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|標題:||Cloning and Characterization of XveI and XveII Restriction- Modification Genes from Xanthomonas campestris pv. vesicatoria
Xanthomonas campestris pv. vesicatoria XveI及 XveII 限制修飾系統基因之選殖與特性研究
By 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
|Appears in Collections:||分子生物學研究所|
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