CHROMOSOME SEGREGATION IN A FILAMENTOUS BACTERIUM

描述（由申请人提供）：在已被详细研究的少数生物体中，杆状细菌的单个圆形染色体高度组织，染色体区域具有特定的亚细胞地址；复制起点位于细胞的旧极，复制终点位于细胞分裂产生的新极附近。其他基因座在细胞中排列的顺序是它们出现在染色体上的顺序，从起点到终点。相比之下，丝状的土壤细菌冷色链霉菌有一个大的线性基因组。我们对链霉菌孢子中线状染色体的结构知之甚少，而在特化气生菌丝同步分裂过程中线状染色体的分离机制也知之甚少。这些菌丝体生物对卫生和生物技术工业具有特殊和至关重要的意义，因为它们产生许多生物活性化合物，如抗生素、抗真菌剂、抗蠕虫剂、除草剂和抗肿瘤药物。多年来，研究人员一直认为，金黄色葡萄球菌的每个孢子室都有一个基因组拷贝。强有力的初步证据表明，传统观念是不正确的。首先，有基因证据。参与DNA分离和凝聚的某些基因的突变似乎产生含有脱氧核糖核酸的孢子，脱氧核糖核酸被解除浓缩，显示出两个裂片。此外，从野生型菌株中可以分离到明显的遗传二倍体。其次，有细胞学证据。用共聚焦激光扫描显微镜测量荧光染料与野生型类核结合的强度，分析结果支持每个孢子有两个基因组拷贝的假设。同样，荧光原位杂交（FISH）结果的初步分析表明，每个野生型孢子有两个基因组拷贝。杂交探针到起源区域显示两个荧光焦点位于孢子的两端。此外，分离蛋白- EGFP融合，结合位点在起源附近，定位于成熟孢子两极的两个焦点，正如使用FISH观察到的起源区域一样。一个候选基因，编码一个沃克型atp酶，参与建立染色体的位置在孢子内已被确定。本研究拟开展实验，完成初步的遗传学实验，扩展孢子基因组结构的细胞学分析。额外的基因编码蛋白质所需的发育相关的基因组分离将鉴定细菌的两个杂交分析。几个已知的编码分离蛋白的基因的转录将被测试对发育调控基因的依赖性。

DESCRIPTION (provided by applicant): In the few organisms where it has been studied in detail, the single circular chromosome of a rod-shaped bacterium is highly organized and regions of the chromosome have particular subcellular addresses; origins of replication are located at the old cell poles, replication termini are near the new poles derived from cell division. Other loci are arranged in the cell in the order that they appear on the chromosome, origin to terminus. In contrast, the filamentous soil-dwelling bacterium Streptomyces coelicolor has a large linear genome. Little is known about the architecture of the linear chromosome within the Streptomyces spore and the mechanisms which govern its segregation during synchronous division of specialized aerial hyphae are poorly understood. These mycelial organisms are of particular and vital importance to health and biotechnology industries because they produce many biologically active compounds such as antibiotics, antifungals, antihelminthics, herbicides and antitumor drugs. For years researchers have assumed that there is a single copy of the genome per spore compartment of S. coelicolor. Strong preliminary evidence suggests that conventional wisdom is incorrect. First, there is genetic evidence. Mutants of certain genes involved in DNA segregation and condensation appear to produce spores containing DNA that is decondensed showing two lobes of DNA. In addition, apparent genetic diploids can be isolated from the wild type strain. Second, there is cytological evidence. Confocal laser scanning microscopy was used to measure the intensity of fluorescent dye bound to the wild type nucleoid, and the analysis supports the hypothesis of two genome copies per spore. Similarly, preliminary analysis of fluorescent in situ hybridization (FISH) results point to two copies of the genome per wild type spore. Hybridizing a probe to the origin region shows two fluorescent foci located at opposite ends of the spore. Furthermore, a partition protein- EGFP fusion, with binding sites near the origin, localizes in two foci to the poles of maturing spores, as was observed for the origin region using FISH. A candidate gene, encoding a Walker-type ATPase, involved in the establishment of the chromosome position within the spore has been identified. This study proposes experiments to complete the preliminary genetic experiments and extend the cytological analysis of the genome architecture in the spore. Additional genes encoding proteins required for development-associated genome segregation will be identified by bacterial two hybrid analyses. Transcription of several known genes encoding segregation proteins will be tested for their dependence on developmental regulatory genes. PUBLIC HEALTH RELEVANCE: We wish to understand how DNA is segregated and packaged in the special filamentous organisms that produce the majority of biologically active compounds used to treat infection and cancer. These are major producers of the compounds we all rely on for our health and quality of life. Basic knowledge of fundamental cell functions of these organisms may lead to better production of known and novel compounds and lower the cost of buying medicines.