correspond to tsp closer to the promoter. No bands were found in extracts incubated in the absence of the off virus fragments (not shown). Taking into account the inaccuracies inherent in the method of measuring the sizes of transcripts, particularly those of higher molecular weight, this result agrees well with the tsp determined for RNA transcribed in orf virus infected cells. In that case the tsp were at 9 and 10 nucleotides downstream from the putative promoter sequence as determined by S1 nuclease digestion and primer extension respectively. Prominent higher molecular weight bands were seen in all lanes containing the reaction products seen in Fig. 2 and they are particularly prominent in lanes 5 and 6. The sizes of these bands correspond almost exactly with the sizes of the DNA fragments used as templates and we interpret them to represent either an end-labelling reaction or non-specific run-off transcripts that have initiated at the ends of the fragments. The result obtained is a clear indication that vaccinia virus polymerase is capable of recognizing an orf virus promoter despite the otherwise high G + C
In vitro recognition of an orf virus early promoter in vaccinia virus
227
c o n t e n t o f the f r a g m e n t s used in the assay. This is the first d e m o n s t r a t i o n in a functional assay o f the conservation o f early transcription p r o m o t e r s between orthopoxviruses a n d parapoxviruses.
References 1. Broyles SS, Fesler BS (1990) Vaccinia virus gene encoding a component of the viral early transcription factor. J Virol 64:1523-1529 2. Broyles SS, Yuen L, Shuman S, Moss B (1988) Purification of a factor required for transcription of vaccinia .virus early genes. J Biol Chem 263:10754-10760 3. Cochran MA, Puckett C, Moss B (1985) In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. J Virol 54:30-37 4. Davison AJ, Moss B (1989) Structure of vaccinia virus early promoters. J Mol Biol 210:749-769 5. Davison AJ, Moss B (1989) Structure of vaccinia virus late promoters. J Mol Biol 210: 771-784 6. Fleming SB, Fraser KM, Mercer AA, Robinson AJ (1991) Vaccinia virus-like early transcriptional control sequences flank an early gene in orf virus. Gene 97:207-212 7. Fraser KM, Hill DF, Mercer AA, Robinson AJ (1990) Sequence analysis of the inverted terminal repetition in the genome of the parapoxvirus, orf virus. Virology 176: 379389 8. Gershon PD, Moss B (1990) Early transcription factor subunits are encoded by vaccinia virus late genes. Proc Natl Acad Sci USA 87:44014405 9. Golini F, Kates JR (1985) A soluble transcription system derived from purified vaccinia virions. J Virol 53:205-213 10. Hanggi M, Bannwarth W, Stunnenberg HG (1986) Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation. EMBO J 5:1071-1076 11. Kates JR, McAuslan BR (1967) Poxvirus DNA-dependent RNA polymerase. Proc Natl Acad Sci USA 58:134-t41 12. Lee-Chen GY, Bourgeois N, Davidson K, Condit RC, Niles EG (1988) Structure of the transcription initiation and termination sequences of seven early genes in the vaccinia virus HindIII D fragment. Virology 163:64-79 13. Mars M, Beaud G (1987) Characterization of vaccinia virus early promoters and evaluation of their informational content. J Mol Biol 198:619-631 t4. Matthews REF (1982) Classification and nomenclature of viruses. Intervirology 17: 1-199 15. Moss B (1990) Regulation of vaccinia virus transcription. Annu Rev Biochem 59: 661688 16. Munyon W, Paoletti E, Grace JTJ (1967) RNA polymerase activity in purified infectious vaccinia virus. Proc Natl Acad Sci USA 58:2280-2287 17. Rohrmann G, Moss B (1985) Transcription of vaccinia virus early genes by a templatedependent soluble extract of purified virions. J Virol 56:349-355 18. Rosel JL, Earl PL, Weir JP, Moss B (1986) Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment. J Virol 60: 436-449 19. Weir JP, Moss B (1987) Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase. Virology 158:206-210
