Date of Award

1998

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Beverly C. Delidow

Second Advisor

Todd L. Green

Third Advisor

Terry W. Fenger

Fourth Advisor

Sheldon N. Finver

Fifth Advisor

Donald A. Primerano

Sixth Advisor

Dr. Leonard Deutsch

Abstract

Sporulation is a developmental process in which temporally distinct sets of genes regulate the processes of meiosis and ascospore formation. Sporulation-specific genes are classified into early, middle, and late genes based on their peak time of expression during sporulation. Regulation of two late genes, SPR1 and SPR2, was studied. The function of SPR2 is unknown. Its regulatory region contains two Mid Sporulation Elements (MSE) repeated in tandem that are sufficient to confer sporulation-specific regulation to a CYCl-lacZ reporter. These sequences have UAS (upstream activation sequence) properties, as each 14 base pair element is sufficient to confer sporulation-specific regulation independent of orientation. A greater than 5-fold higher induction is observed with the tandem MSEs compared to the individual elements. Mutations within a GC rich region indicate that these sequences are necessary for MSE function.

The SPR1 gene encodes a 1,3-β-glucanase that is believed to be involved in spore wall maturation. The 5’ regulatory region of SPR1 was studied by constructing a series of translational and transcriptional fusions. Studies using the translational fusions suggest that sequences from -268 to +1 are sufficient to confer sporulation-specific regulation, and that SPR1 is controlled by a TATA-less promoter that lies between -151 to +1. In addition, homology searches revealed two sequences with weak matches to known activation sequences: (a) sequences from -293 to -279 have 10/15 matches to the UAS of SPS4; (b) sequences from -252 to -236 have 12/17 matches to the MSE of SPR2. Internal deletion and transcriptional fusion studies, taken together, indicate that both the UAS-like and MSE-like sequences are necessary but not sufficient for temporal regulation of SPR1. This evidence suggests that SPR1 is regulated by a composite promoter with essential elements lying downstream of -268 and other weaker positive elements which are dispersed from -268 to -762.

Studies with mutants of ime2, ume6 and ndt80 suggest that all three proteins are positive regulators of the SPR1 and SPR2 genes. Distinct banding patterns observed during mobility shift assays with extracts from vegetative and sporulating cells, suggest that different protein complexes bind the regulatory sequences during vegetative growth and sporulation. Recombinant Ndt80p shows DNA protein interactions with both SPR1 and SPR2 probes. This evidence suggests that Ndt80p regulates SPR2 via the MSE, while it regulates SPR1 via other sequences.

Subject(s)

Genetic regulation – Research.

Promoters (Genetics).

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