A frameshift mutation affecting the carboxyl terminus of the simian virus 40 large tumor antigen results in a replication- and transformation-defective virus.

Lewis, Eleanor T.; Chen, Suzie; Kumar, Amar; Blanck, George; Pollack, Robert; Manley, J L

doi:10.1073/pnas.80.23.7065

Cited by 19 publications

(9 citation statements)

References 34 publications

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“…Based upon its reactivity with HAF but not with NHS and its absence in mock-infected cell lysates, we identified the 80K protein as T*-ag. This result is in agreement with previous reports which indicated that T*-ag migrates faster in polyacrylamide gels than WT T-ag, even though its predicted molecular weight is slightly higher (29,61). The difference in apparent molecular weight allowed us to examine membrane preparations for evidence of T*-ag.…”

Section: Resultssupporting

confidence: 83%

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

Jarvis¹,

Cole²,

Butel³

1986

Mol. Cell. Biol.

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The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine-or proline-containing peptides, or by two-dimensional tryptic peptide mapping of methionine-containing peptides. The carboxy-terminal, methionine-containing tryptic peptide was identified in the two-dimensional maps and was shown to be identical in nT-ag and mT-ag. Thus, a structural basis for the recognition and differential localization of T-ags could not be demonstrated. The carboxy terminus of the T-ag encoded by mutant dL42413 is derived from the alternate open reading frame of the simian virus 40 early region, in analogy with the theoretical early gene product, T*-ag. We used this mutant to identify peptides unique to T*-ag. None of these peptides were detected in maps of mT-ag; only wild-type T-ag-specific peptides were found. These findings suggest that T*-ag does not represent the membrane-associated form of T-ag, but that mT-ag is encoded within the same reading frame used for nT-ag.

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Section: Resultssupporting

confidence: 83%

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

Jarvis¹,

Cole²,

Butel³

1986

Mol. Cell. Biol.

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“…BIOL. (8,10,22,40). In light of these findings and those reported by Petit et al (33), that (30) or human fetal glial cells (25).…”

Section: Resultsmentioning

confidence: 62%

Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

Neufeld¹,

Ripley²,

Henderson³

et al. 1987

Mol. Cell. Biol.

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Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori-transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SVWHF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre-and postimmortalization, should be useful in defining molecular changes associated with immortalization.

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“…4a) by means of oligonucleotide-directed mutagenesis (41) to alter the most highly conserved portion of the heptamer (8). cTNT-129 carrying the M-CAT 1 mutation directed lower CAT activity (by a factor of 50) than wild-type cTNT-129 in skeletal muscle cells (Fig.…”

Section: A Ctnt-129mentioning

confidence: 99%

A conserved CATTCCT motif is required for skeletal muscle-specific activity of the cardiac troponin T gene promoter.

Mar

Ordahl

1988

Proc. Natl. Acad. Sci. U.S.A.

171

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Transcription of the cardiac troponin T (cTNT) gene is restricted to cardiac and embryonic skeletal muscle tissue. A DNA segment containing 129 nucleotides upstream from the cTNT transcription initiation site (cTNT-129) directs expression of a heterologous marker gene in transfected embryonic skeletal muscle cells but is inactive in embryonic cardiac or fibroblast cells. By using chimeric promoter constructions, in which distal and proximal segments of cTNT-129 are fused to reciprocal segments of the herpes simplex virus thymidine kinase (HSV tk) gene promoter, the DNA segment responsible for this cell specificity can be localized to the cTNT distal promoter region, located between 50 and 129 nucleotides upstream of the transcription initiation site. The ability of the cTNT distal promoter region to confer skeletal muscle-specific activity upon a heterologous promoter is abolished when it is displaced 60 nucleotides upstream, indicating that its ability to direct skeletal muscle-specific transcription probably requires proximity to other components of the transcription initiation region. Two copies of the heptamer, CATTCCT ("muscle-CAT" or "M-CAT" motif), reside within the 80-nucleotide cTNT distal promoter region. A 3-nucleotide mutation in one of these copies inactivates the cTNT promoter in skeletal muscle cells. Therefore, the M-CAT motif is a distal promoter element required for expression of the cTNT promoter in embryonic skeletal muscle cells. Since the M-CAT motif is found in other contractile protein gene promoters, it may represent one example of a muscle-specific promoter element.

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A frameshift mutation affecting the carboxyl terminus of the simian virus 40 large tumor antigen results in a replication- and transformation-defective virus.

Cited by 19 publications

References 34 publications

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

A conserved CATTCCT motif is required for skeletal muscle-specific activity of the cardiac troponin T gene promoter.

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