DNA Initiation Gene, dnaB, of Bacillus Subtilis

将研究枯草芽孢杆菌染色体复制起始的调控。它仍然没有被很好地理解。其机制可能涉及细胞膜内由特定DNA片段和蛋白质组成的多组分结构。在蛋白质成分方面，将研究dna操纵子基因编码的蛋白质的结构和功能。dna蛋白的功能对染色体的复制起始至关重要。该基因及其侧翼区域已被克隆并测序。dnaB基因被证明是由三到四个基因组成的操纵子的第一个基因，dnaB, Y-， z -可能还有其他基因。针对前三种的特异性抗血清已经制备出来，并将用于分离由dna操纵子基因编码的蛋白质。抗体也将用于体外起始的研究。将尝试分离Y-和z -基因的突变体。对于参与复制起始的DNA片段，我们将研究枯草芽孢杆菌染色体复制起始区与质粒pUB110的两种膜结合的性质。i型膜结合是耐盐的，依赖于dnaB+基因功能，而ii型膜结合是盐敏感的，不依赖于dnaB+基因功能。认为I型结合在起始和分型的生化过程中是必不可少的。II结合对于pUB110和染色体原体结合到膜上的一个结构上是很重要的，这对于开始复制和子染色体的分裂都是必不可少的。i型结合位点似乎位于枯草芽孢杆菌染色体和pUB110的复制起点或附近。ii型结合在pUB110分子的四个区域和位于距枯草芽孢杆菌染色体复制起源20.50 kb范围内的purA16位点附近的两个区域发现。我们将进一步分析这些绑定的属性和功能意义。

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.