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Papilloma virus-induced tumors contain a virus-specific transcript
VIROLGGY
117, 257-261 (1982)
Papilloma
Virus-Induced
U. K. FREESE,’
Tumors Contain a Virus-Specific
P. SCHULTE,
Transcript
AND H. PFISTER
Institut fiir Virologie, Zentrum fiir Hygiene, Universitit Freiburg, D78W Freiburg, West Gemnany
Hermann-Herder-Strasse
11,
Received July 28, 1981; accepted October 19, 1981 Papillomavirus-specific. transcription was investigated in the absence of virus particle production in BPV l-induced hamster tumors. The analysis revealed a single virus-specific polyadenylated transcript of 1300-base length, which was mapped on the genome of BPV 1. Hybridization with 32P-labeled probes confined the transcribed region to two HpuII fragments, which are adjacent to the BamHI cleavage site within the 1.4 X lo6 dalton BumHI/EcoRI fragment. Related DNA sequences could be detected within the genomes of HPV 1 and HPV 4 using relaxed hybridization conditions.
tissue tumors in horses (5), mice, and hamsters (9). Fibromas or fibrosarcomas of hamsters grow progressively and eventually lead to death of the animal; in about 10% of the cases, they metastasize to internal organs (10, 11). The tumors contain BPV 1 or 2 DNA in high copy number but do not produce virus particles (5). The establishment of the biological system for this study is presented elsewhere (12). Briefly hamsters were subcutaneously inoculated with BPV 1 virus particles. After 15 months animal l/l developed a fibrosarcoma at the site of administration that could be transplanted to two new animals (2/l and 2/2). The tumors contained about 30 genome equivalents of extrachromosomal BPV 1 DNA per cell. No papillomavirus particles were detected electronmicroscopically and no BPV 1 capsid antigen was revealed by indirect immunofluorescence tests with type-specific antiBPV 1 rabbit antiserum. Total RNA was prepared after mechanical homogenization of fresh tumor biopsies in denaturing buffer, by phenol-chloroform-isoamyl alcohol extraction and selective ethanol precipitation from guanidinium-thiocyanate solution at low pH in the cold (18-15). Each preparation was run on a methyl mercury hydroxide gel (16). The RNA pattern was revealed by ethidium bromide staining and proved
Papilloma viruses induce benign epithelial or fibroepithelial tumors in their natural hosts, most probably by transforming an epidermal germinal cell by abortive infection (I). The tumors show tissue differentiation-dependent expression of viral genes. Replication of viral DNA starts within the stratum spinosum, and only in the keratinizing cell layers of the epidermis, virus particle production occurs. Mature capsids are found in the nuclei of cells of the stratum granulosum and in the stratum corneum. Late viral proteins are rather well known from studies of purified virus particles (2, 3). Due to the lack of an in vitro system for virus propagation, nothing is known so far about early viral functions, which are responsible for DNA replication and possibly for cell transformation. These functions, however, are of special interest in view of the carcinogenic potential of some papilloma virus types, as for example of Shope papilloma virus in rabbits (4), of bovine papillomavirus (BPV) types 1,2, and 4 (5,6), and of human papillomavirus (HPV) type 5 (7’). BPV 1 and 2 induce cutaneous fibropapillomas in cattle (8). In contrast to most papillomavirus types they show a rather broad host range and induce connective ’ To whom reprint
requests should be addressed. 257
0042-6822/82/030257-05$02.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form resewed.