228 J. C. Vos etal.: In vitro recognition of an orf virus early promoter in vaccinia virus 20. Wittek R, Kuenzle CC, Wyler R (1979) High G + C content in parapoxvirus DNA. J Gen Virol 43:231-234 21. Yuen L, Davison AJ, Moss B (1987) Early promoter-binding factor from vaccinia virions. Proc Natl Acad Sci USA 84:6069-6073 Authors' address: Dr. A. J. Robinson, Medical Reserach Council Virus Research Unit and Centre for Gene Research, University of Otago, P.O. Box 56, Dunedin, New Zealand. Received June 24, 1991
Vi rology
Arch Virol (1992) 123:223-228
© Springer-Verlag 1992 Printed in Austria
In vitro recognition of an orf virus early promoter in a vaccinia virus extract Brief R e p o r t J. C. Vos 1, A. A. Mercer 2, S. B. Fleming2, and A. J. R o b i n s o n 2
1European Molecular Biology Laboratories, Heidelberg, Federal Republic of Germany 2Medical Research Council Virus Research Unit and Centre for Gene Research, University of Otago, Dunedin, New Zealand Accepted July 26, 1991
Summary. DNA fragments containing varying lengths of the 5' end of an orf virus early gene (ORF 3) and its associated promoter were introduced into sodium deoxycholate-solubilized vaccinia virus extracts capable of initiating transcription in vitro from vaccinia virus early promoters. After separation of the radiolabelled products of the reactions on a 5% polyacrylamide/7 M urea gel, discrete transcripts were detected the sizes of which were consistent with initiation of transcription from the off virus early promoter. This is the first demonstration in a functional assay of the conservation of early transcriptional promoters between an orthopoxvirus and a parapoxvirus.
Poxviruses are a family of complex, DNA-containing viruses that replicate in the cytoplasm of the infected cell. Vaccinia virus, a member of the orthopoxvirus genus, is the prototype of the family and it has been found that viral mRNA is transcribed, capped, methylated and polyadenylated by enzymes coded for by the virus rather than the host cell [for a review see 15]. Sequence analysis of the viral genome, mapping of transcriptional start points (tsp), and mutational analyses have revealed that vaccinia virus transcriptional promoters are A + Trich and that the particular nucleotide sequence determines whether or not they are active early (in the presence of inhibitors of protein synthesis or DNA replication i.e. before the initiation of viral DNA replication) [3, 4, 13, 19] or late (after the initiation of DNA replication) [5, 10, 18] in the replication cycle. The structures of these promoters are unlike those of the host cell and are not recognized by the cellular RNA polymerases.
224
J.C. Vos et al.
Recognition and binding of the RNA polymerase complex to the early promoter sequence are mediated by a protein termed vaccinia early transcription factor (VETF) [2, 21] which consists of two subunits produced late in infection [1, 8] and is packaged in the virion. In fact all of the enzymatic machinery necessary for early transcription is packaged in the virion and preparations of viral cores or permeabilized virions will make viral RNA from their resident viral genomes under suitable reaction conditions [11, 16]. In a later development it was found that virions treated with sodium deoxycholate will synthesize viral RNA from added DNA templates [9, 17]. Early but not late genes are transcribed and mRNAs produced are capped and sometimes polyadenylated. Early transcription initiates in vitro at or close to the initiation site found in vivo [9, 12, 17]. Orf virus is a member of the poxvirus family [14] and it has a genome which is 64% G + C, unlike vaccinia virus which has a genome with a G + C content of 36% [20]. Sequencing of a 6 kb region of the orf virus genome has revealed a number of open reading frames (ORFs) that are preceded by A + T-rich sequences with similarities to vaccinia virus early promoters [7]. S1 and primer extension analysis of one of these ORFs (ORF 3) showed that the tsp for the gene were on an A or a G, 9-10 nucleotides downstream from the 3' end of the sequence 5'-CAAAGTGAAAAAGGAC in a region of the genome which is