Doctoral Dissertations

Date of Award

12-1985

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Francis T. Kenney

Committee Members

Audrey Stevens, Ken Volkin, Wen Yyang, Frank Larimer

Abstract

Gene 33 Is a previously undescribed gene whose expression above basal levels in Sprague-Dawley rat liver cells is regulated independently by three hormones; hydrocortisone (glucocorticoids), insulin, and glucagon. This gene was isolated from a lambda bacteriophage recombinant library of rat liver DNA and characterized. The isolated molecular clone of gene 33 is 13.1 kbp in length and analysis of its subcloned fragments indicate that the gene is single copy in the haploid rat genome. Restriction endonuclease mapping and Northern transfer analysis of gene 33 subclones revealed that the 5' end of the gene is represented in a 2 kbp EcoRl/Hindlll fragment. DNA sequence analysis of 999 bp of this fragment demonstrated that gene 33 is typical of other eukaryotic structural genes in that a TATA box, surrounded by GC rich sequences, is found at -31. An inverted CAAT box is found at -89. Inspection of 480 bp of 5'-flanking sequences revealed four areas of homology to puta tive glucocorticoid receptor binding sites; three are found in the sense strand, one is found in the antisense strand, and two are overlapping. Inspection of 519 bp of the RNA coding region revealed two direct repeats, one inverted repeat, one putative donor splice site, and one putative acceptor splice site. An N-terminal amino acid sequence of 19 residues was derived from the DNA sequence. Data and reagents gained as a result of this study will be invaluable for future studies of gene 33 hormonal regulation.

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