Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 Feb;5(2):281–290. doi: 10.1128/mcb.5.2.281

Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication.

P A Norton, J M Coffin
PMCID: PMC366710  PMID: 2983187

Abstract

We have developed a convenient and sensitive assay of eucaryotic gene expression which uses the Escherichia coli lacZ gene product, beta-galactosidase, as a nonselectable marker. This system has been applied to the analysis of Rous sarcoma virus replication and gene expression. Avian cells were transfected with plasmids encoding in-frame gene fusions of the N-terminal portion of the gag gene to a 'lacZ gene, which requires both transcriptional and translational initiation signals; these were supplied by the virus long terminal repeat and leader region. Readily detectable quantities of beta-galactosidase were synthesized in transfected cells; it was demonstrated that the levels of enzyme activity induced in such cultures increased linearly with the input DNA concentration and also correlated with mRNA levels. By using a Rous sarcoma virus-derived vector containing the src gene and a related virus as a helper, it was shown that lac sequences were compatible with all phases of the virus life cycle. gag-lacZ fusion proteins were immunoprecipitable from cultures which stably expressed lacZ as well as src. Virus rescued from stably transfected cultures resulted in continued lac and src expression in recipient cells. One particular construction was efficiently transmitted as virus, although it lacked sequences thought to be important for encapsidation of RNA into virions. The data presented here demonstrate the use of lacZ as a marker of retrovirus gene expression and replication.

