PROMOTER FUNCTIONS IN THE YEAST DEBARYOMYCES HANSENII

本研究的长期目标是了解盐诱导的酵母菌（Debaryomyces hansenii）基因激活的分子机制。为此，我们已经分离出两个假定的编码NAD+甘油-3磷酸脱氢酶（GPDI）的克隆，已知GPDI是盐调控的。我们将采用两种方法来研究GPD1的调控。第一种方法涉及分离甘油代谢缺陷突变体。这将通过化学诱变来完成，并通过percoii -山梨醇梯度进行选择。这些突变的基因将通过与为此目的构建的基因组文库的互补来选择。利用一步基因破坏技术分离GPD1缺失突变体。第二种方法需要研究启动子区域，特别参考顺式作用。第二种方法需要研究启动子区域，特别参考顺式作用序列，并最终利用这些序列分离反式激活子。交互。GPD1的转录和翻译研究将在northern blots（以GPD1基因为探针）和western blots（目前正在培养抗甘油-3磷酸脱氢酶的多克隆抗体）的帮助下进行。最后，通过构建GPD1启动子-绿色荧光蛋白基因盒来研究GPD1的调控作用。从长远来看，这项研究将有助于我们了解致病微生物如何在人体体液中生存（考虑到人体体液富含钠和氯离子）。作为一个额外的优势，在本研究分析的盐启动子的控制下，任何蛋白质都可以在汉斯脱哈里菌中表达。

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.