Author

Waltz

Date of Award

12-2003

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Dr. John Geiser

Second Advisor

Dr. Bruce Bejcek

Third Advisor

Dr. Rob Eversole

Access Setting

Masters Thesis-Open Access

Abstract

Cytoplasmic dynein is a minus-end directed microtubule motor that recently has been described as a member of the AAA+ ATPase family. Dyn1p contains four ATP-binding consensus sequences. To understand the role of each ATP-binding site we have begun to systematically mutate the consensus sequence at each ATP-binding site. Thirty-two mutant alleles have been constructed and integrated into the yeast Saccharomyces cerevisiae. We have examined each mutant for ability to grow in rich media, to produce binucleate cells at 12°C, ability to grow in the absence of CIN8, and ability of Dyn1p to bind microtubules. Attempts to localize cytoplasmic dynein by direct immunofluorescence were not successful when expressed from its promoter. We have thus expressed Dyn1p from the galactose inducible promoter, GALI. Dynein, microtubules and DNA were visualized using standard immunofluorescence techniques. Our results show that wild-type Dyn1p binds at the minus-end of microtubules. No Dyn1p staining is seen within the nucleus. A yeast strain carrying a mutant allele of dynein lacking the microtubule-binding site does not bind to microtubules. Strains mutated in each of the four ATP-binding sites show intermediate phenotypes between wild-type and the strains lacking the microtubule-binding site.

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Biology Commons

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