INHIBITION OF PROTEIN SYNTHESIS FROM DNA REPLICATION GENES BY THE PRODUCT OF GENE 1, A dna GENE OF BACILLUS SUBTILIS PHAGE φ29

抄録

The DNA polymerase (Pol, 66 kDa, product of gene 2) and the primer protein (PP, 31 kDa, product of gene 3) of Bacillus subtilis phage φ29 are indispensable, together with the other phage-encoded DNA binding proteins, for the protein-primed DNA replication of φ29. Immediately after infection, genes of these essential proteins are transcribed in a polycistronic fashion. For the further investigation of these essential proteins, genes 2 and 3 of φ29 were cloned together into an Escherichia coli expression-vector system with T7 promoter. At the same time, gene 1, located downstream of gene 2, was incidentally cloned in the same segment with genes 2 and 3 due to the site of the restriction enzymes used. Though gene 1 belongs to dna genes, its function is still unclear. Upon induction of these cloned genes, we observed a very limited amount of Pol and PP in comparison to the product of gene 1 (gp1). To determine the reason for this large difference in gene expression between these three polycistronic genes, highly expressed gene, gene 1 was removed. As the deletion of gene 1, a reasonably high level of expression from the genes of both Pol and PP appeared. Efficient expression was also observed when gene 1 was substituted with its suppressor-sensitive allele, demonstrating that gp1 affected the expression of its upstream genes 2 and 3 in E. coli. This evidence is consistent with the very small amount of both Pol and PP detected in the φ29-infected cells of B. subtilis, and implies that gp1 acts as a regulator of protein synthesis of the DNA replication genes.