GENE EXPRESSION DURING SPORULATION

对三种芽孢杆菌的表达调控进行了分析 枯草杆菌孢子形成基因座，SpoIIA，SpoVA和SpoVI，选择的目的是 阐明整个基因表达网络的调控 产孢子。SPO基因座控制区与TO的转录融合 大肠杆菌lac Z基因将用于提供一种简便的检测方法 表情。它们将被用来测试添加营养素的效果 (分解代谢)对基因表达的影响；甲基化细胞可能被用来克服 渗透性屏障。将研究平移型SPO-Lac Z融合 看看操纵子内的基因有时是否可以独立于 ；将特别注意SPIIAC，它为一种 RNA聚合酶Sigma因子样蛋白。 SPO-lac Z基因拷贝数的控制扩增 转录融合，通过使用整合载体，将被用于 根据酶活性滴定基因拷贝数。使用 正交场交替凝胶电泳将使准确的 在很大范围内确定拷贝数。这种方法将是 用来研究在不同的遗传和生理背景下， 表达受到抑制、激活或不受调控。这个 完整的spIIAC基因扩增对其他产孢量的影响 赛事也将接受测试。 携带多个SPO-Lac融合副本的菌株将被用于 分离负性和结构性突变体，以确定新的基因 调节SPO基因的表达，特别是锻炼的基因 时间控制。将使用可整合的质粒来寻找突变， 转座子和化学诱变。调控基因将被克隆 以便对它们进行深入的分析。进一步的研究将确定这些产品 调控基因及其在SPO基因座上的位置(S)。

It is proposed to analyze the control of expression of three Bacillus subtilis sporulation loci, spoIIA, spoVA and spoVI, chosen so as to elucidate the controls of the whole network of gene expression during sporulation. Transcriptional fusions of the spo locus control region to the E. coli lac Z gene will be used to provide an easy assay for expression. They will be used to test the effect of addition of nutrients (catabolites) on gene expression; toluenized cells may be used to overcome permeability barriers. Translational spo-lac Z fusions will be studied to see if genes within an operon can sometimes be regulated independently of each other; particular attention will be paid to spoIIAC which codes for an RNA polymerase sigma-factor-like protein. Controlled amplification of the number of copies of spo-lac Z transcriptional fusions, by use of integrational vectors, will be used to titrate gene copy number against enzyme activity. Use of orthogonal-field-alternation gel electrophoresis will enable accurate determination of copy numbers over a wide range. This approach will be used to see if, in different genetic and physiological backgrounds, expression is subject to repression, activation, or is unregulated. The effect of amplification of the intact spoIIAC gene on other sporulation events will also be tested. Strains harbouring multiple copies of spo-lac fusions will be used to isolate negative and constitutive mutants so as to identify new genes that regulate spo gene expression, and in particular genes that exercise temporal control. Mutations will be sought using integratable plasmid, transposon, and chemical mutagenesis. The regulatory genes will be cloned so as to analyze them in depth. Further studies will identify the products of regulatory genes and their site(s) on the spo loci.