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dc.contributor.advisorYang, Ming-Deen_US
dc.contributor.authorYu, Yu-Zhenen_US
dc.description.abstractBy utilizing several kinds of restriction enzymes to digest Xanthomonas campestirs pv. vesicatoria #7-1 (Xcv7-1) genomic DNA, we found that Xcv7-1 genomic DNA was resistant to PstI and SmaI digestion. In order to clone the DNA fragments that carried the potential PstI or SmaI-like restristion-modification system (R-M system) genes, Xcv7-1 genomic DNA library was constructed on pBK-CMV plasmid and those plasmids that completely resistant to PstI or SmaI digestion were isolated. One of the R-M system obtained from Xcv7-1 strain was an isoschizomer of PstI and designated as XveI. The two genes were aligned in a tail-to-head orientation with the methytransferase (MTase) preceeding and overlapping the endonuclease (ENase) by two base pairs. The deduced amino acid sequences of XveI indicated that the ENase and MTase contained 364 amino acids and 597 amino acids, respectively. Comparison of the amino acid sequences of ENase and MTase of XveI to other isoschizomers revealed that XveI share 93% and 50% identities with XphI and RleI, respectively. On the other hand, XveI exhibited less than 20% identity to the other two isoschizomers, PstI and BsuBI. The amino acid sequence of M. XveI contained a conserved sequence motif, NPPF, which is characteristic of N6-methylase. The other R-M system cloned from Xcv7-1 in this study, XveII, is an isoschizomer of SmaI. The two genes were aligned in a tail-to-tail orientation and overlapping by 12 base pairs. The XveII MTase encoded a 299-amino acid protein. Comparisons of predicted amino acid sequences with the published sequences indicated that M.XveII was similar to M.SmaI, M.XcyI, M.Cfr9I and M.Pac25I. They share about 44-55% identity. The amino acid sequence of M. XveII contained two conserved motifs, TSPPY and F- G-G, which have been identified as catalytic and SAM binding domains of N4-methylase, respectively. However, R. XveII shared only 38% identity to R.SmaI. A TAA stop codon was found at position 162 of the R.XveII and encoded a 18.3 kDa polypeptide instead of a 30.7 kDa protein (271 amino acids). No enzymatic activity of R.XveII was observed, indicating that it is an inactive protein in vitro. The specificity of M.XveI and M.XveII were also determined and the results showed that M.XveI methylated at the A of CTGCAG and M.XveII at the 3rd C of CCCGGG, respectively. Moreover, the endonuclease and methylase genes of XveI and XveII were PCR amplified and subcloned into pET or pQE vectors. The results demonstrated that R.XveII and M.XveII were overexpressed and the expression of R.XveII was further identified by maxicell method.en_US
dc.subjectRestriction-Modification systemen_US
dc.titleCloning and Characterization of XveI and XveII Restriction- Modification Genes from Xanthomonas campestris pv. vesicatoriaen_US
dc.titleXanthomonas campestris pv. vesicatoria XveI及 XveII 限制修飾系統基因之選殖與特性研究zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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