Studies on the regulation of DNA replication of Rtsl and P

a .与Dr.S.Austin小组的联合研究Rtsl RepA蛋白由288个氨基酸组成，是质粒复制起始所必需的反式作用蛋白。为了研究RepA的功能域，构建了Rts1 RepA与噬菌体P1启动蛋白的杂交蛋白，其中n端部分来自Rts1 RepA， c端部分来自P1 RepA (RepAXn)， n端部分来自P1 RepA， c端部分来自Rts1 RepA （RepALXn）。我们已经报道了RepAXn函数在J.Bacteriol中的应用。177: 4028-4035, 1995，表明氨基酸残基113 ~ 129对ori体外结合很重要，残基177 ~ 206区是ori体内活化和ori结合域所必需的。构建了4个RepALXn蛋白；RepALX12 （P1 RepA 256 aa/Rts1 RepA 31 aa）、RepALX13（205/82）、RepALX15（144/143）和RepALX17 (112/175) RepALX15和RepALX17有效激活了Rts1 ori。这证实了我们之前的发现，即177 - 206aa区域对Rts1 ori的激活至关重要。P1基因被RepALX12和RepALX13激活，这表明P1 RepA的结构域与rts1 RepA非常相似。b .与Dr.A.Kaji组联合研究，我们在rts1基因组上确定了抑制宿主细胞生长的相关位点。该基因座由higA和higB基因组成，并对其功能进行了分析。HigB产物（92 aa）对宿主细胞具有杀伤活性，HigA蛋白（104 aa）抑制了HigB的功能。研究结果将发表在《生物化学》杂志上。Biophys.Res.Commun。(印刷中)。

A.The joint research with Dr.S.Austin groupThe Rtsl RepA protein, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rtsl RepA with the initiator protein of phage P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA (RepAXn), and the N-terminal from P1 RepA and the C-terminal from Rts1 RepA (RepALXn). We already reported the RepAXn functions in J.Bacteriol. 177 : 4028-4035, 1995, suggesting that the aminoacid residues 113 to 129 was important for ori binding in vitro, and the residues 177 to 206 region was required for ori activation in vivo as well as the ori binding domain.Four RepALXn proteins were constructed ; RepALX12 (P1 RepA 256 aa/Rts1 RepA 31 aa), RepALX13 (205/82), RepALX15 (144/143), and RepALX17 (112/175) RepALX15 and RepALX17 activated Rts1 ori efficiently. This evidenced our previous finding that the 177 to 206 aa region is essential for Rts1 ori activation. The P1 ori was activated by RepALX12 and RepALX13, which suggests a quite similar domain structure of P1 RepA to rts1 RepA.B.The joint research with Dr.A.Kaji groupWe determined the locus related to the inhibition of the host cell growth on the Rts1 genome. The locus, named hig, consists of higA and higB genes, and their functions were analyzed. The HigB product (92 aa) displayd a killer activity to the host cells and the HigA protein (104 aa) suppressed the HigB function. The results will be appeared in Biochem. Biophys.Res.Commun. (in press).