Construction of an <i>Escherichia coli lac</i> Repressor-Based System to Study Human Cytomegalovirus Gene Expression

<p>An <em>Escherichia coli lac</em> repressor-based system was developed to study the roles of human cytomegalovirus (HCMV) genes during viral replication. To this end, a recombinant HCMV expressing the <em>lac</em> repressor was generated (RV<em>lac</em>), and an HCMV-specific promoter was targeted for conditional expression by inserting the <em>lac</em> operator sequence. The promoter of a nonessential gene was chosen in order to be able to assess parameters of repression and derepression of the operator-containing promoter in the endogenous locus, without having virus growth dependent on the specific inducer isopropylthiogalactoside (IPTG). The feasibility of this approach to conditionally express an HCMV promoter was demonstrated by analyzing <em>lac</em> operator-containing, HCMV US9 promoters in CAT reporter constructs, using RV<em>lac</em> and IPTG in transient assays. This study demonstrates that efficient repression mediated by the <em>lac</em> repressor can be achieved, and this repression is efficiently reversed by IPTG. In addition, the operator-containing US9 promoters were inserted into the endogenous locus to investigate the impact of the operator insertion on basal promoter expression in the context of the virus, before these constructs are used to study conditional expression in the viral genome. It was observed that US9 endogenous promoter expression was not affected significantly by the operator insertions. Because attempts to isolate a recombinant virus containing the operator-containing US9 promoter and expressing the <em>lac</em> repressor were unsuccessful, future studies should target the insertion of the operator-containing US9 promoter and the <em>lac</em> repressor gene into two separate recombinant viruses. These viruses could be used in coinfection experiments to address conditional US9 gene expression. Furthermore, alternative sites for insertion of the operator sequence within the US9 promoter should also be evaluated. After demonstrating the feasibility of this approach in the context of the viral genome, the system can then be adapted to target putative essential HCMV genes.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Year dc:date.available

1999

Author dc:creator

• Cageao-Luchetti, Laura Fernanda

Contributors dc:contributor

• Richard M. Stenberg
• Timothy J. Bos
• Ann E. Campbell

• Escherichia coli
• Human cytomegalovirus
• Viral replication
• Lac repressor
• Microbiology
• Molecular Biology

Identifier

9780599652620

OAI identifier oai:identifier

oai:digitalcommons.odu.edu:biomedicalsciences_etds-1015