258
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5
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FIG. 1. Blot analysis of hamster tumor RNA which was prepared according to published procedures (IS1.5). Briefly, hamster tumors or control tissue were mechanically homogenized grossly in a “Starmix” followed by a second finer homogenization in a “Potter-S” tight fitting assembly, each step in the presence of extraction buffer (4 M guanidiniumthiocyanate, 25 mM NaaCitrate, pH 7.0, 0.5% Sarcosyl, 0.1 M 2-mercaptoethanol). The homogenized tissues were then extracted with phenol/chloroform/isoamylalcohol and chloroform/isoamylalcohol alone. RNA was precipitated and purified from contaminating DNA as described (18). The resulting pellets were dissolved in SDS-buffer (10 mM Tris.HCl, pH 7.5, 1mM EDTA, 0.1 M 2-mercaptoethanol, 0.5% SDS), reprecipitated with ethanol, washed twice, and stored finally at -20” in ethanol. Poly(U)-Sepharose chromatography was essentially as described (21). Ten micrograms of total RNA per slot were run on denaturing 1.2% agarose gels in 20 mM Mops, pH 7.0, 5 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde electrophoresis buffer (17’), blotted and hybridized with nick-translated 3zP-labeled BPV 1 DNA (18). For R-loop conditions (20) the melting temperature of BPV 1 DNA was experimentally determined (80). RNAs: lanes 1,4-10, hamster tumor RNA, lane 2, hamster skin; lane 3, hamster organ mix control RNA. Poly(U)-Sepharose chromatography (21): lane 5, flowthrough poly(A)-minus fraction; lane 6, poly(A)plus fraction. 32P-labeled DNA probes (for map details see Fig. 2): lanes 1-4, total BPV 1 DNA, lanes 5-7, K 5, the smaller BPV 1 BumHI/EcoRI DNA fragment, cloned in the bacterial plasmid pBR322; lane 8 K 14, the larger BPV 1 BamHI/EcoRI DNA fragment, cloned in the bacterial plasmid pBR322; lane 9, the BamHI/HhaI subgenomic DNA fragment, Ak& lane 10, the HpaII/EcoRI subgenomic DNA fragment, Bk5.Hybridization conditions were as described (18) for all lanes with the exception of lane 4, where R-loop conditions were used (20, 80). Hybridization temperature in the R-loop experiment was 52’ for K5 with a strand separation temperature of 50’ (data not shown).
that the RNA was undegraded (data not shown). RNA preparations from tumors l/l, 2/ 1, and 2/2 were tested for the presence of virus-specific transcripts. RNAs were run on denaturing gels (17) and tranferred to nitrocellulose paper (18). Hybridization with 32P-labeled BPV 1 DNA revealed one band, corresponding to RNA of 1300 nucleotides. This band was not detected with control RNAs from hamster skin or from a hamster tissue mix of muscle, liver, and spleen (Fig. 1, lanes 1, 2, and 3). To prove the RNA nature of the hybridizing band we performed one experiment (Fig. 1, lane 4) under conditions where only DNA-RNA hybrids are stable (2” above the melting temperature of the the DNA) (19,20). The tumor RNA was fractionated by poly-(U)Sepharose chromatography (21). Virusspecific RNA was only found in the fraction binding to poly-(U)-Sepharose, indicating that the RNA is polyadenylated (Fig. 1, lanes 5 and 6). For physical mapping of the RNA subgenomic fragments of BPV 1 DNA were prepared after digest with various restriction endonucleases (22). Hybridization with 32P-labeled probes confined the transcribed region to two HpuII fragments which are adjacent to the BamHI cleavage site and lay within the 2150-bp BarnHI/ EcoRI fragment (Fig. 1, lanes 7-10, and Fig. 2). The two HpaII fragments have a size of 1260 bp. Assuming a poly-(A) tail of 200 bases this size would be only slightly larger than that of the colinear part of the messenger RNA molecule excluding the possibility of a splice of greater than about 160 bases. We were interested to see whether this region is unique for BPV 1. A relationship between DNAs from different papillomavirus types was established recently by relaxed hybridization conditions (28, 24) where stable hybrids are formed between single strands showing up to 30% mismatch. HPV 1 DNA was prepared from purified virus particles (25) and cloned HPV 4 DNA (2.4) was kindly provided by P. Howley. Southern blots were hybridized at 50” below the melting temperature of the DNA with 32P-labeled HhaI fragment AK5 (Fig. 2) to detect HPV 1 and HPV 4
-K5
-
!
