MOLECULAR GENETIC ANALYSIS OF YEAST SPHINGOLIPIDS

鞘脂在自然界中广泛存在，但其功能 不太了解。 真菌含有鞘脂， 与动物鞘脂的区别在于存在 磷酸肌醇。 酵母中鞘脂的生物合成途径 示意性地，前体--长链神经酰胺-- 鞘脂 酿酒酵母含有少量 并提供了一个独特的机会，使用分子 遗传技术与生物化学技术相结合， 研究鞘脂。 由于我们的努力，酵母是唯一的 已知存在长链缺陷突变体的真核细胞 碱基合成，并且生物合成途径中的基因具有 被孤立。 鞘脂生物合成后期的突变体 在任何真核生物中都没有分离出来;然而，我们提出他们的 通过我们开发的新富集程序分离。 为了了解鞘脂生物合成途径的细节， 其调节作用，以及这些脂质在S.酿酒酵母， 我们将描述负责长链碱基的基因 通过使用长链营养缺陷型（Lcb-）合成已经 孤立的，以及额外的获得了有效的 选择方法;其中一个基因已经被克隆 （LCB 1）。 我们建议通过使用 通过类似的选择获得的温度敏感突变体 法 指导鞘脂合成的基因将被克隆，我们将 确定它们的转录本大小，转录方向， 转录起始位点和核苷酸序列。 调控 将对这些基因的转录进行检查。 的功能 酵母鞘脂将通过分离和 表征第二位点抑制基因和通过过表达 克隆的鞘脂生物合成基因。 酵母鞘脂为合理设计提供了一个局部焦点 用于减轻人类真菌感染的抗真菌剂 感染.

Sphingolipids are widespread in nature but their functions are poorly understood. Fungi contain sphingolipids that are distinguished from animal sphingolipids by the presence of phosphoinositol. The pathway of sphingolipid biosynthesis in yeast is schematically, precursors---long chain base ceramide-- sphingolipids. Saccharomyces cerevisiae contains a small number of sphingolipids and offers a unique opportunity to use molecular genetic techniques in conjunction with biochemical techniques to study sphingolipids. As a result of our efforts yeast are the only eucaryote for which there are know mutants defective in long chain base synthesis and for which a gene in the biosynthetic pathway has been isolated. Mutants in later steps of sphingolipid biosynthesis have not been isolated in any eucaryote; however we propose their isolation by a novel enrichment procedure we have developed. To understand the details of the sphingolipid biosynthetic pathway, its regulation, and the function of these lipids in S. cerevisiae, we will characterize the genes responsible for long chain base synthesis by use of long chain base auxotrophs (Lcb-) already isolated as well as additional ones obtained by an effective selection method; one of these genes has already been cloned (LCB1). We propose to isolate genes later in this pathway by use of temperature sensitive mutants obtained by a similar selection method. Genes that direct sphingolipid synthesis will be cloned and we will determine their transcript size, direction of transcription, transcription start sites, and nucleotide sequence. The regulation of transcription of these genes will be examined. The functions of yeast sphingolipids will be probed by isolating and characterizing second-site suppressor genes and by overexpression of the cloned sphingolipid biosynthesis genes. Yeast sphingolipids provide a local focus for the rational design of antifungal agents for use in alleviating human fungal infections.