Masters Theses

Jon S. Evans

Date of Award

12-1988

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Jerry P. Weir

Abstract

The expression of the Herpes Simplex Virus fusion gene was studied by linking the promoter-regulatory region to the coding sequences of β-galactosidase. A 1983 base pair Sph I-Bam HI fragment containing the fusion gene and 894 base pairs upstream from the start site of translation was cloned into the phage M13mpl8 and a new Xba I site was created by oligonucleotide directed site-specific mutagenesis in order to isolate the promoter-regulatory region of the fusion gene. The 894 base pair Sph I-Xba I fragment was cloned into the Herpes Simplex Virus insertion vector pGal8 and the resultant clone, pGal8/fp, was co-transfected into Vero cells with wild type Herpes Simplex Virus Type 1 DNA. Homologous recombination between pGal8/fp and viral DNA resulted in a recombinant virus that expressed beta-galactosidase from the promoter of the fusion gene. Vero cells were infected with the purified recombinant in the presence or absence of phosphonoacetic acid, an inhibitor of viral DNA synthesis, and assayed for β-galactosidase activity. Activity was first observed at 6 hrs, reached a peak at 9.5 hours, and was enhanced in the presence of phosphonoacetic acid, indicating that the chimeric gene was regulated as a Herpes Simplex Virus early gene. RNA was isolated from cells infected in the presence of phosphonoacetic acid at 9.5 hours and in the absence of phosphonoacetic acid at 24 hours post-infection and used to determine the start site of transcription by primer extension analysis. Oligonucleotides complementary to the 5' DNA sequences of either the β-galactosidase gene or the structural portion of the authentic fusion gene were hybridized to RNA, extended with reverse transcriptase, and were electrophoresed alongside a sequence ladder generated by either the plasmid pGal8/fp and the β-galactosidase primer or the plasmid Bam HI L with the fusion gene primer. The start sites of transcription in the first case were found to be at 59, 61, and 65 bases upstream from the start site of translation, and in the second case at 59 and 61 bases upstream from the translational start site. In both cases, the strongest signal was seen with the RNA extracted from cells harvested at 9.5 hours post-infection. Lastly, a deletion mutant containing only the the promoter region up to 205 base pairs upstream of the first ATG was constructed from the pGal8/fp clone, designated pGal8/fpΔ7C, and used to generate a second recombinant virus. Initial plaque assays revealed the presence of thymidine kinase negative virus that expressed β-galactosidase, indicating that the sequences deleted are not essential for the expression of the fusion gene.

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