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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 Feb;77(2):847–851. doi: 10.1073/pnas.77.2.847

Two species of full-length cDNA are synthesized in high yield by melittin-treated avian retrovirus particles.

L R Boone, A Skalka
PMCID: PMC348378  PMID: 6153806

Abstract

A method of activating endogenous cDNA synthesis in avian retroviruses that results in the formation of two species of full-length cDNA in high yield is described. Tests of biological activity show infectivity of at least the same order of magnitude as for full-length cDNA made by other procedures. Melittin, the major component of bee venom, is used as an alternative to nonionic detergents to make the viral envelope permeable and thus activate the endogenous RNA-dependent DNA polymerase. This compound is a toxic peptide known to interact with phospholipid membranes. It appears to be less disruptive to the viral structure than detergents, resulting in a more efficient transcription of the viral genome. Preliminary tests indicate that this method will also prove useful for studying enzymatic activities associated with other enveloped viruses.

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Selected References

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  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. Blattner F. R., Blechl A. E., Denniston-Thompson K., Faber H. E., Richards J. E., Slightom J. L., Tucker P. W., Smithies O. Cloning human fetal gamma globin and mouse alpha-type globin DNA: preparation and screening of shotgun collections. Science. 1978 Dec 22;202(4374):1279–1284. doi: 10.1126/science.725603. [DOI] [PubMed] [Google Scholar]
  3. Böhlen P., Stein S., Dairman W., Udenfriend S. Fluorometric assay of proteins in the nanogram range. Arch Biochem Biophys. 1973 Mar;155(1):213–220. doi: 10.1016/s0003-9861(73)80023-2. [DOI] [PubMed] [Google Scholar]
  4. Clayman C. H., Mosharrafa E., Faras A. J. In vitro synthesis of infectious transforming DNA by the avian sarcoma virus reverse transcriptase. J Virol. 1979 Jan;29(1):242–249. doi: 10.1128/jvi.29.1.242-249.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coffin J. M. Structure, replication, and recombination of retrovirus genomes: some unifying hypotheses. J Gen Virol. 1979 Jan;42(1):1–26. doi: 10.1099/0022-1317-42-1-1. [DOI] [PubMed] [Google Scholar]
  6. Dawson C. R., Drake A. F., Helliwell J., Hider R. C. The interaction of bee melittin with lipid bilayer membranes. Biochim Biophys Acta. 1978 Jun 16;510(1):75–86. doi: 10.1016/0005-2736(78)90131-1. [DOI] [PubMed] [Google Scholar]
  7. Friedrich R., Moelling K. Effect of viral RNase H on the avian sarcoma viral genome during early transcription in vitro. J Virol. 1979 Sep;31(3):630–638. doi: 10.1128/jvi.31.3.630-638.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gallo R. C., Miller N. R., Saxinger W. C., Gillespie D. Primate RNA tumor virus-like DNA synthesized endogenously by RNA-dependent DNA polymerase in virus-like particles from fresh human acute leukemic blood cells. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3219–3224. doi: 10.1073/pnas.70.11.3219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gianni A. M., Weinberg R. A. Partially single-stranded form of free Moloney viral DNA. Nature. 1975 Jun 19;255(5510):646–648. doi: 10.1038/255646a0. [DOI] [PubMed] [Google Scholar]
  10. Gilboa E., Goff S., Shields A., Yoshimura F., Mitra S., Baltimore D. In vitro synthesis of a 9 kbp terminally redundant DNA carrying the infectivity of Moloney murine leukemia virus. Cell. 1979 Apr;16(4):863–874. doi: 10.1016/0092-8674(79)90101-6. [DOI] [PubMed] [Google Scholar]
  11. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  13. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  14. Hsu T. W., Sabran J. L., Mark G. E., Guntaka R. V., Taylor J. M. Analysis of unintegrated avian RNA tumor virus double-stranded DNA intermediates. J Virol. 1978 Dec;28(3):810–818. doi: 10.1128/jvi.28.3.810-818.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Junghans R. P., Duesberg P. H., Knight C. A. In vitro synthesis of full-length DNA transcripts of Rous sarcoma virus RNA by viral DNA polymerase. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4895–4899. doi: 10.1073/pnas.72.12.4895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kacian D. L., Myers J. C. Synthesis of extensive, possibly complete, DNA copies of poliovirus RNA in high yields and at high specific activities. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2191–2195. doi: 10.1073/pnas.73.7.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kacian D. L., Watson K. F., Burny A., Spiegelman S. Purification of the DNA polymerase of avian myeloblastosis virus. Biochim Biophys Acta. 1971 Sep 24;246(3):365–383. doi: 10.1016/0005-2787(71)90773-8. [DOI] [PubMed] [Google Scholar]