BPV 1
FIG. 2. Physical map of BPV 1. The map displays the cleavage sites of various enzymes. The two EamHI/EcoRI fragments, cloned separately in pBR322, are indicated as K 14 and K 5. The map position of the transcript is marked within K 5.
sequences, which are related to the transcribed region of BPV 1. The cross-reacting sequence was mapped with various restriction endonucleases. The maps are shown in Fig. 3. To conclude, a virus-specific transcript was identified in BPV l-induced hamster tumors which could code at best for a pro-
tein with a molecular weight of about 40,000. Similar data were independently obtained recently by Amtmann und Sauer (26). As the band on the northern blots is rather broad we cannot exclude a doublet in this region. Both RNAs would be transcribed, however, from the same DNA sequence so that we were dealing with a
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FIG. 3. HPV 1 and HPV 4 DNA sequences, which are related to the transcribed region of BPV 1. Left part: Representative analysis of HPV 1 DNA cleaved with restriction enzymes BgZII (No. l), HindII/III (No. 2), and HoeHI (No. 3). The corresponding blots were hybridized with 32P-labeled BPV 1 H&I fragment Ak5 (see Fig. 2) under relaxed conditions as described (2.0 Right part: Physical map of HPV 1 (Bgl II according to Ref. (31) and HPV 4 (24) with the homologous fragments in black (blot data from HPV 4 not shown).
slight transcript heterogeneity. As there is no evidence for synthesis of structural proteins in hamster tumors it is tempting to assume that we are dealing with an early viral function which might be responsible for the induction of cell proliferation. It is interesting to note that the coding region for this transcript is located within the subgenomic BPV 1 DNA fragment which was shown to be sufficient for transformation in transfection experiments (27). Recently DNA-RNA reassociation analysis of RNA from Shope papillomavirus-induced skin carcinomas gave evidence for virus-specific transcription covering 6-12s of the viral genome (28) which is in the same size range as in the BPV l/hamster tumor system. So far, no proteins could be identified in the papillomavirus system which are comparable to the T antigens of polyoma and SV40 viruses (29). In vitro translation of the viral message from hamster tumors might be one approach to characterize such a protein of papillomaviruses. Hybridization experiments under nonstringent conditions point to a more widespread distribution of the coding region among papillomaviruses. Identification of similar regions in various HPV types could deliver more specific probes to look for the viral transcripts in human tumors.
ACKNOWLEDGMENTS We wish to thank Dr. Harald sur Hausen for helpful discussions and for critical reading of the manuscript. The excellent technical assistance of Mrs. Edith Kofler and Miss Inge Hettich is gratefully appreciated. We are indebted to Miss Beate Fink for providing cloned BPV 1 DNA. This work was supported by the Deutsche Forsehungsgemeinschaft (SFB 31, Medizinische Virologie: Tumorentstehung und Entwicklung). REFERENCES M., and CROISCancer”, Vol. 4, Cold Spring Harbor Conferences on Cell Proliferation H. H. Hiatt, J. D. Watson, and J. A. Winsten, eds.), pp. 1043-1068. Cold Spring Harbor, Laboratory, Cold Spring Harbor, N. Y., 1977.
1. ORTH, G., BREITBURD, F., FAVRE, SANT, O., In “Origins of Human
2. FAVRE, M., BREITBURD, F., CROISSANT, O., AND ORTH, G., J. Viral 15, 1239-1247 (1975). 3. PFISTER, H., GISSMANN, L., and ZUR HAUSEN, H., Virobgy 83, 131-137 (1977). 4. SYVERTON, J. T., Ann. N. Y Acd Sci. 54, 1126
(1952). 5. LANCASTER, W. D., THEILEN, G. H., and OLSON, L., Intervirology 11, 227-233 (1979). 6. CAMPO, M. S., MOAR, M. H., JARRET, W. F. H., and LAIRD, H. M. Nature &mdon) 286, 180182 (1980). 7. ORTH, G., JABLONSKA, S., JARZABEK-~HORZELSKA, M., OBALEK, S., RZESA, G., FAVRE, M., and CROISSANT, O., Cancer Res 39,1074-1082 (1979).
