STRUCTURE & EXPRESSION OF MAMMAL ALC DEHYDROGENASE GENES

申请人摘要：本研究的长期目标是 了解调节哺乳动物乙醇脱氢酶的机制 (ADH)基因表达，以及生理和病理 ADH表达改变的后果。 我们假设 ADH基因表达的差异 ADH同工酶的差异，影响酒精的代谢， 酒精消费的后果。 更好地了解 调节ADH基因的表达将大大增加我们的 了解酒精代谢。 这次更新继续我们的 顺式作用DNA序列的探索， 调节ADH基因表达，并将我们的研究扩展到分离 并检测与这些位点结合的转录因子。 我们将确定顺式作用元件是重要的调节 人类和小鼠I类ADH基因的组织特异性表达 ADH 1、ADH 2、ADH 3和Adh 1。 这些内容将由一个 结合蛋白质结合分析（如DNA酶I足迹和 凝胶阻滞）和功能研究（瞬时转染， 报道构建体）。 密切相关的I类之间的比较 基因将揭示顺式作用中微妙机会的影响， 序列，并将阐明组织特异性的演变。 的 ADH 4、ADH 5、ADH 6和ADH 7基因具有不同的表达模式， 并产生酶（分别为PI-ADH、X-ADH、ADH 6和SIGMA-ADH） 影响重要的代谢过程，包括 许多其他醇如视黄醇。 我们会研究 这些基因的表达与我们对I类的研究平行， 基因. 我们将尝试确定哪些转录因子与顺式- 我们通过检查已经描述的因素， and by searching搜索for novel新factors因素. 我们努力的一个主要方向是 克隆与顺式作用元件结合的转录因子。 我们将用寡核苷酸筛选cDNA表达文库 含有顺式作用元件。 由于一些顺式作用的 元件出现新的，我们期望克隆新的转录因子。 将分析转录因子以确定其结合 特异性，并鉴定蛋白质内的功能结构域。 这些研究将有助于我们对基因的基本理解， 调节，组织特异性和发育，以及我们的理解 的遗传因素之间的差异，在个人 酒精的代谢、药理和病理作用 消费

APPLICANT'S ABSTRACT: The long term Goals of this research are to understand the mechanisms which regulate mammalian alcohol dehydrogenase (ADH) gene expression, and the physiological and pathological consequences of alterations in ADH expression. It is our hypothesis that differences in the expression of the ADH genes can, like differences in ADH isozymes, affect the metabolism of alcohols and the consequences of alcohol consumption. A greater understanding of the regulation of the expression of the ADH genes will add much to our understanding of alcohol metabolism. This renewal continues our exploration of the cis-acting DNA sequences that are important in regulating ADH gene expression, and expands our study to the isolation and examination of the transcription factors that bind to these sites. We will identify cis-acting elements that are important in the regulation of tissue-specific expression of the human and mouse class I ADH genes ADH1, ADH2, ADH3 and Adh1. These elements will be studied by a combination of protein-binding analyses (such as DNaseI footprinting and gel retardation) and functional studies (transient transfections with reporter constructs). Comparisons among the closely related class I genes will reveal the effects of subtle chances in the cis-acting sequences, and will illuminate the evolution of tissue specificity. The ADH4, ADH5, ADH6 and ADH7 genes have different patterns of expression, and produce enzymes (PI-ADH, X-ADH, ADH6 and SIGMA-ADH, respectively) that influence important metabolic processes, including metabolism of many other alcohols such as retinol. We will examine the regulation of expression of these genes in parallel with our studies of the class I genes. We will attempt to determine which transcription factors bind to the cis- acting elements we identify, both by examining already described factors and by searching for novel factors. A major thrust of our efforts will be to clone transcription factors that bind to the cis-acting elements. We will screen cDNA expression libraries with oligonucleotides containing a cis- acting element. Since several of the cis-acting elements appear novel, we expect to clone novel transcription factors. Transcription factors will be analyzed to determine their binding specificity, and to identify functional domains within the protein. These studies will contribute both to our basic understanding of gene regulation, tissue specificity and development, and to our understanding of the genetic factors underlying differences among individuals in the metabolic, pharmacological and pathological effects of alcohol consumption.