Herpes Simplex Virus Ribonucleotide Reductase DNA and Requirements for mRNA 3' End Formation

McLauchlan, John (1986) Herpes Simplex Virus Ribonucleotide Reductase DNA and Requirements for mRNA 3' End Formation. PhD thesis, University of Glasgow.

Abstract

This study was undertaken to examine the organisation and structures of mRNAs mapping at the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase locus. From comparisons between equivalent HSV-2 and HSV-1 nucleotide sequences, putative transcription control signals were identified, and functional analysis of certain control elements was performed. The positions of translated regions within the mRNAs encoding ribonucleotide reductase were identified as were conserved amino acid domains between viral and cellular reductases. During the course of these comparisons, a conserved DNA sequence, YGTGTTYY (Y = pyrimidine), located 30nuc downstream from the polyadenylation (AATAAA) signal of both viral and cellular genes, was identified. Functional tests showed that the YGTGTTYY signal was required for efficient formation of mRNA 3' termini. Arrangement of mRNAs and DNA Sequence Comparisons at the HSV-2 Ribonucleotide Reductase Locus. Four overlapping mRNAs have been identified at the ribonucleotide reductase locus. These mRNAs are unspliced and share common 5' and 3' termini. Two early mRNAs with sizes of 4.5kb and 1.2kb have a common 3' terminus and encode HSV-2 polypeptides Vmw138 and Vmw38 respectively which are almost certainly components of the viral ribonucleotide reductase. The other two mRNAs are late, 5' co-terminal species with sizes of 6.4kb and 1.7kb which appear to encode an identical 54,000 mol. wt. protein; the 6.4kb transcript is 3' co-terminal with the early mRNAs. As a consequence of this mRNA arrangement, the 5' termini of the 4.5kb and 1.2kb mRNAs and the 3' terminus of the 1.7kb mRNA are located within larger, overlapping transcripts. The genome region adjacent to the 6.4kb and 1.7kb mRNAs specifies three late transcripts which are synthesised in the opposite orientation; the 5' terminal region of one of these species overlaps with the 6.4kb and 1.7kb mRNAs. The 3' terminus of the late 1.7kb mRNA is located within the transcribed region of the 6. 4kb species. Previously, it has been proposed that read-through of HSV poly A sites is due to inefficient processing at certain 3' termini. The ability of sequences flanking the 1.7kb mRNA 3' terminus to produce functional mRNA was tested using a plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) protein coding sequences fused to an HSV-2 immediate early promoter. CAT activities produced in HeLa cells by a plasmid carrying the 1.7kb mRNA 3' terminal sequences were comparable to activities obtained with a plasmid containing the 3' processing signals from a non-internal poly A site. Read-through of HSV poly A sites may therefore reflect a general reduction in mRNA 3' processing efficiency rather than differences in the 3' processing signals of individual genes. The HSV-1 transcripts equivalent to the 4.5kb and 1.2kb mRNAs have sizes of 5. 0kb and 1. 2kb and encode HSV-1 polypeptides Vmw136 and Vmw38 respectively. Nucleotide sequence comparisons between the 5' flanking regions of these equivalent HSV-2 and HSV-1 mRNAs have revealed the following homologies: 1) upstream from the 4.5kb and 5.0kb mRNA 5' termini, blocks of conserved sequences are present which resemble transcription control signals at the promoter regions of other genes; TATA box homologues, C-rich tracts and A+C-rich elements have been identified. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Virology, Biochemistry
• Date of Award: 1986
• Depositing User: Enlighten Team
• Unique ID: glathesis:1986-77329
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jan 2020 11:53
• Last Modified: 14 Jan 2020 11:53
• URI: https://theses.gla.ac.uk/id/eprint/77329