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COMMUNICATIONS
8. LANCASTER, W. D., and OLSON, C., Virology 89, 372-379 (1978). 9. BOIRON, M., LEVY, J. P., THOMAS, M., FRIEDMANN, J. C., and BERNARD, J., Nature (Londmz) 201, 423-424 (1964). 10. CHEVILLE, N. F., Cancer Res. 26,2334-2339 (1966). Il. ROBL, M. G., and OLSON, C., Cancer Rex 28,15961604 (1968). 12. PFISTER, H., FINK, B., and THOMAS, C., Virology, 115,414-418 (1981). 13. CHIRGWIN, M. J., PRZYBYLA, A. E., MACDONALD, R. J., and RUTTER, W. J., Biochemistry 18, 5294-5299 (1979). 14. ALJFFRAY, C., and ROUGEON, F., Eur. J. B&hem. 107, 303-314 (1980). 15. HOLMES, D. S., and BONNER, J. Biochem 12,23302338 (1973). 16. BAILEY, J., and DAVIDSON, N., Anal. Biochem 70, 75-85 (1976). 17. MANIATIS, T., personal communication. 18. THOMAS, P. S., Proc. Nat. Amd. Sci. USA 77,52015205 (1980). 19. DOERFLER, W., STABEL, S., IBELGAUTS, H., SUTTER, D., NEUMANN, R., GRONENBERG, J., SCHEIDTMANN, K. H., DEURING, R., and WINTERHOFF, U., Cold Spring Harbor Symp. @ant. Biol. 44, 551-562 (1979). 20. CASEY, J., and DAVIDSON, N., Nucleic Acids Res. 4, 1539-1552 (1977).
261
21. NEVINS, J. R., In “Methods in Enzymology” (L. Grossmann and K. Moldave, eds.), Vol. 65, pp. 76%785., Academic Press, New York, 1980. 22. KOLLER, B., DELIUS, H., BUNEMANN, H., and M~ER, W., Gene 4,227-239 (1978). 2.9. LAW, M. F., LANCASTER, W. D., and HOWLEY, P. M., J. Viral 32, 199-207 (1979). 24. HEILMANN, C. A., LAW, M. F., ISRAEL, M. A., and HOWLEY, P. M., J. Viral 36, 395-407 (1980). 2.5. GISSMANN, L., PFISTER, H., and ZUR HAUSEN, H., Virology 76, 569-580 (1977). 26. AMTMANN, E., and SAUER, G., personal communication. 27. LOWY, D. R., DVORETZKY, I., SHOBER, R., LAW, M. F., ENGEL, L., and HOWLEY, P. M., Nature (Londoni 287, 72-74 (1980). 28. WETTSTEIN, F. O., and STEVENS, J. G., Virology 109, 448-451 (1981). 29. TOPP, W. C., LANE, D., and POLLACK, R., In “Molecular Biology of Tumor Viruses” (J. Tooze, ed.), 2nd ed., Vol. 2, pp. 205-296. Academic Press, New York, 1981. 30. ROSBASH, M., BLANK, D., FAHRNER, K., HEREFORD, L., RICCIARDI, R., ROBERTS, B., RUBY, S., and WOOLFORD, J., In “Methods in Enzymology” (R. Wu, ed.), Vol. 68, pp. 454-469. Academic Press, New York, 1979. 31. DANOS, O., KATINKA, M., and YANIV, M., Eur. J. B&hem. 109, 457-461 (1980).
