Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21077
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
作者: Yu, Yu-Jen
于玉珍
關鍵字: 限制脢;Endonuclease;甲基轉移脢;限制修飾系統;Methyltransferase;Restriction-Modification system
出版社: 分子生物研究所
摘要: 
Abstract
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
method.

中 文 摘 要
利用不同的限制切割九株 Xanthomonas 菌株染色體 DNA,結果發現
Xanthomonas campestris pv. vesicatoria #7-1 (Xcv7-1) 之染色體
DNA 不會被 及 SmaI 限制所作用,因此本研究利用構築的 Xcv7-1 染
色體基因庫,來篩選 Xcv7-1 可能具有的限制修飾系統基因。在方法上
將帶基因庫的質體 DNA 分別以 PstI 或 SmaI 切割後,轉形送入宿主
中,挑出存活的菌落,其中即可能含有與 Pst及 SmaI 作用類似的限制修
飾系統基因。本研究由此方法篩選到了與 PstI 作用類 似的 XveI 限制
修飾系統及與 SmaI 作用類似之 XveII 限制修飾系統。在 XveI 系統中
甲基轉移基因位在限制基因之前,二個基因的轉錄方向是相同的,並
於 甲基轉移基因轉譯終止處與限制基因起始處重疊了二個鹼基對
。XveI 限制 基因共可轉錄出 364 個胺基酸,分子量為 39.7 kDa
,而甲基轉移為 597 個 胺基酸,分子量為 64.8 kDa。經胺基酸序列
比對後,與 XphI 限制修飾系統的相 同性為 93%,與 RleI 限制修飾系
統具有 49-50% 的相同性,而與其他同功 PstI及 BsuBI 限制修飾系統
的相同性則低於 20% 以下。在 XveI 甲基轉移中可以 找到 NPPF 保
留性區域,是屬於 N6-甲基轉移。
另外從 Xcv7-1 分離出來的與 SmaI 作用類似限制修飾系統基因稱為
XveII,其二個基因轉錄的方向是相反的,且在彼此轉譯終止處重疊了 12
個鹼基 對。XveII 甲基轉移含 299 個胺基酸,其分子量為 33.7 kDa
,將甲基轉移基 因進行比對後,與 SmaI、Cfr9I、XcyI 及 Pac25I 甲
基轉移約有 44% 至 55% 的相同性。XveII 甲基轉移中則具有二個
保留性區域,分別為 TSPPY 及 F-G- G,是與甲基轉移活性與 SAM
輔助因子鍵結區域,屬於 N4-甲基轉移。然 而在限制只與 SmaI
限制具有 38% 的相同性,應可轉譯出 271 個 胺基酸, 分子量為
30.7 kDa。但在 XveII 限制中第 162 個胺基酸出現 TAA 終止子,
而只能轉譯出一 18.3 kDa 的蛋白,且經由生體外的活性測試,此蛋白是
不具有限 制的活性。
在偵測 XveI 與 XveII 甲基轉移的特異性方面,初步的結果推測 XveI
甲基 轉移可在 CTGCAG 的 A 鹼基上進行甲基化,而 XveII 甲基轉
移則會在 CCCGGG 的第三個 C 上進行甲基化。進一步利用 PCR
方式分別增幅出此四個 基因,並黏接於 pET 或 pQE 表現載體上,
進行蛋白的大量表現,結果位於 pQER-XveII 及 pQEM-XveII 上之
xveII 基因可利用 SDS-PAGE 分析,觀察到蛋 白的大量表現。由此二
重組質體所表現的蛋白且經由 Maxicell 作進一步的確認。
URI: http://hdl.handle.net/11455/21077
Appears in Collections:分子生物學研究所

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