Studies on the early step of bacteriophage N4 infection and purification and characterization of type Ⅱrestriction endonuclease stu Ⅰ

Abstract

In this thesis, the content will be classified into two main parts; [1] Studies on the early step of bacteriophage N4 infection, [2] Purification and characterization of Stu I endonuclease. For the first part, the novel genes from bacterial strains Proteus vulgaris and Brevibacterium albidum, which render the host resistant to both phages of N4 and a $\lambda$ lib, a randomly selected $\lambda$ derivative from the human genomic library, has been analyzed. The following experimental results obtained from the study of the genes supported the possibility that phage N4 shares some components of host cells with $\lambda$ in the early step of its infection processes. (i) The clones transformed by one of the recombinant plasmids, pRMG101 (contains the genes from P. vulgaris) or pRMG216 (contains the genes from B. albindum), were resistant to both $\lambda$ lib and N4, but sensitive to $\varphi$80vir, T4, and T7. (ii) Naked phage DNAs of $\lambda$lib and N4 were transfected and propagated successfully even when the host cell carried one of the two plasmid DNAs (pRMG101AB and pRMG216). (iii) When pBR322 DNA was used instead of pUC9 DNA to carry the genes from Proteus vulgaris, the degree of resistance of the E. coli host having this plasmid was reduced both in N4 and $\lambda$ phages. It seems to be due to the smaller copy number of pBR322 than that of pUC9. (iv) E. coli K-12/pRMG101AB and E. coli K12/pRMG216 lost their resistance to N4 and $\lambda$lib simultaneously when they experienced some physiological changes by grown in maltose medium. On the basis of above implications, the interrelationship between N4 and the host components mediating $\lambda$ infection in the early step was investigated. N4 phage grows normally on the various lamB mutants of E. coli, which are resistant to $\lambda$ because of the abnormality or the absence of LamB protein, the receptor molecule for $\lambda$. This result indicates that LamB is not the only required component for N4 to adsor...