Date of Award

Winter 1990

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Clyde Denis

Abstract

Transcription of the glucose-repressible ADH (ADH2 locus) in Saccharomyces cerevisiae is controlled by two regulatory pathways. The general transcriptional factors CCR4, CRE1, and CRE2 constitute the first pathway while the second pathway is comprised of the trans-activators ADR1 and CCR1. Both ADR1 and CCR1 act through upstream activation sequences (UAS) found in the 5$\sp\prime$-regulatory region of the ADH2 structual gene. In contrast, the action of CCR4, CRE1, and CRE2 is likely to be at sequences near the TATAA element of ADH2.

The CCR4 locus was precisely mapped on the left arm of chromosome I where it had previously been localized. Plasmid constructions bearing sequences from chromosome I in the vicinity of CCR4 were tested for their ability to complement a defective ccr4 allele. A functional copy of CCR4 was identified and the DNA sequenced to reveal a 2511-bp open reading frame that predicts a protein with an estimated mass of 94.6-kDal. The CCR4 protein showed similarity with a family of proteins containing a leucine-rich tandem repeat motif. The repeats are characterized by the 24 amino acid repeating sequence P-X-X-o-X-X-L-X-X-L-X-X-L-X-L-X-X-N-X-o (where X = any residue; o = aliphatic residues L, I, or V) and have been suggested to represent a domain involved in protein binding. Deletion analysis of CCR4 indicates that these repeats are required for its proper function. The amino terminus of CCR4 showed similarities to a variety of transcription factors including TFIID, the TATAA binding factor from humans and Drosophila.

Additional studies indicated that CCR4 mRNA and protein levels were not regulated by carbon source availability or the allelic state of the CRE genes. These results suggest that the interactions observed between CCR4 and the CRE genes occur directly or indirectly at a protein level.

The possible role that CCR4 plays in the transcriptional regulation of the ADH2 locus based on (1) the sequence similarities seen between CCR4 and other proteins and (2) the functional characterization of deletions and disruptions created within the coding sequences of CCR4 are discussed.

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