117, 257-261 (1982)
Papilloma
Virus-Induced
U. K. FREESE,’
Tumors Contain a Virus-Specific
P. SCHULTE,
Transcript
AND H. PFISTER
Institut fiir Virologie, Zentrum fiir Hygiene, Universitit Freiburg, D78W Freiburg, West Gemnany
Hermann-Herder-Strasse
11,
Received July 28, 1981; accepted October 19, 1981 Papillomavirus-specific. transcription was investigated in the absence of virus particle production in BPV l-induced hamster tumors. The analysis revealed a single virus-specific polyadenylated transcript of 1300-base length, which was mapped on the genome of BPV 1. Hybridization with 32P-labeled probes confined the transcribed region to two HpuII fragments, which are adjacent to the BamHI cleavage site within the 1.4 X lo6 dalton BumHI/EcoRI fragment. Related DNA sequences could be detected within the genomes of HPV 1 and HPV 4 using relaxed hybridization conditions.
tissue tumors in horses (5), mice, and hamsters (9). Fibromas or fibrosarcomas of hamsters grow progressively and eventually lead to death of the animal; in about 10% of the cases, they metastasize to internal organs (10, 11). The tumors contain BPV 1 or 2 DNA in high copy number but do not produce virus particles (5). The establishment of the biological system for this study is presented elsewhere (12). Briefly hamsters were subcutaneously inoculated with BPV 1 virus particles. After 15 months animal l/l developed a fibrosarcoma at the site of administration that could be transplanted to two new animals (2/l and 2/2). The tumors contained about 30 genome equivalents of extrachromosomal BPV 1 DNA per cell. No papillomavirus particles were detected electronmicroscopically and no BPV 1 capsid antigen was revealed by indirect immunofluorescence tests with type-specific antiBPV 1 rabbit antiserum. Total RNA was prepared after mechanical homogenization of fresh tumor biopsies in denaturing buffer, by phenol-chloroform-isoamyl alcohol extraction and selective ethanol precipitation from guanidinium-thiocyanate solution at low pH in the cold (18-15). Each preparation was run on a methyl mercury hydroxide gel (16). The RNA pattern was revealed by ethidium bromide staining and proved
Papilloma viruses induce benign epithelial or fibroepithelial tumors in their natural hosts, most probably by transforming an epidermal germinal cell by abortive infection (I). The tumors show tissue differentiation-dependent expression of viral genes. Replication of viral DNA starts within the stratum spinosum, and only in the keratinizing cell layers of the epidermis, virus particle production occurs. Mature capsids are found in the nuclei of cells of the stratum granulosum and in the stratum corneum. Late viral proteins are rather well known from studies of purified virus particles (2, 3). Due to the lack of an in vitro system for virus propagation, nothing is known so far about early viral functions, which are responsible for DNA replication and possibly for cell transformation. These functions, however, are of special interest in view of the carcinogenic potential of some papilloma virus types, as for example of Shope papilloma virus in rabbits (4), of bovine papillomavirus (BPV) types 1,2, and 4 (5,6), and of human papillomavirus (HPV) type 5 (7’). BPV 1 and 2 induce cutaneous fibropapillomas in cattle (8). In contrast to most papillomavirus types they show a rather broad host range and induce connective ’ To whom reprint
requests should be addressed. 257
0042-6822/82/030257-05$02.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form resewed.
