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The study on the structure and expression of the glycine methyltransferase gene from rat normal and tumor livers

大鼠正常及肿瘤肝脏甘氨酸甲基转移酶基因的结构及表达研究

基本信息

批准号：
60570109
负责人：
OGAWA Hirofumi
金额：
$ 0.96万
依托单位：
Toyama Medical and Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

项目摘要

1. The N-terminal of rat liver glycine methyltransferase (EC2.1.1.20) is blocked. The peptide containing the N-terminal residue was obtained from various protease-digests through HPLC, and its sequence was determined as Acetylval-Asp-Ser-Val-Tyr-Arg by means of Edman degradation, HPLC and mass spectrometric analysis.2. A lot of cDNA clones for this enzyme were screened from a lambda gtll library containing liver cDNA inserts by plaque hybridization, using the previously obtained cDNA clone as a probe. Sequence analysis of the largest insert DNA showed that the cDNA consisted of 4 bp of 5' noncoding region, an open reading frame of 882 bp, and 102 bp of 3' noncoding region. The cDNA-deduced amino acid sequence contained both the amino and carboxyl terminal sequences.3. The genomic DNA clone for glycine methyltransferase was obtained from a lambda Charon 4A library by in situ plaque hybridization. Five clones were found to have an insert of 6500 bp long. Sequence determination indicated that the gene consisted of 6 exons split by 5 introns as compared to the cDNA sequence. The sequences of exon/intron splice junctions clearly obeyed the "GT...AG" rule. Sl nuclease mapping and primer extension analysis allowed us to propose that the A residue located 19 bp upstream from the translation initiation codon is the site of transcription initiation. "TATA", "CAT" and "GC" boxes,and the complementary sequence to the enhancer core element were located at the expected positions upstream of the transcription initiation site.

1.阻断大鼠肝甘氨酸甲基转移酶（EC 2.1.1.20）的N-末端。通过高效液相色谱法从多种蛋白酶解产物中分离得到含有N-末端残基的多肽，经Edman降解、高效液相色谱和质谱分析确定其序列为Acetylval-Asp-Ser-Val-Tyr-Arg.用预先获得的cDNA克隆作为探针，通过噬斑杂交从含有肝cDNA插入片段的λ gt 11文库中筛选出该酶的大量cDNA克隆。最大插入片段的序列分析表明，该cDNA由4 bp的5'端非编码区、882 bp的开放阅读框和102 bp的3'端非编码区组成。cDNA推导的氨基酸序列含有氨基端和羧基端序列.甘氨酸甲基转移酶的基因组DNA克隆通过原位噬斑杂交从λ Charon 4A文库获得。发现5个克隆具有6500 bp长的插入片段。序列测定表明，该基因由6个外显子和5个内含子组成。外显子/内含子剪接点的序列明显服从“GT... AG”规则。S1核酸酶作图和引物延伸分析使我们能够提出位于翻译起始密码子上游19 bp处的A残基是转录起始位点。“TATA”、“CAT”和“GC”盒以及增强子核心元件的互补序列位于转录起始位点上游的预期位置。