DNA Initiation Gene, dnaB, of Bacillus Subtilis

我们将研究枯草芽孢杆菌中染色体复制启动的调控。它仍然没有被很好地理解。其机制可能涉及嵌入细胞膜的多组分结构，该结构由特定的DNA片段和蛋白质组成。至于蛋白质组分，将研究dna B操纵子基因编码的蛋白质的结构和功能。Dna B蛋白的功能对于染色体的复制启动是必不可少的。该基因及其侧翼区已被克隆和测序。Dna B基因被证明是操纵子的第一个基因，操纵子由三到四个基因组成，dna B，Y-，Z-，可能还有基因。针对前三种病毒的特异性抗血清已经制备好，并将用于分离由dna B操纵子基因编码的蛋白质。抗体也将用于体外启动的研究。将尝试分离Y基因和Z基因的突变。关于参与复制起始的DNA片段，我们将研究枯草杆菌染色体复制起始区和质粒pUB110两种类型的膜结合的性质。I型膜结合是耐盐的，依赖于dna B+基因的功能，而II型结合是盐敏感的，不依赖dna B+基因的功能。I型结合被认为是起始和类型生化过程中必不可少的。II型结合对于pUB110和染色体原始与膜上的结构结合是重要的，该结构对复制的启动和子代染色体的分割都是必不可少的。在枯草杆菌染色体和pUB110中，I型结合位点似乎都位于复制起始处或附近。在pUB110分子的四个区域和距离枯草杆菌染色体复制起点20.50kb的PurA16基因座附近的两个区域发现了II型结合。这些绑定的性质和功能意义将进一步分析。

The regulation of initiation of chromosome replication in Bacillus subtilis will be investigated . It still is not yet well understood. The mechanism likely involves a multi- component structure inbedded in the cellular membrane which consists of specific DNA fragments and proteins. As for the protein components, the structure and function of the proteins encoded by the dnaB operon genes will be worked out. The function of the dnaB protein is essential for the replication initiation of the chromosome. The gene and its flanking regions have been cloned and sequenced. The dnaB gene turned out to be the first gene of an operon which consists of three or four genes, dnaB, Y-,Z-and possibly genes. Specific antisera against the first three have been prepared and will be used to isolate proteins encoded by the genes of the dnaB operon. Antibodies will be also used for the studies of the in vitro initiation. Attempts will be made to isolate mutants of the Y- and Z-genes. With regard to the DNA fragments involved in the replication initiation, we will study the nature of the two types of membrane binding of the replication origin areas of the B. subtilis chromosome and a plasmid pUB110. The type-I membrane binding is salt-resistant and dependent on the dnaB+ gene function, whereas type-II binding is salt-sensitive and independent of the dnaB+ gene function. It is believed that type I binding is essential for biochemical processes of initiation and type.II binding is important for pUB110 and the chromosomal original to be bound to a structure on the membrane that is essential for both initiation of replication and the partition of daughter chromosomes. The type -I binding site seems to be located at or near the origin of replication both in the B. subtilis chromosome and pUB110. Type-II binding is found in four areas of the pUB110 molecule and in two areas near the purA16 locus that is located within 20.50 kb from the replication origin of the B.subtilis chromosome. The properties and functional significance of these bindings will be analayzed further.