The long term goals of this investigation are to understand the molecular mechanisms in the activation of salt-induced genes in the yeast Debaryomyces hansenii. To this end we have already isolated two putative clones encoding for NAD+glycero-3 phosphate dehydrogenase (GPDI) which is known to be salt regulated. Two approaches will be employed to study regulation of GPD1. The first approach involves the isolation of mutants defective in glycerol metabolism. This will be accomplished by chemical mutagenesis and selected with a percoII-sorbitol gradient. The gene(s) for these mutants will be selected by complementation with a genomic library being constructed for that purpose. GPD1 deletion mutants will be isolated with the help for one step gene disruption technique. The second approach entails the study of the promoter region with special reference to cis-acting. The second approach entails the study of the promoter region with special reference to cis-acting sequences and eventually utilizing these sequences to isolate the trans-activator. The interaction. Transcription and translation studies of GPD1 will be done with the help of northern blots (with the GPD1 gene is a probe) and western blots (with polyclonal antibodies against glycerol-3 phosphate dehydrogenase being raised at the moment). Finally, regulation of GPD1 will be studied by the construction of the GPD1 promoter-green fluorescent protein gene cassette. In the long term this research will help our understanding on how pathogenic microorganisms survive in human body fluids (considering that human body fluids are rich in sodium and chloride ions). As an added advantage it is feasible to express any protein in Deharyomyces hansenii under the control of the salt promoters analyzed in this study.
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