Full text

PDF
281

Images in this article

284Image
on p.284
287Image
on p.287

Selected References

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

  1. An G., Hidaka K., Siminovitch L. Expression of bacterial beta-galactosidase in animal cells. Mol Cell Biol. 1982 Dec;2(12):1628–1632. doi: 10.1128/mcb.2.12.1628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bandyopadhyay P. K., Watanabe S., Temin H. M. Recombination of transfected DNAs in vertebrate cells in culture. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3476–3480. doi: 10.1073/pnas.81.11.3476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bizub D., Katz R. A., Skalka A. M. Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis. J Virol. 1984 Feb;49(2):557–565. doi: 10.1128/jvi.49.2.557-565.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Canaani E., Helm K. V., Duesberg P. Evidence for 30-40S RNA as precursor of the 60-70S RNA of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1973 Feb;70(2):401–405. doi: 10.1073/pnas.70.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  7. Conklin K. F., Coffin J. M., Robinson H. L., Groudine M., Eisenman R. Role of methylation in the induced and spontaneous expression of the avian endogenous virus ev-1: DNA structure and gene products. Mol Cell Biol. 1982 Jun;2(6):638–652. doi: 10.1128/mcb.2.6.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper G. M., Okenquist S. Mechanism of transfection of chicken embryo fibroblasts by Rous sarcoma virus DNA. J Virol. 1978 Oct;28(1):45–52. doi: 10.1128/jvi.28.1.45-52.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cullen B. R., Lomedico P. T., Ju G. Transcriptional interference in avian retroviruses--implications for the promoter insertion model of leukaemogenesis. Nature. 1984 Jan 19;307(5948):241–245. doi: 10.1038/307241a0. [DOI] [PubMed] [Google Scholar]
  10. Cullen B. R., Skalka A. M., Ju G. Endogenous avian retroviruses contain deficient promoter and leader sequences. Proc Natl Acad Sci U S A. 1983 May;80(10):2946–2950. doi: 10.1073/pnas.80.10.2946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Emerman M., Temin H. M. High-frequency deletion in recovered retrovirus vectors containing exogenous DNA with promoters. J Virol. 1984 Apr;50(1):42–49. doi: 10.1128/jvi.50.1.42-49.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foster D. A., Hanafusa H. A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens. J Virol. 1983 Dec;48(3):744–751. doi: 10.1128/jvi.48.3.744-751.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hall C. V., Jacob P. E., Ringold G. M., Lee F. Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet. 1983;2(1):101–109. [PubMed] [Google Scholar]
  15. Hayward W. S. Size and genetic content of viral RNAs in avian oncovirus-infected cells. J Virol. 1977 Oct;24(1):47–63. doi: 10.1128/jvi.24.1.47-63.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hughes S., Kosik E. Mutagenesis of the region between env and src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors. Virology. 1984 Jul 15;136(1):89–99. doi: 10.1016/0042-6822(84)90250-2. [DOI] [PubMed] [Google Scholar]
  17. Joyner A. L., Bernstein A. Retrovirus transduction: segregation of the viral transforming function and the herpes simplex virus tk gene in infectious Friend spleen focus-forming virus thymidine kinase vectors. Mol Cell Biol. 1983 Dec;3(12):2191–2202. doi: 10.1128/mcb.3.12.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kalnins A., Otto K., Rüther U., Müller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J. 1983;2(4):593–597. doi: 10.1002/j.1460-2075.1983.tb01468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Katz R. A., Omer C. A., Weis J. H., Mitsialis S. A., Faras A. J., Guntaka R. V. Restriction endonuclease and nucleotide sequence analyses of molecularly cloned unintegrated avian tumor virus DNA: structure of large terminal repeats in circle junctions. J Virol. 1982 Apr;42(1):346–351. doi: 10.1128/jvi.42.1.346-351.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kawai S., Koyama T. Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biological properties of mutant TK15 and mutant-induced transformants. J Virol. 1984 Jul;51(1):147–153. doi: 10.1128/jvi.51.1.147-153.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koyama T., Harada F., Kawai S. Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biochemical properties of mutant TK15 and mutant-induced transformants. J Virol. 1984 Jul;51(1):154–162. doi: 10.1128/jvi.51.1.154-162.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Luciw P. A., Bishop J. M., Varmus H. E., Capecchi M. R. Location and function of retroviral and SV40 sequences that enhance biochemical transformation after microinjection of DNA. Cell. 1983 Jul;33(3):705–716. doi: 10.1016/0092-8674(83)90013-2. [DOI] [PubMed] [Google Scholar]
  24. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  25. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  26. Mermer B., Malamy M., Coffin J. M. Rous sarcoma virus contains sequences which permit expression of the gag gene in Escherichia coli. Mol Cell Biol. 1983 Oct;3(10):1746–1758. doi: 10.1128/mcb.3.10.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mitsialis S. A., Caplan S., Guntaka R. V. An upstream regulatory domain of avian tumor virus long terminal repeat is required for the expression of a procaryotic neomycin gene in eucaryotic cells. Mol Cell Biol. 1983 Nov;3(11):1975–1984. doi: 10.1128/mcb.3.11.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nielsen D. A., Chou J., MacKrell A. J., Casadaban M. J., Steiner D. F. Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5198–5202. doi: 10.1073/pnas.80.17.5198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. PERRIN D. Immunological studies with genetically altered beta-galactosidases. Ann N Y Acad Sci. 1963 May 8;103:1058–1066. doi: 10.1111/j.1749-6632.1963.tb53757.x. [DOI] [PubMed] [Google Scholar]
  30. Palmiter R. D., Gagnon J., Vogt V. M., Ripley S., Eisenman R. N. The NH2-terminal sequence of the avian oncovirus gag precursor polyprotein (Pr76gag). Virology. 1978 Dec;91(2):423–433. doi: 10.1016/0042-6822(78)90388-4. [DOI] [PubMed] [Google Scholar]
  31. Pugatsch T., Stacey D. W. Identification of a sequence likely to be required for avian retroviral packaging. Virology. 1983 Jul 30;128(2):505–511. doi: 10.1016/0042-6822(83)90279-9. [DOI] [PubMed] [Google Scholar]
  32. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  33. Sealy L., Moscovici G., Moscovici C., Bishop J. M. Site-specific mutagenesis of avian erythroblastosis virus: v-erb-A is not required for transformation of fibroblasts. Virology. 1983 Oct 15;130(1):179–194. doi: 10.1016/0042-6822(83)90126-5. [DOI] [PubMed] [Google Scholar]
  34. Sealy L., Privalsky M. L., Moscovici G., Moscovici C., Bishop J. M. Site-specific mutagenesis of avian erythroblastosis virus: erb-B is required for oncogenicity. Virology. 1983 Oct 15;130(1):155–178. doi: 10.1016/0042-6822(83)90125-3. [DOI] [PubMed] [Google Scholar]
  35. Shank P. R., Linial M. Avian oncovirus mutant (SE21Q1b) deficient in genomic RNA: characterization of a deletion in the provirus. J Virol. 1980 Nov;36(2):450–456. doi: 10.1128/jvi.36.2.450-456.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shimotohno K., Temin H. M. Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus. Cell. 1981 Oct;26(1 Pt 1):67–77. doi: 10.1016/0092-8674(81)90034-9. [DOI] [PubMed] [Google Scholar]
  37. Shimotohno K., Temin H. M. Loss of intervening sequences in genomic mouse alpha-globin DNA inserted in an infectious retrovirus vector. Nature. 1982 Sep 16;299(5880):265–268. doi: 10.1038/299265a0. [DOI] [PubMed] [Google Scholar]
  38. Sompayrac L. M., Danna K. J. Efficient infection of monkey cells with DNA of simian virus 40. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7575–7578. doi: 10.1073/pnas.78.12.7575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sorge J., Hughes S. H. Splicing of intervening sequences introduced into an infectious retroviral vector. J Mol Appl Genet. 1982;1(6):547–559. [PubMed] [Google Scholar]
  40. Sorge J., Ricci W., Hughes S. H. cis-Acting RNA packaging locus in the 115-nucleotide direct repeat of Rous sarcoma virus. J Virol. 1983 Dec;48(3):667–675. doi: 10.1128/jvi.48.3.667-675.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Varmus H. E. Form and function of retroviral proviruses. Science. 1982 May 21;216(4548):812–820. doi: 10.1126/science.6177038. [DOI] [PubMed] [Google Scholar]
  42. Vogt V. M., Wight A., Eisenman R. In vitro cleavage of avian retrovirus gag proteins by viral protease p15. Virology. 1979 Oct 15;98(1):154–167. doi: 10.1016/0042-6822(79)90534-8. [DOI] [PubMed] [Google Scholar]
  43. Watanabe S., Temin H. M. Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5986–5990. doi: 10.1073/pnas.79.19.5986. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES