MOLECULAR GENETICS OF SULFUR METABOLISM IN NEUROSPORA

神经孢子虫硫代谢的分子遗传学

• 批准号: 3466386
• 负责人: JOHN V PAIETTA
• 金额: $ 10.4万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466386
• 关键词: DNA footprinting; Neurospora; arylsulfatases; cysteine; fungal genetics; gene interaction; genetic manipulation; genetic transcription; messenger RNA; microorganism metabolism; molecular cloning; mutant; nucleic acid sequence; regulatory gene; structural genes; sulfur compounds; tissue /cell culture

A major sulfur regulatory system in Neurospora crassa will be characterized at the molecular level by using a genetically- defined set of trans-acting regulatory genes and a structural gene they control. The studies are aimed at achieving an understanding of the regulatory interactions and overall organization of this multigene network. The analysis will also lead to a better understanding of how a cell regulates its sulfur status and of human inborn errors of metabolism. The system consists of the cys-3+ regulatory gene (positive regulator), three negative regulatory genes (designated scon-1+, scon-2+, scon-3+), and the arylsulfatase structural gene (ars+). The cys-3+ and ars+ genes have been have been cloned. The scon+ negative regulatory genes will be analyzed on a genetic and molecular level. Epistasis tests and function in heterokaryons will aid in establishing the hierarchy of control. At least on of the scon+ genes will be cloned by sib selection. All the cloned genes will be fully characterized (i.e., their sequence, transcripts, and gene products). Detailed studies will use the cloned genes to determine the transcriptional and post-transcriptional controls in this multigene system. Transcriptional regulation will be confirmed by nuclear transcription assays. Steady state and kinetic mRNA measurements will also be done. Posttranscriptional control will be detected by constructing gene fusions followed by gene replacement and assay. These mRNA analyses will be done with the regulatory mutants available and under different growth conditions (i.e., sulfur derepressing and repressing) so that both genetic and metabolic effects can be assessed. Potential cis- regulatory sequences will be mutagenized in vitro and re- introduced by gene replacement in order to directly confirm and test their function. The regulatory gene products will be produced in vitro and using gel mobility assays along with DNA footprinting further confirm the regulatory sequences. The interaction of cysteine (the metabolic effector) with the regulatory gene products will be examined by equilibrium dialysis. The prediction that one (or more) of the regulatory gene products will demonstrate DNA and/or cysteine binding properties will be directly tested. If time allows, the study of functional domain in the regulatory gene products will be carried out by the in vitro alteration of the genes coding sequences followed by both in vivo and in vitro assessment of function.

粗糙脉孢菌中的一个主要硫调节系统将是 在分子水平上，通过使用基因- 一组确定的反式作用调控基因和一个结构基因 他们控制。 这些研究旨在了解 监管互动和整体组织， 多基因网络 分析还将导致更好的 了解细胞如何调节其硫状态， 人类先天性代谢缺陷。 该系统由 cys-3+调节基因（正调节子），3个负调节子 调节基因（命名为scon-1+，scon-2+，scon-3+），以及 芳基硫酸酯酶结构基因（ARS+）。 cys-3+和ars+基因 已经被克隆了。 scon+负调控基因 将在基因和分子水平上进行分析。 上位性检验 异核体的功能将有助于建立 控制层次。 至少有一个scon+基因将是 通过同胞选择克隆。 所有克隆的基因都将完全 其特征在于（即，它们的序列、转录本和基因 产品）。 详细的研究将使用克隆的基因来确定 转录和转录后调控 多基因系统 转录调控将得到证实 通过核转录分析。 稳态和动力学mRNA 还将进行测量。 转录后调控将 通过构建基因融合体， 替换和分析。 这些mRNA分析将用 可获得的调控突变体和不同生长条件下的调控突变体 条件（即，硫去抑制和抑制）， 可以评估遗传和代谢效应。 潜在顺式- 调节序列将在体外诱变， 通过基因置换引入，以直接确认和 测试其功能。 调控基因产物将 在体外产生并使用凝胶迁移率测定沿着DNA 足迹法进一步证实了调控序列。 的 半胱氨酸（代谢效应物）与 调节基因产物将通过平衡透析来检查。 预测一个（或多个）调控基因产物 将证明DNA和/或半胱氨酸结合特性， 直接测试。 如果时间允许， 调控基因产物将通过体外 改变基因编码序列，然后在体内 和功能的体外评估。