Detection and enumeration of transformation-defective strains of avian sarcoma virus with molecular hybridization

doi:10.1016/0042-6822(77)90250-1

Virology

Volume 76, Issue 2, February 1977, Pages 675-684

https://doi.org/10.1016/0042-6822(77)90250-1 Get rights and content

Abstract

Serial propagation of avian sarcoma viruses generates deletions in the viral gene responsible for cellular transformation (src). We have devised an assay for these deletion mutants which utilizes molecular hybridization and exploits the availability of DNA (cDNA_sarc) complementary to the nucleotide sequences affected by the deletion in src. Our procedure is also applicable to deletions in other viral genes and offers several advantages over conventional bioassays for the deletion mutants; moreover, it can be used to detect deletions in virus-specific intracellular nucleic acids. In order to illustrate the utility of the assay, we demonstrate that all 20 copies of the proviral DNA for avian sarcoma viruses in XC cells contain src, and we show that single avian cells can contain functioning proviruses for both avian sarcoma virus and a congenic deletion mutant. It should now be possible to use molecular hybridization to study the mechanism by which deletions in src are generated.

References (21)

M.L. Birnstiel et al.
Kinetic complexity of RNA molecules
J. Mol. Biol.
(1972)
J.M. Bishop et al.
The low molecular weight RNAs of Rous sarcoma virus. II. The 7S RNA
Virology
(1970)
A.C. Garapin et al.
RNA-directed DNA synthesis by virions of Rous sarcoma virus. Further characterization of the templates and the extent of their transcription
Virology
(1973)
J. Hillova et al.
Sarcoma and transformation-defective viruses produced with infectious DNA(s) from Rous sarcoma virus (RSV)-transformed chicken cells
Virology
(1974)
D.E. Kennell
Principles and practices of nucleic acid hybridization
G. Ringold et al.
Production of mouse mammary tumor virus by cultured cells in the absence and presence of hormones: Assay by molecular hybridization
Virology
(1975)
D. Stehelin et al.
Purification of DNA complementary to nucleotide sequence required for neoplastic transformation of fibroblasts by avian sarcoma viruses
J. Mol. Biol.
(1976)
P.K. Vogt
Spontaneous segregation of nontransforming viruses from cloned sarcoma viruses
Virology
(1971)
J.A. Wyke et al.
Temperaturesensitive avian sarcoma viruses: A physiological comparison of twenty mutants
Virology
(1973)
P.H. Duesberg et al.
Differences between the ribonucleic acids of transforming and nontransforming avian tumor viruses

There are more references available in the full text version of this article.

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¹: Present address: I.R.S.C., B.P. 8, 94800-Villejuif, France.

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Detection and enumeration of transformation-defective strains of avian sarcoma virus with molecular hybridization

Abstract

J. Mol. Biol.

Virology

Virology

Virology

Virology

J. Mol. Biol.

Virology

Virology

Differences between the ribonucleic acids of transforming and nontransforming avian tumor viruses