258
SHORT COMMUNICATIONS 1
2
3
4
5
6
7
6
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FIG. 1. Blot analysis of hamster tumor RNA which was prepared according to published procedures (IS1.5). Briefly, hamster tumors or control tissue were mechanically homogenized grossly in a “Starmix” followed by a second finer homogenization in a “Potter-S” tight fitting assembly, each step in the presence of extraction buffer (4 M guanidiniumthiocyanate, 25 mM NaaCitrate, pH 7.0, 0.5% Sarcosyl, 0.1 M 2-mercaptoethanol). The homogenized tissues were then extracted with phenol/chloroform/isoamylalcohol and chloroform/isoamylalcohol alone. RNA was precipitated and purified from contaminating DNA as described (18). The resulting pellets were dissolved in SDS-buffer (10 mM Tris.HCl, pH 7.5, 1mM EDTA, 0.1 M 2-mercaptoethanol, 0.5% SDS), reprecipitated with ethanol, washed twice, and stored finally at -20” in ethanol. Poly(U)-Sepharose chromatography was essentially as described (21). Ten micrograms of total RNA per slot were run on denaturing 1.2% agarose gels in 20 mM Mops, pH 7.0, 5 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde electrophoresis buffer (17’), blotted and hybridized with nick-translated 3zP-labeled BPV 1 DNA (18). For R-loop conditions (20) the melting temperature of BPV 1 DNA was experimentally determined (80). RNAs: lanes 1,4-10, hamster tumor RNA, lane 2, hamster skin; lane 3, hamster organ mix control RNA. Poly(U)-Sepharose chromatography (21): lane 5, flowthrough poly(A)-minus fraction; lane 6, poly(A)plus fraction. 32P-labeled DNA probes (for map details see Fig. 2): lanes 1-4, total BPV 1 DNA, lanes 5-7, K 5, the smaller BPV 1 BumHI/EcoRI DNA fragment, cloned in the bacterial plasmid pBR322; lane 8 K 14, the larger BPV 1 BamHI/EcoRI DNA fragment, cloned in the bacterial plasmid pBR322; lane 9, the BamHI/HhaI subgenomic DNA fragment, Ak& lane 10, the HpaII/EcoRI subgenomic DNA fragment, Bk5.Hybridization conditions were as described (18) for all lanes with the exception of lane 4, where R-loop conditions were used (20, 80). Hybridization temperature in the R-loop experiment was 52’ for K5 with a strand separation temperature of 50’ (data not shown).
that the RNA was undegraded (data not shown). RNA preparations from tumors l/l, 2/ 1, and 2/2 were tested for the presence of virus-specific transcripts. RNAs were run on denaturing gels (17) and tranferred to nitrocellulose paper (18). Hybridization with 32P-labeled BPV 1 DNA revealed one band, corresponding to RNA of 1300 nucleotides. This band was not detected with control RNAs from hamster skin or from a hamster tissue mix of muscle, liver, and spleen (Fig. 1, lanes 1, 2, and 3). To prove the RNA nature of the hybridizing band we performed one experiment (Fig. 1, lane 4) under conditions where only DNA-RNA hybrids are stable (2” above the melting temperature of the the DNA) (19,20). The tumor RNA was fractionated by poly-(U)Sepharose chromatography (21). Virusspecific RNA was only found in the fraction binding to poly-(U)-Sepharose, indicating that the RNA is polyadenylated (Fig. 1, lanes 5 and 6). For physical mapping of the RNA subgenomic fragments of BPV 1 DNA were prepared after digest with various restriction endonucleases (22). Hybridization with 32P-labeled probes confined the transcribed region to two HpuII fragments which are adjacent to the BamHI cleavage site and lay within the 2150-bp BarnHI/ EcoRI fragment (Fig. 1, lanes 7-10, and Fig. 2). The two HpaII fragments have a size of 1260 bp. Assuming a poly-(A) tail of 200 bases this size would be only slightly larger than that of the colinear part of the messenger RNA molecule excluding the possibility of a splice of greater than about 160 bases. We were interested to see whether this region is unique for BPV 1. A relationship between DNAs from different papillomavirus types was established recently by relaxed hybridization conditions (28, 24) where stable hybrids are formed between single strands showing up to 30% mismatch. HPV 1 DNA was prepared from purified virus particles (25) and cloned HPV 4 DNA (2.4) was kindly provided by P. Howley. Southern blots were hybridized at 50” below the melting temperature of the DNA with 32P-labeled HhaI fragment AK5 (Fig. 2) to detect HPV 1 and HPV 4
-K5
-
!
