Date of Award

9-1-1989

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Department of Chemistry

First Advisor

Dr. Cecil B. Pickett

Abstract

In situ hybridization using a cDNA probe complementary to glutathione S-transferase Yb-j mRNA was utilized to isolate a glutathione S-transferase Yb-| gene from a rat liver genomic library. DNA sequence analysis incorporating "gene walking" strategies revealed that the Yb-j gene spans approximately 5.5 Kb, is comprised of eight exons separated by seven introns and is 99% homologous to the Ybi cDNA clone pGTA/C44. The transcription start site, which maps 29 bp downstream of the TATA element, was determined by primer extension analysis. Gene fragments containing the Yb-| promoter (and various deletions of the promoter region) were inserted upstream of the structural gene encoding chloramphenicol acetyl transferase (CAT). When these chimeric genes were transfected into mammalian cells, CAT assays revealed that the Yb^ minimal promoter is contained within an 80 bp DNA fragment which contains a TATA element, a transcription initiation site and 50 bp of sequence upstream of the TATA element. Contrary to in vivo studies which have shown that Yb-| gene activity is induced in response to phenobarbital and 3-methylcholanthrene administration, no elevation in activity was observed when mammalian hepatoma cells which had been transfected with the chimeric genes were treated with these xenobiotics. These data suggests that the regulatory element which confers xenobiotic inducibility is absent from the first 1700 bp of the Yb-j gene.

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