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. 1981 Mar;78(3):1518–1522. doi: 10.1073/pnas.78.3.1518

Molecular cloning of avian myelocytomatosis virus (MC29) transforming sequences.

J A Lautenberger, R A Schulz, C F Garon, P N Tsichlis, T S Papas
PMCID: PMC319162  PMID: 6262810

Abstract

Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo and transforms fibroblasts and hematopoietic target cells in vitro. We have used recombinant DNA technology to isolate and to characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2-kilobase cloned DNA insert contains approximately 4 kilobases of viral sequences and 5.2 kilobases of quail cellular sequences. The viral sequences contain all of the MC29-specific sequences and 5' helper-related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of transformed cells with high efficiency.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Bister K., Hayman M. J., Vogt P. K. Defectiveness of avian myelocytomatosis virus MC29: isolation of long-term nonproducer cultures and analysis of virus-specific polypeptide synthesis. Virology. 1977 Oct 15;82(2):431–448. doi: 10.1016/0042-6822(77)90017-4. [DOI] [PubMed] [Google Scholar]
  3. Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E., Furlong L., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. doi: 10.1126/science.847462. [DOI] [PubMed] [Google Scholar]
  4. Chang E. H., Maryak J. M., Wei C. M., Shih T. Y., Shober R., Cheung H. L., Ellis R. W., Hager G. L., Scolnick E. M., Lowy D. R. Functional organization of the Harvey murine sarcoma virus genome. J Virol. 1980 Jul;35(1):76–92. doi: 10.1128/jvi.35.1.76-92.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Copeland N. G., Cooper G. M. Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29. J Virol. 1980 Mar;33(3):1199–1202. doi: 10.1128/jvi.33.3.1199-1202.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeLorbe W. J., Luciw P. A., Goodman H. M., Varmus H. E., Bishop J. M. Molecular cloning and characterization of avian sarcoma virus circular DNA molecules. J Virol. 1980 Oct;36(1):50–61. doi: 10.1128/jvi.36.1.50-61.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duesberg P. H., Bister K., Moscovici C. Avian acute leukemia virus MC29: conserved and variable RNA sequences and recombination with helper virus. Virology. 1979 Nov;99(1):121–134. doi: 10.1016/0042-6822(79)90043-6. [DOI] [PubMed] [Google Scholar]
  8. Duesberg P. H., Bister K., Vogt P. K. The RNA of avian acute leukemia virus MC29. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4320–4324. doi: 10.1073/pnas.74.10.4320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Enquist L. W., Madden M. J., Schiop-Stanley P., Vande Woude G. F. Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector. Science. 1979 Feb 9;203(4380):541–544. doi: 10.1126/science.216076. [DOI] [PubMed] [Google Scholar]
  10. Graf T., Beug H. Avian leukemia viruses: interaction with their target cells in vivo and in vitro. Biochim Biophys Acta. 1978 Nov 17;516(3):269–299. doi: 10.1016/0304-419x(78)90011-2. [DOI] [PubMed] [Google Scholar]
  11. Hanafusa T., Hanafusa H., Miyamoto T. Recovery of a new virus from apparently normal chick cells by infection with avian tumor viruses. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1797–1803. doi: 10.1073/pnas.67.4.1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Highfield P. E., Rafield L. F., Gilmer T. M., Parsons J. T. Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones. J Virol. 1980 Oct;36(1):271–279. doi: 10.1128/jvi.36.1.271-279.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
  14. Hu S. S., Lai M. M., Vogt P. K. Genome of avian myelocytomatosis virus MC29: analysis by heteroduplex mapping. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1265–1268. doi: 10.1073/pnas.76.3.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hughes S. H., Payvar F., Spector D., Schimke R. T., Robinson H. L., Payne G. S., Bishop J. M., Varmus H. E. Heterogeneity of genetic loci in chickens: analysis of endogenous viral and nonviral genes by cleavage of DNA with restriction endonucleases. Cell. 1979 Oct;18(2):347–359. doi: 10.1016/0092-8674(79)90054-0. [DOI] [PubMed] [Google Scholar]
  16. Hughes S. H., Shank P. R., Spector D. H., Kung H. J., Bishop J. M., Varmus H. E., Vogt P. K., Breitman M. L. Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites. Cell. 1978 Dec;15(4):1397–1410. doi: 10.1016/0092-8674(78)90064-8. [DOI] [PubMed] [Google Scholar]
  17. Leder P., Tiemeier D., Enquist L. EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from higher organisms: the lambdagtWES system. Science. 1977 Apr 8;196(4286):175–177. doi: 10.1126/science.322278. [DOI] [PubMed] [Google Scholar]
  18. Lowy D. R., Rands E., Scolnick E. M. Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. J Virol. 1978 May;26(2):291–298. doi: 10.1128/jvi.26.2.291-298.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mellon P., Pawson A., Bister K., Martin G. S., Duesberg P. H. Specific RNA sequences and gene products of MC29 avian acute leukemia virus. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5874–5878. doi: 10.1073/pnas.75.12.5874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sheiness D. K., Hughes S. H., Varmus H. E., Stubblefield E., Bishop J. M. The vertebrate homolog of the putative transforming gene of avian myelocytomatosis virus: characteristics of the DNA locus and its RNA transcript. Virology. 1980 Sep;105(2):415–424. doi: 10.1016/0042-6822(80)90042-2. [DOI] [PubMed] [Google Scholar]
  21. Sheiness D., Bishop J. M. DNA and RNA from uninfected vertebrate cells contain nucleotide sequences related to the putative transforming gene of avian myelocytomatosis virus. J Virol. 1979 Aug;31(2):514–521. doi: 10.1128/jvi.31.2.514-521.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sheiness D., Bister K., Moscovici C., Fanshier L., Gonda T., Bishop J. M. Avian retroviruses that cause carcinoma and leukemia: identification of nucleotide sequences associated with pathogenicity. J Virol. 1980 Mar;33(3):962–968. doi: 10.1128/jvi.33.3.962-968.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  24. Souza L. M., Strommer J. N., Hillyard R. L., Komaromy M. C., Baluda M. A. Cellular sequences are present in the presumptive avian myeloblastosis virus genome. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5177–5181. doi: 10.1073/pnas.77.9.5177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Thomas M., White R. L., Davis R. W. Hybridization of RNA to double-stranded DNA: formation of R-loops. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2294–2298. doi: 10.1073/pnas.73.7.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tsichlis P. N., Coffin J. M. Role of the C region in relative growth rates of endogenous and exogenous avian oncoviruses. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 2):1123–1132. doi: 10.1101/sqb.1980.044.01.121. [DOI] [PubMed] [Google Scholar]
  27. Wittek R., Barbosa E., Cooper J. A., Garon C. F., Chan H., Moss B. Inverted terminal repetition in vaccinia virus DNA encodes early mRNAs. Nature. 1980 May 1;285(5759):21–25. doi: 10.1038/285021a0. [DOI] [PubMed] [Google Scholar]

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