BPV 1
FIG. 2. Physical map of BPV 1. The map displays the cleavage sites of various enzymes. The two EamHI/EcoRI fragments, cloned separately in pBR322, are indicated as K 14 and K 5. The map position of the transcript is marked within K 5.
sequences, which are related to the transcribed region of BPV 1. The cross-reacting sequence was mapped with various restriction endonucleases. The maps are shown in Fig. 3. To conclude, a virus-specific transcript was identified in BPV l-induced hamster tumors which could code at best for a pro-
tein with a molecular weight of about 40,000. Similar data were independently obtained recently by Amtmann und Sauer (26). As the band on the northern blots is rather broad we cannot exclude a doublet in this region. Both RNAs would be transcribed, however, from the same DNA sequence so that we were dealing with a
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FIG. 3. HPV 1 and HPV 4 DNA sequences, which are related to the transcribed region of BPV 1. Left part: Representative analysis of HPV 1 DNA cleaved with restriction enzymes BgZII (No. l), HindII/III (No. 2), and HoeHI (No. 3). The corresponding blots were hybridized with 32P-labeled BPV 1 H&I fragment Ak5 (see Fig. 2) under relaxed conditions as described (2.0 Right part: Physical map of HPV 1 (Bgl II according to Ref. (31) and HPV 4 (24) with the homologous fragments in black (blot data from HPV 4 not shown).
slight transcript heterogeneity. As there is no evidence for synthesis of structural proteins in hamster tumors it is tempting to assume that we are dealing with an early viral function which might be responsible for the induction of cell proliferation. It is interesting to note that the coding region for this transcript is located within the subgenomic BPV 1 DNA fragment which was shown to be sufficient for transformation in transfection experiments (27). Recently DNA-RNA reassociation analysis of RNA from Shope papillomavirus-induced skin carcinomas gave evidence for virus-specific transcription covering 6-12s of the viral genome (28) which is in the same size range as in the BPV l/hamster tumor system. So far, no proteins could be identified in the papillomavirus system which are comparable to the T antigens of polyoma and SV40 viruses (29). In vitro translation of the viral message from hamster tumors might be one approach to characterize such a protein of papillomaviruses. Hybridization experiments under nonstringent conditions point to a more widespread distribution of the coding region among papillomaviruses. Identification of similar regions in various HPV types could deliver more specific probes to look for the viral transcripts in human tumors.
ACKNOWLEDGMENTS We wish to thank Dr. Harald sur Hausen for helpful discussions and for critical reading of the manuscript. The excellent technical assistance of Mrs. Edith Kofler and Miss Inge Hettich is gratefully appreciated. We are indebted to Miss Beate Fink for providing cloned BPV 1 DNA. This work was supported by the Deutsche Forsehungsgemeinschaft (SFB 31, Medizinische Virologie: Tumorentstehung und Entwicklung). REFERENCES M., and CROISCancer”, Vol. 4, Cold Spring Harbor Conferences on Cell Proliferation H. H. Hiatt, J. D. Watson, and J. A. Winsten, eds.), pp. 1043-1068. Cold Spring Harbor, Laboratory, Cold Spring Harbor, N. Y., 1977.
1. ORTH, G., BREITBURD, F., FAVRE, SANT, O., In “Origins of Human
2. FAVRE, M., BREITBURD, F., CROISSANT, O., AND ORTH, G., J. Viral 15, 1239-1247 (1975). 3. PFISTER, H., GISSMANN, L., and ZUR HAUSEN, H., Virobgy 83, 131-137 (1977). 4. SYVERTON, J. T., Ann. N. Y Acd Sci. 54, 1126
(1952). 5. LANCASTER, W. D., THEILEN, G. H., and OLSON, L., Intervirology 11, 227-233 (1979). 6. CAMPO, M. S., MOAR, M. H., JARRET, W. F. H., and LAIRD, H. M. Nature &mdon) 286, 180182 (1980). 7. ORTH, G., JABLONSKA, S., JARZABEK-~HORZELSKA, M., OBALEK, S., RZESA, G., FAVRE, M., and CROISSANT, O., Cancer Res 39,1074-1082 (1979).
