Isolation, characterization and baculovirus-mediated expression of the cDNA encoding cytosine DNA methyltransferase from Pisum sativum.
Isolation, characterization and baculovirus-mediated expression of the cDNA encoding cytosine DNA methyltransferase from Pisum sativum.
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豌豆胞嘧啶 DNA 甲基转移酶 cDNA 的分离、表征和杆状病毒介导的表达。
DOI：
10.1093/nar/26.5.1214
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发表时间：
1998
影响因子：
14.9
通讯作者：
Adams,RL
中科院分区：
文献类型：
--
作者：
Pradhan,S;Cummings,M;Roberts,RJ;Adams,RL
从豌豆 cDNA 文库中分离出一系列与拟南芥胞嘧啶 5 DNA 甲基转移酶 (C-5 MTase) 互补的重叠克隆。组装的核酸序列包含4761bp的开放阅读框，编码1554个氨基酸的蛋白质。与其他真核 C-5 MTase 一样，推断的蛋白具有与催化 C 端结构域相连的假定调节 N 端区域，该结构域具有原核 C-5 MTase 中发现的 10 个保守基序中的 8 个。豌豆 C-5 MTase 与拟南芥 C-5 MTase 在核苷酸水平上有 65% 的同一性，在蛋白质水平上有 61% 的同一性。豌豆酶的催化结构域与拟南芥有 78% 的同一性，与鼠类和人类 C-5 MTase 具有约 52% 的同一性，包括催化中心脯氨酸-半胱氨酸二肽的相对位置。使用 cDNA 的保守区域作为探针，我们鉴定了 5 kb 的转录本。使用上述探针对豌豆基因组 DNA 进行 Southern 印迹分析表明存在单个基因。使用来自不同发育阶段和不同组织的poly(A)+ RNA，我们观察到表达主要局限于植物的快速分裂组织。该组装的 cDNA 在杆状病毒系统中表达，产生约 174 kDa 的蛋白质。表达的蛋白质可以以 AdoMet 依赖性方式交联到含有 FdC 的双链寡核苷酸底物，以代替 CG 或 CAG/CTG 序列中的靶胞嘧啶。
A series of overlapping clones complementary to the Arabidopsis cytosine-5 DNA methyltransferase (C-5 MTase) has been isolated from pea cDNA libraries. The assembled nucleic acid sequence contains an open reading frame of 4761 bp encoding a protein of 1554 amino acids. Like other eukaryotic C-5 MTases, the inferred protein has a presumed regulatory N-terminal region linked to a catalytic C-terminal domain, which has eight of the ten conserved motifs found in prokaryotic C-5 MTases. The pea C-5 MTase has 65% identity at the nucleotide level and 61% identity at the protein level, with the Arabidopsis C-5 MTase. The catalytic domain of the pea enzyme shares 78% identity with Arabidopsis and approximately 52% identity with murine and human C-5 MTases, including the relative position of the proline-cysteine dipeptides of the catalytic centre. Using the conserved region of the cDNA as a probe, we have identified a transcript of 5 kb. Southern blot analysis of pea genomic DNA with the above probe indicates the presence of a single gene. Using poly(A)+ RNA from different developmental stages and different tissues, we have observed that expression is confined mostly to the rapidly dividing tissues of the plant. Expression of this assembled cDNA in a baculovirus system gives a protein of approximately 174 kDa. The expressed protein can be cross-linked, in an AdoMet-dependent manner, to duplex oligonucleotide substrates containing FdC in place of target cytosines in either CG or CAG/CTG sequences.