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R G Fenton

Publications and source records attributed to R G Fenton.

44 records · Page 3Linked to original sources
PubMed1982

Changes in the topography of early region transcription during polyoma virus lytic infection.

We have studied the pattern of transcription of the early region of polyoma virus DNA after the onset of the late phase of lytic infection of mouse cells. Following initiation of viral DNA synthesis, the early/late switch is accompanied not only by efficient production of late mRNAs but also by the appearance of previously unidentified early-strand RNAs which have certain structural features in common with the classical early mRNAs. Stable poly(A)+RNAs have been identified by blot analysis and S1 nuclease mapping that are not detected early during infection or in polyoma virus-transformed cells. One group consists of transcripts whose 5' ends map 150-200 nucleotides upstream from the major early 5' ends (at positions 148 and 153 on the polyoma virus genome) but whose splicing pattern and poly(A) addition sites are indistinguishable from those of mRNAs produced early in infection. The 5' exons of these early region transcripts contain an open translational reading frame that extends from nucleotide positions 5,255 to 124 and is capable of encoding a basic protein of 53 amino acids. Transcription of these RNAs does not appear to be negatively regulated by large tumor antigen. A transcript of 1,800 nucleotides appears to map predominantly between 93 and 26 map units and does not contain sequences present in the early mRNA 5' exons. These data suggest that, after the onset of polyoma DNA replication, the activation of new early-strand promoters leads to the expression of previously untranscribed viral DNA sequences.

Antigens, Viral↗
PubMed1981

Viral gene expression in polyoma virus-transformed rat cells and their cured revertants.

We have studied transcription of integrated viral DNA sequences in a variety of ts-a polyoma virus-transformed rat cells and cured revertants (which had undergone excision of variables amounts of integrated viral DNA) to characterize the structure of viral mRNA's produced in these lines under conditions in which integrated DNA is stable. Our results indicate that cells containing intact early region sequences, either in single-copy or tandem insertions, produce mRNA's indistinguishable from those observed early in lytic infections; sequences complementary to the polyoma late region were not transcribed from integrated viral DNA. Cured revertants no longer encoded full-length early mRNA's , but produced viral transcripts whose 3' ends mapped at an alternative early region polyadenylic acid attachment site at 99 map units or extended in to flanking host sequences. The phenotype of these revertant cells correlated with the abundance of these transcripts, suggesting that the transforming function(s) of polyoma virus controls the cellular phenotype in a dose-dependent manner. Unexpected results were obtained from studies of cells containing tandem repeats of defective viral DNA in which the polyadenylic acid attachment signal at 25.8 map units and surrounding sequences were deleted. In these cases, polyadenylated mRNA's were observed that contained sequences complementary to the early strand of the polyoma late region. These mRNA's (some larger than 8 kilobases) originated at the viral early promoter, extended into the late region, and continued into the early region of the contiguous repeat in the tandem. The multimeric mRNA's produced contained defective early regions in tandem with late region sequences. S1 analysis indicated that whereas the 5' early region sequences of readthrough transcripts were spliced in the usual manner, internal early region repeats were either unspliced or used only one of the small early region splices. When deletions in the viral readthrough transcripts were observed. This suggests that sequences nearby the AAUAAA sequence at 26 map units may control transcription termination of the polyoma early region.

Animals↗
PubMed1981

Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells.

To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to "position" effects or to the high efficiency of recombination of large DNA fragments.

Animals↗
PubMed1977

Methylated nucleosides in globin mRNA from mouse nucleated erythroid cells.

Poly(A)-containing mRNAs labeled with [methyl-3H]methionine were isolated from nucleated erythroid cells obtained from the spleens of anemic mice. The RNAs were further separated into non-globin poly(A)-containing RNAs and highly purified globin mRNA by globin cDNA-cellulose affinity chromatography. DEAE-Sephadex column chromatography of the T2 ribonuclease digestion products of the cDNA-purified globin mRNA fraction yielded methylated resistant fragments with charges of -4.7 (Cap 1) and -5.3 (Cap 2). Digestion of the non-globin RNA fraction revealed a similar pattern with the addition of a methylated mononucleotide identified as 6-methyladenosine at -2 charges. Alkaline phosphatase treatment of the T2 resistant fragments reduced their charges by approximately 2, which is consistent with the removal of one terminal phosphate. Treatment of the globin T2 and alkaline phosphatase-resistant fragments withpenicillium P1 nuclease and alkaline phosphatase yielded a P1-resistant core structure in both fragments. In addition to the core, 2'-O-methylcytidine (Cm) was released from the more negatively charged globin fragment. The P1-resistant cores of the cap structures eluted from DEAE-Sephadex with the known standard m2G5'ppp5'Am and were found to be pyrophosphatase-sensitive establishing a 5'-5'-triphosphate linkage. The pyrophosphatase and alkaline phosphatase digestion products of the globin Cap 1 and Cap 2 core structures were analyzed by high voltage electrophoresis and paper chromatography and found to be 7-methyiguanosine (m7G) and the dimethylated nucleoside 6-methyl-2'-O-methyladenosine (N6mAm). A small amount of the singularly methylated adenosine, 2'-O-methyladenosine (Am) was also observed. The predominant sequences of the methylated nucleosides in the globin cap structures are therefore m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm.

Animals↗
PubMedDate not supplied

Melanoma-specific cytotoxicity induced by a tyrosinase promoter-enhancer/herpes simplex virus thymidine kinase adenovirus.

The ability to specifically and efficiently express selected genes in tumor cells is an important goal for cancer gene therapy. Transcriptional targeting of adenovirus to tumor cells, thereby limiting their expression to specific cell types, represents one experimental approach to this problem. We have previously shown that a recombinant adenovirus containing the murine tyrosinase promoter coupled to a dimer of the tyrosinase-enhancer element can target the expression of beta-galactosidase cDNA to melanoma cells. We now report that this same promoter/enhancer cassette can efficiently drive the expression of the herpes simplex virus thymidine kinase gene in melanoma cells. Infection of melanoma cells with the AdmTyr-tk virus along with subsequent ganciclovir treatment induces S phase cell cycle arrest associated with a profound change in cell size and morphology. Treated cells remain viable for prolonged periods, but clonogenic assays demonstrate that the cell cycle arrest is irreversible. In contrast, nonmelanoma cells are unaffected by this treatment regimen, exhibiting normal growth kinetics, metabolic activity, and cell cycle progression. The therapeutic efficacy of the AdmTyr-tk virus was tested in vivo using a xenograft model of human melanoma. The injection of the AdmTyr-tk virus into established subcutaneous tumor nodules in combination with systemic ganciclovir administration led to a decreased tumor growth rate and to complete tumor regressions in some cases. These studies demonstrate the feasibility of selectively targeting growth-inhibitory genes to melanoma cells in vitro and in vivo.

Adenoviridae↗