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COMMUNICATIONS
8. LANCASTER, W. D., and OLSON, C., Virology 89, 372-379 (1978). 9. BOIRON, M., LEVY, J. P., THOMAS, M., FRIEDMANN, J. C., and BERNARD, J., Nature (Londmz) 201, 423-424 (1964). 10. CHEVILLE, N. F., Cancer Res. 26,2334-2339 (1966). Il. ROBL, M. G., and OLSON, C., Cancer Rex 28,15961604 (1968). 12. PFISTER, H., FINK, B., and THOMAS, C., Virology, 115,414-418 (1981). 13. CHIRGWIN, M. J., PRZYBYLA, A. E., MACDONALD, R. J., and RUTTER, W. J., Biochemistry 18, 5294-5299 (1979). 14. ALJFFRAY, C., and ROUGEON, F., Eur. J. B&hem. 107, 303-314 (1980). 15. HOLMES, D. S., and BONNER, J. Biochem 12,23302338 (1973). 16. BAILEY, J., and DAVIDSON, N., Anal. Biochem 70, 75-85 (1976). 17. MANIATIS, T., personal communication. 18. THOMAS, P. S., Proc. Nat. Amd. Sci. USA 77,52015205 (1980). 19. DOERFLER, W., STABEL, S., IBELGAUTS, H., SUTTER, D., NEUMANN, R., GRONENBERG, J., SCHEIDTMANN, K. H., DEURING, R., and WINTERHOFF, U., Cold Spring Harbor Symp. @ant. Biol. 44, 551-562 (1979). 20. CASEY, J., and DAVIDSON, N., Nucleic Acids Res. 4, 1539-1552 (1977).
261
21. NEVINS, J. R., In “Methods in Enzymology” (L. Grossmann and K. Moldave, eds.), Vol. 65, pp. 76%785., Academic Press, New York, 1980. 22. KOLLER, B., DELIUS, H., BUNEMANN, H., and M~ER, W., Gene 4,227-239 (1978). 2.9. LAW, M. F., LANCASTER, W. D., and HOWLEY, P. M., J. Viral 32, 199-207 (1979). 24. HEILMANN, C. A., LAW, M. F., ISRAEL, M. A., and HOWLEY, P. M., J. Viral 36, 395-407 (1980). 2.5. GISSMANN, L., PFISTER, H., and ZUR HAUSEN, H., Virology 76, 569-580 (1977). 26. AMTMANN, E., and SAUER, G., personal communication. 27. LOWY, D. R., DVORETZKY, I., SHOBER, R., LAW, M. F., ENGEL, L., and HOWLEY, P. M., Nature (Londoni 287, 72-74 (1980). 28. WETTSTEIN, F. O., and STEVENS, J. G., Virology 109, 448-451 (1981). 29. TOPP, W. C., LANE, D., and POLLACK, R., In “Molecular Biology of Tumor Viruses” (J. Tooze, ed.), 2nd ed., Vol. 2, pp. 205-296. Academic Press, New York, 1981. 30. ROSBASH, M., BLANK, D., FAHRNER, K., HEREFORD, L., RICCIARDI, R., ROBERTS, B., RUBY, S., and WOOLFORD, J., In “Methods in Enzymology” (R. Wu, ed.), Vol. 68, pp. 454-469. Academic Press, New York, 1979. 31. DANOS, O., KATINKA, M., and YANIV, M., Eur. J. B&hem. 109, 457-461 (1980).