otherwise over 80% G + C [6]. Davison and Moss [4] have shown that the vaccinia virus early promoter consists of a 16 base-pair critical region followed by a non-critical 11 base-pair spacer region which is in turn followed by a seven base-pair region within which initiation occurs, usually on a purine. The sequence of the critical region of a vaccinia virus strong early promoter, based on mutational analysis of the 7.5kDa promoter, was 5'-AAAAgTaGAAAataTA where the upper case letters represent nucleotides which could not be substituted without reducing, usually drastically, the promoter activity and the lower case letters represent nucleotides that could be substituted without affecting, and sometimes enhancing, activity. The ORF 3 promoter is a strong promoter in off virus but a comparison of its sequence with that of the vaccinia virus strong promoter suggests that it might not be a strong vaccinia virus promoter and that there might be some differences in the regulation of transcription between the two viruses. We have yet to compare directly the activity of off virus promoters in vaccinia virus, but show here that a soluble extract ofvaccinia virus virions will initiate transcription in vitro at the ORF 3 promoter. Vaccinia virus WR virions and soluble extracts for in vitro transcription were prepared as described by Rohrmann and Moss [ 17]. To prepare templates, a 756 bp SmaI-HpaI fragment from the BamHI E fragment of off virus DNA, which contained the ORF 3 promoter [7], was cloned into the Sinai site of plasmid pTZ18R (Pharmacia, Uppsala, Sweden). A number of clones were isolated and one with the orientation shown in Fig. 1 A was used to prepare the templates. After digestion with PvuII-ApaI, PvuII-RsaI, PvuII-NotI or PuvII alone, fragments of 397, 502, 662, and 694 bp were purified from agarose gels
In vitro recognition of an off virus early promoter in vaccinia virus
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P-P I p-r p-a p-n Fig. 1. Origin of fragments used in the in vitro run-off assays. A The bar represents the relevant section of the plasmid pTZ18R containing a 756 bp SrnaI-HpaI fragments from the orf virus BamHI E fragment. The hatched portion represents pTZ18R DNA, the stippled portion, orf virus ORF 3 from the putative promoter region (P, solid arrow), and the open region, the flanking regions of orf virus DNA. Restriction endonuclease cutting sites are indicated above the bar with the relevant nucleotide from either pTZ18R (sequence available from Pharmacia, Uppsala, Sweden) or orf virus DNA [7]. PvuII (p), Sinai (s), ApaI (a), RsaI (r), NotI (n), and HpaI (h). Represented below the bar are the fragments used in the assays with their sizes in bp. B The nucleotide sequence of the off virus putative promoter region is shown. Underlined is the A + T-rich sequence that shows strong homology with the critical region of vaccinia virus early promoters [4]. The tsp of ORF 3 transcripts in off virus infected cells as determined by Sl ($1) and primer extension analysis (pe) [6]. pp, p-n, p-r, p-a The tsp calculated for transcripts initiated in vitro on the fragments shown in (B) by vaccinia virus RNA polymerase
using glass powder (Fig. 1 A). Reaction mixtures in 30 gl final volumes contained 3 gl extract (5 ng/ml), 50ng fragment, 5 gCi of [32p]GTP, 1 m M ATP, CTP, and UTP, 0.05 m M GTP, 50 m M KCt, 5 m M MgC12, 0.1 m M EDTA, 25 m M Hepes (pH 7.9) and 7.5% glycerol and were incubated for 45 min at 30 °C. The components of the reaction were treated with proteinase K (1 mg/ml final concentration), ethanol precipitated and then separated in a 5% polyacrylamide gel containing 7 M urea in 0.1 M Tris-borate (pH 8.0), 1 m M EDTA. Radiolabelled fragments of an HpaII digest of the plasmid pBR 322 were used as size markers. After autoradiography of the gel (Fig. 2), discrete products corresponding to 190 (PvuII-ApaI), 305 (PvuII-RsaI), 475 (PvuI-NotI), and 510 (PvuI alone) nucleotides, were found. The two shorter run-off transcripts correspond to tsp
226
J.C. Vos et al.
