The Construction of Expression Vectors of Several Variants of the JC Virus
Aurelia, Jacqueline L.
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Expression vectors for the transcriptional analysis of several variants of the JCV promoter were constructed at the Wayne state University School of Medicine. JCV is a virus that causes progressive multifocal leukoencephalopathy (PML). Recombinant DNA technologies, polymerase chain reaction (PCR) , bacterial transformations, small-scale plasmid preparations, and the Dideoxy Sanger method of DNA sequencing were used to create the constructs. Restriction endonucleases were used to excise the regulatory region from the entire viral genome, and to linearize the expression vector. PCR was employed, using oligonucleotides as primers, the entire viral genomic DNA as template, and Tag DNA polymerase to amplify a specific short sequence within the regulatory region of JCV. Fragments were isolated by agarose gel electrophoresis; their concentrations were determined by measuring their optical densities. The complete promoter region of JCV N-4 was placed upstream of a chloramphenicol acetyl transferase gene in plasmid pGKOCat for one construct. In another construct, a 66 base pair putative enhancer region was placed upstream of the SV40 promoter in plasmid pGKA10Cat. Two other constructs were assembled by positioning the Adenovirus Major Late (AdML) initiator adjacent to a truncated and mutated JCV core promoter in pGKOCat. These final cloned constructs will be used to measure the ability of the regulatory region to control transcription.