REGULATION OF NITROGEN METABOLISM IN YEAST

在几乎所有复杂的大分子中发现的主要元素之一 活细胞是氮。氮对合成特别重要。 并不令人惊讶的是细胞拥有 调节氮代谢的主要控制机制，确保 为生长不断提供充足的这种养分。这些实验 在这份提案中概述的是旨在研究分子 酵母氮代谢的水平及其调控 酿酒。酵母为这些研究提供了一个很好的模型系统 因为生化分析和先进的分子分析都很容易 这种有机体中的遗传学。 已分离出不能在富营养介质上生长的酵母突变株 (YPD)，但在以氨为氮源的最低限度培养基上正常生长。 添加单一氨基酸(苯丙氨酸、苏氨酸、丝氨酸、 异亮氨酸、缬氨酸、谷氨酸、谷氨酰胺)在低浓度下对 这些突变体生长在最小的氨板上。然而，它们会增长 通常依赖于贫乏的氮源(例如，脯氨酸)，不受 添加这些氨基酸。这些结果提示YPD-表型 是由这些菌株的氮代谢失衡引起的， 我已经将它们命名为AAT(氨基酸毒性)1和AAT2。一个完整的 AAT酵母菌株氮代谢的生化分析 试图确定这些突变体中的特定病变。 尤其是关键酶(如谷氨酸脱氢酶)的水平， 氨基酸池和氨基酸转运将被确定。监管 将对这些反应进行研究，以确定氮的水平 在这些菌株中的抑制作用。 我还建议开始对这些菌株进行基因分析。克隆人拥有 与aat1和aat2的ypd表型互补 酵母。这些将被进一步表征，然后用来获得 有关AAT基因及其编码的蛋白质的信息 酵母分子遗传学技术。

One of the major elements found in nearly all complex macromolecules of living cells is nitrogen. Nitrogen is particularly important for synthesis of proteins and nucleic acids and it is not surprising that cells possess major control mechanisms to regulate nitrogen metabolism and ensure a constant adequate supply of this nutrient for growth. The experiments outlined in this proposal are designed to investigate, at the molecular level, regulation of nitrogen metabolism in the yeast Saccharomyces cerevisiae. Yeast provides an excellent model system for these studies because of the ease of both biochemical analysis and advanced molecular genetics in this organism. Yeast mutants have been isolated that are unable to growth on rich media (YPD) but grow normally on minimal media using ammonia as nitrogen source. Addition of single amino acids (phenylalanine, threonine, serine, isoleucine, valine, glutamate, glutamine) at low concentrations is toxic to these mutants growing on minimal ammonia plates. However, they grow normally on poor nitrogen sources (e.g., proline) and are unaffected by addition of these amino acids. These results suggest the YPD- phenotype has been caused by an imbalance of nitrogen metabolism in these strains and I have named them AAT (amino acid toxicity) 1 and AAT2. A complete biochemical analysis of nitrogen metabolism in aat yeast strains is proposed in an attempt to identify the specific lesion in these mutants. In particular, the levels of key enzymes (e.g., glutamate dehydrogenase), amino acid pools and amino acid transport will be determined. Regulation of these reactions will be studied to determine the level of nitrogen repression in these strains. I also propose to start a genetic analysis of these strains. Clones have been isolated that complement the YPD- phenotype of both aat1 and aat2 yeast. These will be further characterized and then used to obtain information about the AAT genes and the proteins they encode by the techniques of yeast molecular genetics.