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Biomedical subjects

E May

Publications and source records attributed to E May.

At least 127 records · Page 7Linked to original sources
PubMed1978

Visualization of mouse DNA transcriptional complexes in mouse kidney cells infected with SV 40 virus.

Electron microscopic studies of transcribed cellular deoxynucleoprotein (DNP) fibers from mouse kidney cells abortively infected with Simian Virus 40 (SV 40) revealed two types of transcriptional complexes. Tandemly repeating units representing ribosomal transcriptional events, although few in number because of the procedure employed, clearly confirmed results of other techniques which revealed the unusually long length of the untranscribed spacer intercepts in mouse nucleolar chromatin. In non-nucleolar arrays the density of the ribonucleoprotein (RNP) fibrils varied, as did the length and configuration of the associated DNP fibers. A statistical correlation between a "smooth" appearance of a transcribed portion of a DNP fiber and a high density of nascent RNP fibrils was observed.

Animals↗
PubMed1977

Mapping of transcription sites of simian virus 40-specific late 16S and 19S mRNA by electron microscopy.

Simian virus 40 (SV40) late 19S and 16S mRNAs were annealed to complementary regions of partially melted viral double-stranded SV40(LHpa II) DNA or SV40(LBam HI) DNA. The RNA-DNA hybrid regions within the DNA molecules were visualized as loops [with SV40(LBam HI) DNA] in the electron microscope. The data confirm the previous localizations of the 3' and 5' ends of 16S SV40 mRNA and of the 3' end of late 19S SV40 mRNA. The 5' end of the major stable SV40 late 19S mRNA has been positioned at 0.755 map unit. Thus, the sequences of viral DNA from 0.655 to 0.755 map unit, including the replication origin, are not converted into major stable species of late viral mRNA.

Chromosome Mapping↗
PubMed1977

Characterization of simian virus 40 tsA58 transcriptional intermediates at restrictive temperatures: relationship between DNA replication and transcription.

When nuclei from simian virus 40 (SV40)-infected cells are lysed with Sarkosyl and the chromatin is pelleted, the supernatant fluid contains a nucleoprotein complex capable of synthesizing viral RNA (Laub and Aloni, Virology 75:346-354, 1976; Gariglio and Mousset, FEBS Lett. 56:149-155, 1975). The level of activity of the RNA polymerase in the complex increased during infection in parallel with the amount of viral DNA that had been synthesized. If cells infected at 33 degrees C with the SV40 mutant tsA 58 were shifted to the nonpermissive temperature of 40 degrees C at any time between 18 and 48 h postinfection, no viral DNA replication was detected after 45 min and no new rounds of synthesis were initiated after 20 to 30 min. However, after this shift, polymerase activity associated with the nucleo-protein complex did continue to increase for 5 h, at which time it reached a plateau. There was an increase of RNA synthesized from both the early (E) and late (L) SV40 DNA strands, and there was a threefold increase in the ratio of early-to-late RNA species after the shift. In comparable experiments with cells infected with wild-type virions, no increase in polymerase activity occurred because of the temperature change alone. At 33 degrees C, the relative amount of RNA transcribed from the wild-type E-strand was less than tsA 58 at 33 degrees C and did not increase after a shift to 40 degrees C. The tsA 58 transcriptional complexes extracted from cells grown at 33 degrees C sedimented heterogeneously in sucrose gradients, with a peak near 26S. There were no detectable alterations in the sedimentation properties of the complexes when tsA 58-infected cells were shifted to 40 degrees C for 2 h. We conclude that continued synthesis of viral DNA is not an obligatory prerequisite for maintenance of late viral transcription nor is the sedimentation of the transcriptional complex at 26S related to actively replicating DNA molecules serving as templates for transcription. Further, an increase in late transcription can occur under conditions where reinitiation of viral DNA synthesis is prevented. The increase in the synthesis of early and late RNA at the restrictive temperature without concurrent DNA synthesis is discussed in relationship to the function of the A gene product.

Cell Line↗
PubMed1977

Regulation of early and late simian virus 40 transcription: overproduction of early viral RNA in the absence of a functional T-antigen.

