REGULATION OF SEQUENTIAL GENE EXPRESSION IN BACILLUS

分化细胞使用什么机制来激活特定的 基因的正确序列，以确保从一个细胞的有序发展， 另一种类型？ 对这个问题的可能答案将由 研究在癌症晚期激活基因的控制机制， 枯草芽孢杆菌中的内生孢子形成。 在孢子形成期间 这种生物在形态上经历了连续的变化， 生理学可以调节，至少在早期阶段， 修饰细胞的RNA聚合酶。 监管因素， 激活在孢子形成后期转录的基因， 定时机制，允许这些因素发挥作用，在这个特定的 发展阶段不详。 我们将研究进一步的RNA聚合酶修饰是否可以发挥作用， 通过多次分离这种酶， 在内生孢子形成的最后一半， 组成和体外转录特异性。 此外，RNA 在这些时间段内唯一合成的基因将被检测到。 DNA 与这些RNA互补的序列将被克隆并用作 用于体外转录的模板。 转录酶将 在“晚期”期间分离的部分纯化的RNA聚合酶， 孢子形成”，或者，如果这些酶不能转录克隆的序列， 然后从已经发展到这个阶段的细胞中提取粗蛋白质， 晚期 导致“后期转录”的因素包括 使用它们在体外转录中的活性鉴定和纯化 作为指导。 然后分析假定的调节因素，以确定如何 它们只能在特定阶段发挥作用， 发展计划。 将制备单克隆抗体， 预期的新的RNA聚合酶亚基和/或其他晚期转录 用作免疫探针的因子，以确定这些因子何时 存在于细胞中以及影响其合成的条件。 的 抗体也将用于开发基于抗体的快速 这些蛋白质的纯化系统。 多克隆抗体和 将制备寡核苷酸探针并用于克隆结构 编码晚期转录因子的基因。 一旦克隆，这些 然后可以通过基因融合和体外转录来分析基因 技术.

What mechanisms do differentiating cells use to activate particular sets of genes in the proper sequence to ensure an orderly progression from one cell type to another? Possible answers to this question will be sought by examining the control machanisms that activate genes at late stages of endospore formation in the bacterium Bacillus subtilis. During sporulation this organism undergoes a sequential alteration in morphology and physiology that may be regulated, at least during the early stages, by modification of the cell's RNA polymerase. The regulatory factors that activate genes transcribed at late stages of spore formation as well as the timing mechanism that permits these factors to function at this particular stage of development are unknown. We will investigate whether further RNA polymerase modifications could play a role in late gene transcription by isolating this enzyme at several times during the final half of endospore formation to examine its subunit composition and in vitro transcriptional specificity. In addition, RNA's that are uniquely synthesized during these periods will be detected. DNA sequences that are complementary to these RNA's will be cloned and used as templates for in vitro transcription. The transcribing enzymes will be either partially purified RNA polymerases that were isolated during "late sporulation" or, if these enzymes fail to transcribe the cloned sequences, then crude protein extracts prepared from cells that had progressed to this late stage. The factors responsible for "late transcription" would be identified and purified using their activity in the in vitro transcription assay as a guide. The putative regulatory factors would then be analyzed to determine how they are able to perform their function only at a particular stage in the developmental program. Monoclonal antibodies will be prepared against anticipated novel RNA polymerase subunits and/or other late transcription factors for use as immunological probes to determine when these factors are present in cells as well as conditions that influence their synthesis. The antibodies would also be used in the development of an antibody-based rapid purification system for these proteins. Polyclonal antibody and oligonucleotide probes will be prepared and used to clone the structural genes that code for the late transcription factors. Once cloned, these genes could then be analyzed by gene fusion and in vitro transcription techniques.