GENE EXPRESSION DURING SPORULATION

分析了三种芽孢杆菌的表达调控 枯草芽孢杆菌孢子形成位点，spoIIA、spoVA和spoVI，选择以 阐明整个基因表达网络的控制， 孢子形成 spo基因座控制区与 急诊coli lac Z基因将用于提供一种简单的检测方法， 表情 它们将被用来测试添加营养素的效果 （catalysts）对基因表达的影响;甲苯化细胞可用于克服 渗透屏障 翻译spo-lac Z融合将被研究， 看看操纵子中的基因是否有时可以独立于 将特别注意spoIIAC，它编码一个 RNA聚合酶σ因子样蛋白。 spo-lac Z拷贝数的受控扩增 通过使用整合载体的转录融合物将用于 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 在宽范围内确定拷贝数。 这种方法将是 用来观察在不同的遗传和生理背景下， 表达受到抑制、激活或不受调节。 的 完整spoIIAC基因扩增对其他孢子形成的影响 事件也将受到考验。 携带多拷贝Spo-lac融合物的菌株将用于 分离阴性和组成型突变体，以便鉴定 调节spo基因表达，特别是运动基因， 时间控制 将使用可整合质粒寻找突变， 转座子和化学诱变。 调控基因将被克隆 以便深入分析。 进一步的研究将确定产品 调节基因及其在spo基因座上的位点。

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.