  18. Kaplan H. S., Goodenow R. S., Epstein A. L., Gartner S., Declève A., Rosenthal P. N. Isolation of a type C RNA virus from an established human histiocytic lymphoma cell line. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2564–2568. doi: 10.1073/pnas.74.6.2564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kawai S., Hanafusa H. Genetic recombination with avian tumor virus. Virology. 1972 Jul;49(1):37–44. doi: 10.1016/s0042-6822(72)80005-9. [DOI] [PubMed] [Google Scholar]
  20. Lai M. M., Hu S. S., Vogt P. K. Occurrence of partial deletion and substitution of the src gene in the RNA genome of avian sarcoma virus. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4781–4785. doi: 10.1073/pnas.74.11.4781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  22. McClements W., Hanafusa H., Tilghman S., Skalka A. Structural studies on oncornavirus-related sequences in chicken genomic DNA: two-step analyses of EcoRI and Bgl I restriction digests and tentative mapping of a ubiquitous endogenous provirus digests and tentative mapping of a ubiquitous endogenous provirus. Proc Natl Acad Sci U S A. 1979 May;76(5):2165–2169. doi: 10.1073/pnas.76.5.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  24. Novak U., Friedrich R., Moelling K. Elongation of DNA complementary to the 5' end of the avian sarcoma virus genome by the virion-associated RNA-dependent DNA polymerase. J Virol. 1979 May;30(2):438–452. doi: 10.1128/jvi.30.2.438-452.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rice N. R., Coggins L. Synthesis of long complementary DNA in the endogenous reaction by equine infectious anemia virus. J Virol. 1979 Mar;29(3):907–914. doi: 10.1128/jvi.29.3.907-914.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rothenberg E., Baltimore D. Increased length of DNA made by virions of murine leukemia virus at limiting magnesium ion concentration. J Virol. 1977 Jan;21(1):168–178. doi: 10.1128/jvi.21.1.168-178.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rothenberg E., Baltimore D. Synthesis of long, representative DNA copies of the murine RNA tumor virus genome. J Virol. 1975 Jan;17(1):168–174. doi: 10.1128/jvi.17.1.168-174.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rothenberg E., Smotkin D., Baltimore D., Weinberg R. A. In vitro synthesis of infectious DNA of murine leukaemia virus. Nature. 1977 Sep 8;269(5624):122–126. doi: 10.1038/269122a0. [DOI] [PubMed] [Google Scholar]
  29. Sessa G., Freer J. H., Colacicco G., Weissmann G. Interaction of alytic polypeptide, melittin, with lipid membrane systems. J Biol Chem. 1969 Jul 10;244(13):3575–3582. [PubMed] [Google Scholar]
  30. Shank P. R., Hughes S. H., Kung H. J., Majors J. E., Quintrell N., Guntaka R. V., Bishop J. M., Varmus H. E. Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA. Cell. 1978 Dec;15(4):1383–1395. doi: 10.1016/0092-8674(78)90063-6. [DOI] [PubMed] [Google Scholar]
  31. Sogo J. M., Greenstein M., Skalka A. The circle mode of replication of bacteriophage lambda: the role of covalently closed templates and the formation of mixed catenated dimers. J Mol Biol. 1976 May 25;103(3):537–562. doi: 10.1016/0022-2836(76)90216-3. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Stromberg K. Surface-active agents for isolation of the core component of avian myeloblastosis virus. J Virol. 1972 Apr;9(4):684–697. doi: 10.1128/jvi.9.4.684-697.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thelestam M., Möllby R. Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts. Infect Immun. 1975 Apr;11(4):640–648. doi: 10.1128/iai.11.4.640-648.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thelestam M., Möllby R. Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. Infect Immun. 1975 Aug;12(2):225–232. doi: 10.1128/iai.12.2.225-232.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Varmus H. E., Heasley S., Linn J., Wheeler K. Use of alkaline sucrose gradients in a zonal rotor to detect integrated and unintegrated avian sarcoma virus-specific DNA in cells. J Virol. 1976 May;18(2):574–585. doi: 10.1128/jvi.18.2.574-585.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Varmus H. E., Shank P. R. Unintegrated viral DNA is synthesized in the cytoplasm of avian sarcoma virus-transformed duck cells by viral DNA polymerase. J Virol. 1976 May;18(2):567–573. doi: 10.1128/jvi.18.2.567-573.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Verma I. M. Genome organization of RNA tumor viruses. I. In vitro synthesis of full-genome-length single-stranded and double-stranded viral DNA transcripts. J Virol. 1978 Jun;26(3):615–629. doi: 10.1128/jvi.26.3.615-629.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Williams J. C., Bell R. M. Membrane matrix disruption by melittin. Biochim Biophys Acta. 1972 Nov 2;288(2):255–262. doi: 10.1016/0005-2736(72)90246-5. [DOI] [PubMed] [Google Scholar]

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