The long term goals of this investigation ar to understand the molecular mechanisms involved in the transduction of environmental signals by the yeast Debaryomyces hansenii. For this research we have chosen salt as an external signal. One of the major reasons for this is the fact that Debaryomyces is capable of survival both in the absence of salt as well hypersaline environments. Three approaches will be employed for the isolation of salt-induced genes. The first involves the construction of a subtracted cDNA library for the enrichment of salt-induced cDNA. The second approach will isolate the gene for the glycerol-3-phosphate dehydrogenase utilizing a synthetic oligodeoxynucleotide complementary to a conserved region of the gene. Finally, since we have identified a 150 kD. protein present only in salt-induced cultures, we will purify this protein and raise polyclonal antibodies against it. We will use these antibodies to screen cDNA expression libraries and isolate the corresponding cDNA. This isolated cDNA will be used as a probe to screen genomic libraries for the isolation of the gene to this protein. Once the genes are isolated, they will be sequenced with special emphasis on the 5' non-transcribed sequences. We will analyze these sequences and identify cis-acting regions that could be involved in the regulation. With the help of these regulatory sequences we will also construct vectors that will allow the expression of any gene in Debaryomyces hansenii.
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