We have determined the complete nucleotide sequence of the thymidine Idnase (ATP:thymidine 5' phosphotransferase, EC 2.7.1.21) gene of herpes simplex virus type 1 strain CLO1I from a plasmid clone of viral DNA derived by
CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide 'mapping' and N-terminal analysis.
We identified in herpes simplex virus type 1-infected cells six cytoplasmic transcripts which were complementary to BamHI restriction endonuclease fragment Q. Two transcripts appeared in major amounts compared with the other four. One major transcript of about 1.4 kilobases was the mRNA for the viral thymidine kinase, was synthesized at intermediate times, and was classified as a beta transcript. The other major transcript was synthesized at late times and was classified as a gamma transcript. This late transcript was about 3 kilonucleotides long and was transcribed in the same direction as the gene for thymidine kinase. The 5' end of this late RNA was located by RNA sequence analysis and was 23 nucleotides downstream from the polyadenylation site for the thymidine kinase mRNA. This finding led to the conclusion that the control region for the 3kilobase gamma transcript is contained within the 3' untranslated region of the thymidine kinase transcript.
The intragenic control regions of a eukaryotic tRNA gene have been examined by transcribing mutant forms of a Drosophila tRNAArg gene either by injection into the nucleus of Xenopus oocytes or in extracts prepared from isolated oocyte nuclei. These experiments demonstrate that the selection of the transcription initiation site is a complex mechanism that involves the T-control region, the D-control region, and sequences 5' adjacent to the D-control region. In this study either "half" of the Drosophila tRNAArg gene promoted transcription in Xenopus oocytes. This finding supports a recent model for eukaryotic tRNA gene transcription (Dingermann et al., 1983, J. Biol. Chem. 258, 10395-10402) that proposes transcription initiation is dependent on the ability of specific DNA sequences to sequester two RNA polymerase III transcription factors.
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