MOLECULAR GENETIC ANALYSIS OF YEAST SPHINGOLIPIDS

鞘脂被认为在细胞间相互作用中起作用， 调节动物的生长、分化和肿瘤发生。 最近的发展表明，鞘脂参与信号转导， 转导 尽管取得了很大进展， 鞘脂的具体作用及其作用机制。 一 鞘脂研究的局限性是缺乏实验研究， 适合分子遗传分析的生物体。 期间 在过去的资助期内，我们的研究揭示了酵母菌的潜力， 酿酒厂提供了结合分子遗传分析， 生物化学技术用于鞘脂合成和功能的研究。 这项工作为我们提出的具体目标奠定了基础， 我们将（1）检验鞘脂浓度是 部分通过调节丝氨酸的活性来维持 棕榈酰转移酶，（2）分离肌醇-β-神经酰胺的基因 (IPC)和神经酰胺合成酶，过表达它们，并纯化， 酶的特征。 IPC合成酶将被强调，因为它 催化真菌特有的反应，这应该是一个有效的靶点 抗真菌药物，（3）确定遗传和生化基础， 菌株7 R4绕过制造鞘脂的需要的能力。 的 在该菌株中积累的独特脂质将被表征为 确定它们是否在锚定蛋白的形成中起中介作用 神经酰胺和磷脂酰肌醇-聚糖，（4）使用遗传 研究鞘脂在信号传导中的潜在作用的策略 S.啤酒。 我们的研究结果应该能让我们更好地了解 鞘脂合成、功能和真菌及其他 有机体 此外，它们还应提供一个独特的目标， 设计治疗免疫受损的抗真菌药物 人类 作为我们努力的直接结果， 克隆酵母鞘脂生物合成基因以分离人类同源物。

Sphingolipids are thought to play roles i cell-to-cell interaction, regulation of growth, differentiation, and oncogenesis in animals. Recent developments have shown that sphingolipids participate in signal transduction. Despite great progress, much remains to be learned about the specific roles of sphingolipids and their mechanism of action. One limitation in sphingolipid research has been the lack of experimental organisms that are amenable to molecular genetic analysis. During the past grant period our research revealed the potential that Saccharomyces cerevisiae offers for combining molecular genetic analysis with biochemical techniques to studies of sphingolipid synthesis and function. This work has laid the foundation for our proposed Specific Aims in which we will (1) examine the hypothesis that sphingolipid concentrations is maintained in part by regulating the activity f serine palmitoyltransferase, (2) isolate the gene(s) for inositol-P-ceramide (IPC) and ceramide synthetases, overexpress them, and purify and characterized the enzymes. IPC synthetase will be emphasized since it catalyzed a reaction unique to fungi that should be an efficacious target for antifungal drugs, (3) determine the genetic and biochemical basis for the ability of strain 7R4 to bypass the need to make sphingolipids. The unique lipids that accumulate in this strain will be characterized to determine if they are intermediated in the formation of proteins anchored to ceramide and to phosphatidylinositol-glycans, (4) use a genetic strategy to examine the potential role of sphingolipids in signal transduction in S. cerevisiae. The results of our studies should provide a better understanding of sphingolipid synthesis, function(s), and regulation in fungi and other organisms. In addition, they should provide a unique target for designing antifungal drugs needed for treatment of immune compromised humans. As a direct results of our efforts it should be possible to used clone yeast sphingolipid biosynthetic genes to isolate human homologs.