Virus-specific RNA synthesized in monkey cells after infection by both wild-type simian virus 40 (SV40) and the early SV40 temperature-sensitive mutant tsA58 has been analyzed. The fraction of SV40-specific RNA increased throughout infection with either wild-type SV40 or with tsA58 in direct proportion to the accumulation of progeny DNA molecules, suggesting their role in the late transcriptional process. Cytoplasmic fractions from cells infected at various temperatures (31.5 to 41 degrees C) by wild-type virus and harvested 48 h later contained 4 to 8% virus-specific RNA, of which 5 to 10% was early SV40 RNA. In contrast, though 5 to 8% of the cytoplasmic RNA from tsA 58-infected cells incubated at 31.5 to 37 degrees C for 48 h was virus specific, the percentage of early virus-specific RNA ranged from 25 to 80% as the incubation temperature increased. In tsA58-infected cultures incubated for 48 h at 41 degrees C (a temperature at which essentially no tsA 58 DNA synthesis occurred), only 0.4% of the cytoplasmic RNA was virus specific, but at least 90% of this RNA was early. In experiments where cells were inoculated at 32 degrees C and shifted at 48 h postinfection to 40 degrees C for various times, the percentage of virus-specific pulse-labeled RNA varied from 3.5 to 10.0%. Of the virus-specific RNA, early SV40 RNA ranged from 14 to 65% in tsA 58-infected cultures. Analogous studies with Sarkosyl-extracted viral transcription complexes to incorporate label into nascent (unprocessed) viral RNA yielded essentially identical results. This finding strongly suggests that the overproduction of early SV40 RNA occurs at the level of synthesis. While cytosine arabinoside effectively terminated most viral DNA replication in wild-type-infected cells, the ratio of early to late viral RNA remained less than 1:9. These results demonstrate that: (1) the amount of virus-specific RNA synthesized depends directly on the amount of viral DNA available for use as templates; once viral DNA replication has occurred, presumably providing progeny SV40 DNA molecules for templates, the level of transcription remains high; (ii) termination of viral DNA replication does not terminate late SV40 transcription; (iii) early SV40 RNA is overproduced by tsA 58 at all temperatures, but especially at higher temperatures; and (iv) overproduction of early SV40 RNA appears to be correlated with defectiveness of the tsA mutant T-antigen. These results suggest that T-antigen may regulate its own production either by repressing the synthesis of early viral RNA or by stimulating the synthesis of late SV40 RNA or both.

Antigens, Viral↗
PubMed1977

Properties of simian virus 40 transcriptional intermediates isolated from nuclei of permissive cells.

A nucleoprotein complex that is an intermediate in viral transcription has been isolated from simian virus 40 (SV40)-infected BSC-1 cells after lysing infected nuclei with Sarkosyl. It contain DNA, DNA-dependent RNA polymerase II, and nascent RNA chains. RNA chain elongation continues for several hours in vitro and is dependent on exogenous ribonucleoside triphosphates. The complex sediments in neutral sucrose gradients with a main peak at about 24 to 26S. When the nascent RNA on the complex is treated with RNase A, a fraction of the RNA remains resistant to RNase and is hydrogen bonded to the DNA template. The pulse-labeled RNase-resistant RNA can be chased into RNase-sensitive RNA, indicating that it is located at the 3' terminus of the RNA chain. The rate of RNA displacement from the DNA template is consistent with an average rate of RNA chain elongation of 15 to 30 nucleotides per min. At least 70% of the RNA synthesized in this in vitro system is SV40 specific. Hybridization with the separated strands of SV40 DNA and with fragments of SV40 DNA generated with endonucleases HindII + III indicates that this RNA is complementary to all regions of the "late" SV40 DNA strand. Studies of SV40 RNA synthesis in this partially purified preparation at early and late times after infection should provide a way of locating promoter sites for transcription and identifying the form of SV40 DNA that serves as a template for late transcription.

Animals↗
PubMed1975

Mapping the transcription site of the SV40-specific late 16 S mRNA.

This paper describes the purification of polysomal RNA from SV40-lytically infected CV1 (monkey) cells and separation of the two distinct classes of SV40-specific mRNA sedimenting at 16 S and 19 S. These classes have been hybridized with the whole SV40 DNA genome as well as with the SV40 Hind fragments. The results have permitted the mapping of SV40-specific late 16 S mRNA from approximately 0.945 to 0.175 map units.

Base Sequence↗