Fig. 2. Autoradiograph of the products of in vitro transcription after electrophoresisin a 5% polyacrylamide-7M urea gel. 1 and 2 Molecular weight markers; 3 products from the PvuII-ApaI fragment; 4 the PvuII-RsaI fragment; 5 the PvuII-NotI fragment; 6 the PvuII-PvuII fragment
at 17 and 12nts downstream from the putative promoter sequence for ORF 3 (Fig. 1 B). The longer transcripts correspond to tsp closer to the promoter. No bands were found in extracts incubated in the absence of the off virus fragments (not shown). Taking into account the inaccuracies inherent in the method of measuring the sizes of transcripts, particularly those of higher molecular weight, this result agrees well with the tsp determined for RNA transcribed in orf virus infected cells. In that case the tsp were at 9 and 10 nucleotides downstream from the putative promoter sequence as determined by S1 nuclease digestion and primer extension respectively. Prominent higher molecular weight bands were seen in all lanes containing the reaction products seen in Fig. 2 and they are particularly prominent in lanes 5 and 6. The sizes of these bands correspond almost exactly with the sizes of the DNA fragments used as templates and we interpret them to represent either an end-labelling reaction or non-specific run-off transcripts that have initiated at the ends of the fragments. The result obtained is a clear indication that vaccinia virus polymerase is capable of recognizing an orf virus promoter despite the otherwise high G + C
In vitro recognition of an orf virus early promoter in vaccinia virus
227
c o n t e n t o f the f r a g m e n t s used in the assay. This is the first d e m o n s t r a t i o n in a functional assay o f the conservation o f early transcription p r o m o t e r s between orthopoxviruses a n d parapoxviruses.
References 1. Broyles SS, Fesler BS (1990) Vaccinia virus gene encoding a component of the viral early transcription factor. J Virol 64:1523-1529 2. Broyles SS, Yuen L, Shuman S, Moss B (1988) Purification of a factor required for transcription of vaccinia .virus early genes. J Biol Chem 263:10754-10760 3. Cochran MA, Puckett C, Moss B (1985) In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. J Virol 54:30-37 4. Davison AJ, Moss B (1989) Structure of vaccinia virus early promoters. J Mol Biol 210:749-769 5. Davison AJ, Moss B (1989) Structure of vaccinia virus late promoters. J Mol Biol 210: 771-784 6. Fleming SB, Fraser KM, Mercer AA, Robinson AJ (1991) Vaccinia virus-like early transcriptional control sequences flank an early gene in orf virus. Gene 97:207-212 7. Fraser KM, Hill DF, Mercer AA, Robinson AJ (1990) Sequence analysis of the inverted terminal repetition in the genome of the parapoxvirus, orf virus. Virology 176: 379389 8. Gershon PD, Moss B (1990) Early transcription factor subunits are encoded by vaccinia virus late genes. Proc Natl Acad Sci USA 87:44014405 9. Golini F, Kates JR (1985) A soluble transcription system derived from purified vaccinia virions. J Virol 53:205-213 10. Hanggi M, Bannwarth W, Stunnenberg HG (1986) Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation. EMBO J 5:1071-1076 11. Kates JR, McAuslan BR (1967) Poxvirus DNA-dependent RNA polymerase. Proc Natl Acad Sci USA 58:134-t41 12. Lee-Chen GY, Bourgeois N, Davidson K, Condit RC, Niles EG (1988) Structure of the transcription initiation and termination sequences of seven early genes in the vaccinia virus HindIII D fragment. Virology 163:64-79 13. Mars M, Beaud G (1987) Characterization of vaccinia virus early promoters and evaluation of their informational content. J Mol Biol 198:619-631 t4. Matthews REF (1982) Classification and nomenclature of viruses. Intervirology 17: 1-199 15. Moss B (1990) Regulation of vaccinia virus transcription. Annu Rev Biochem 59: 661688 16. Munyon W, Paoletti E, Grace JTJ (1967) RNA polymerase activity in purified infectious vaccinia virus. Proc Natl Acad Sci USA 58:2280-2287 17. Rohrmann G, Moss B (1985) Transcription of vaccinia virus early genes by a templatedependent soluble extract of purified virions. J Virol 56:349-355 18. Rosel JL, Earl PL, Weir JP, Moss B (1986) Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment. J Virol 60: 436-449 19. Weir JP, Moss B (1987) Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase. Virology 158:206-210
228 J. C. Vos etal.: In vitro recognition of an orf virus early promoter in vaccinia virus 20. Wittek R, Kuenzle CC, Wyler R (1979) High G + C content in parapoxvirus DNA. J Gen Virol 43:231-234 21. Yuen L, Davison AJ, Moss B (1987) Early promoter-binding factor from vaccinia virions. Proc Natl Acad Sci USA 84:6069-6073 Authors' address: Dr. A. J. Robinson, Medical Reserach Council Virus Research Unit and Centre for Gene Research, University of Otago, P.O. Box 56, Dunedin, New Zealand. Received June 24, 1991
