Immunoselection of mutants deficient in cell surface glycoproteins encoded by murine erythroleukemia viruses.

Self Cite

Friend spleen focus-forming virus shuts down its gene expression frequently (ca. 10-3 per generation) in a cis-dominant hereditable fashion in various murine cells but much less frequently in rat cells (less than 10-6 per generation). Thus. nonexpresser variants were isolated at high frequency from murine cell lines by immunoselection directed against virus-encoded cell surface glycoproteins and also simply by subcloning cells from lines which had been cultured for many generations. Studies of independently infected cell clones indicate that shutdown is a property of the cell line rather than of the specific proviral site. Nucleic acid blot analyses suggest that shutdown correlates with decreased transcription. Moreover, preliminary evidence indicates that other murine retroviruses also shut down frequently in murine but not

Section: Methodssupporting

confidence: 51%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Frequent hereditable shutdown of murine retrovirus gene expression in murine cell lines.

Bestwick¹,

Machida²,

Polonoff³

et al. 1984

Self Cite

“…Resistance (18). Virtually all of the radioactivity incorporated into M1.54 was found in the medium after 2 h, whereas [14C]nicotinamide release from CR1, CR4, and HTC was negligible after 2.5 h (Fig.…”

Section: Resultsmentioning

confidence: 99%

Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression.

Firestone¹,

Yamamoto²

1983

We have isolated mutant derivatives of M1.54 (a mammary tumor virus [MTV]infected rat hepatoma [HTC] cell line containing multiple integrated proviruses) that fail to express hormone-inducible cell surface viral glycoproteins. In wildtype M1.54, the synthetic glucocorticoid dexamethasone selectively stimulates the rate of synthesis of MTV RNA. In addition, dexamethasone is essential for posttranslational maturation of three of the four cell surface viral glycoproteins processed from the MTV glycosylated precursor polyprotein; the fourth mature species is produced constitutively. Two mutant phenotypes are described; each contains glucocorticoid receptors that are indistinguishable from the wild-type receptor with respect to hormone affinity, intracellular concentration, nuclear translocation efficiency, DNA-cellulose chromatography, and sedimentation rate. In one class, represented by the mutant line CR1, dexamethasone fails to stimulate the low basal rate of MTV gene transcription; surprisingly, hormonal regulation of tyrosine aminotransferase activity is also defective in CR1, whereas several other cellular responses to dexamethasone are normal. In the second class of mutants, represented by CR4, dexamethasone stimulates synthesis of MTV transcripts indistinguishable from those produced in M1.54, but only the constitutive cell surface viral glycoprotein is expressed. Thus, these mutants define two distinct and novel aspects of glucocorticoid regulated gene expression in HTC cells: CR4 contains a defect in a hormone inducible protein maturation pathway that acts on specific viral (and presumably cellular) precursor polypeptides, whereas the lesion in CR1 appears to affect the expression of a subset of the gene products normally under glucocorticoid control in M1.54.Among vertebrates, glucocorticoid hormones affect the expression of specific genes in virtually every tissue; interestingly, the set of gene products whose synthesis or activities are altered, i.e., the glucocorticoid domain (16), is highly cell type specific (2). The effects of glucocorticoids and other steroids appear to be mediated by hormone-specific intracellular receptor proteins; the hormone-receptor interaction potentiates a change in the receptor (activation) that results in its increased affinity for DNAcontaining sites in the cell nucleus (see reference 44 for review). Purified glucocorticoid-receptor complexes bind in vitro with high affinity to specific sites on cloned mouse mammary tumor virus (MTV) DNA (25), whose expression in vivo is glucocorticoid regulated (see reference 29 for review); this is consistent with the view that selective receptor-DNA interactions participate in defining the stringent gene selectivity of hormone responses.The glucocorticoid-receptor complex stimulates the rate of MTV gene transcription in infected cells (31,41,49) by increasing the efficiency of MTV promoter utilization

“…4) (Table 1) showed that all three DEX-resistant variants in Fig. 4 (6,12). The antibody-coated petri dish method may prove useful as a positive or negative selection for genetic studies involving other antigens found in relatively large numbers on the cell surface.…”

Section: Resultsmentioning

confidence: 97%

Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness.

Danielsen¹,

Peterson²,

Stallcup³

1983

We have devised an immunological procedure to separate cells on the basis of expression of mouse mammary tumor virus (MMTV) gene products. Plastic petri dishes coated with specific antibodies against MMTV proteins bind cells with an efficiency that correlates with the level of MMTV gene expression. Glucocorticoid-sensitive mouse thymoma cell line W7 was infected with MMTV. Clones from the infected population retain the relatively slow cytolytic glucocorticoid response and, in addition, exhibit a rapid induction of MMTV-specific RNA and proteins. By combining our immunological selection with the selection for resistance to hormone-mediated cytolysis, we have isolated variant cells which are resistant to the cytotoxic effect of glucocorticoids but which retain the induction of viral gene products and must therefore have a functional glucocorticoid receptor protein.Like the immunocytes from which they were derived (4), some mouse thymoma cell lines are killed by exposure to physiological concentrations of glucocorticoid hormones for 1 to 3 days (9, 10). Although the mechanism of the killing response is not well defined, the hormone-induced cell death provides a strong selection for hormone-insensitive variants. However, the complexity of the killing response and the absence of an identifiable, induced gene product make the biochemical characterization of variant phenotypes difficult. Selection for hormone resistance has yielded several phenotypic classes of variants in the glucocorticoid receptor protein (11,14,29). These have been extremely useful, but other classes of variants would be of obvious interest.To facilitate the genetic analysis of glucocorticoid responsiveness in lymphoid cells, we have infected them with mouse mammary tumor virus (MMTV), which establishes itself as a stably integrated provirus with glucocorticoid-responsive gene expression (17,26