| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 105 === phiL7 is a lytic phage specifically infecting the gram-negative Xanthomonas campestris pv. campestris (Xcc) that causes black rot in crucifers. Previous studies on this phage have shown that 1) gene p35 of the phage encodes a T7-like RNA polymerase (RNAP), 2) the product of p24 gene is a putative transcriptional regulator, 3) no consensus sequence similar to the E. coli T7 promoter was found in phiL7, and 4) X. campestris pv. campestris strain Xc17 with cloned p24, designated Xc17(p24), was not lysed by infection with phiL7; however, when p35 gene was cloned concomitantly, the anti-lysis effect of P24 was abolished. In addition, cloned fragments from P15 (508 bp) and P23 (486 bp) DNA regions of phiL7 exhibited promoter activity in Xc17 that expresses p24 and p35 concomitantly. In this study, the bacteria two-hybrid system was used to investigate whether P35 and P24 interact directly, while deletion mutation was exploited to understand the promoter regions of P15 and P23 which are regulated by P35 and P24. The results suggest that 1) P24 and P35 indeed interact directly; the Xc17 RNAP-recognized sequence locates in the upstream one-third region (152 bp) of the P15 fragment, while the downstream one-third region of this fragment (262 bp) possesses the sequences recognized by Xc17 RNAP, P35 and P24, 2) the P35-P24 complex has stronger affinity, than does the Xc17 RNAP, for binding to the downstream region of the P15 fragment to give strong promoter activity, 3) in the downstream of the P23 fragment, there is a 100-bp region that is involved in negative regulation, and 4) the P35-P24 complex binding sequence situates in P23C fragment, including bp 244-382 and the adjacent sequences.
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