Date of Award:

5-1-1994

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Biology (Molecular Biology)

Committee Chair(s)

Jon Y. Takemoto

Committee

Jon Y. Takemoto

Committee

Greg J. Podgorski

Committee

Reed P. Warren

Abstract

To study syringomycin's mode of action, a gene that confers sensitivity to specific syringomycin-resistant mutants was cloned and characterized. SYR2 was identified by complementing syringomycin-resistant strains 13N-F2 and 28N-D6 with a wild-type genomic library. Several overlapping clones were isolated. A 2 kilobase DNA fragment was subcloned and sequenced. The sequence revealed an open reading frame of 1047 nucleotides that encodes a protein of 349 amino acids. The sequence was identical to the yeast gene SUR2. Comparisons with sequences in the SwissProtein database showed that Syr2 had no significant homology to know proteins. Syr2 showed significant similarity to a protein encoded by an expressed sequence tag contained in the EMBL nucleotide library. The hydropathy pattern of Syr2 revealed six potential membrane-spanning regions. Gene disruption of SYR2 in S. cerevisiae did not affect viability, but conferred resistance to syringomycin, while gene overexpression showed no distinguishable phenotype. Polyclonal antibodies directed against Syr2 were prepared. Syr2 was detected by Western blot analysis in yeast microsomal extracts and was associated with the endoplasmic reticulum. Disruption of SYR2 altered intact cell ferricyanide-reductase activities stimulated by syringomcin. In summary, SYR2 encodes a protein associated with the endoplasmic reticulum that is essential to the syringomycin response pathway, but not for cell viability.

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