NRK Cell Lines Research Articles

Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus.

BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.

Monovalent antibodies directed to transformation-sensitive membrane components inhibit the process of viral transformation

Monovalent antibodies (Fab) directed to two classes of transformation-sensitive cell surface components, ganglioside and galactoprotein a (Gap a), inhibit the process of oncogenic viral transformation of cells. Mouse 3T3 cells infected with murine sarcoma virus were not transformed in terms of morphology change and enhancement of sugar uptake when the infected cells were cultured in the presence of monovalent antibodies directed to GM(1) ganglioside or to Gap a. Transformation inhibitory activity of these cell surface ligands was not related to cell growth inhibition because the monovalent antibodies to globoside and divalent Con A were growth inhibitory but did not inhibit oncogenic transformation. Neither anti-GM(1) Fab nor anti-Gap a Fab inhibited virus production. The transformation inhibitory activity of antiganglioside and anti-Gap a Fab was additionally assessed by inhibiting the transformed phenotype in NRK cell lines with mutants of avian sarcoma virus that are temperature sensitive for expression of the transformation phenotype (NRK/LA25). In this cell line, the GM(3) ganglioside (not GM(1)) and Gap a were transformation-sensitive cell surface components. The expression at permissive temperature of transformed phenotypes, such as morphology change and capability of growth in 0.3% agar, was inhibited by preincubation of the cells with anti-GM(3) Fab or anti-Gap a Fab.GM(3) labeling of NRK/LA25 cells decreased at permissive temperature, whereas preincubation of cells with anti-Gap a, which induces the inhibition of transformation after a temperature shift, prevented the decline of GM(3) label on the cell surface. The data suggest a possible correlation between GM(3) and Gap a expression. Application of monovalent antibodies to these transformation-sensitive components may prevent changes of these components on cell surfaces, and thus may result in abortion of phenotypic expression of transformation, although the transforming gene (src) has been set active. These results indicate that pericellular structures influence gene expression.