Ogawa H, Konishi K, Takata Y, Nakashima H, Fujioka M
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Eur J Biochem. 1987 Oct 1;168(1):141-51. doi: 10.1111/j.1432-1033.1987.tb13398.x.
The amino terminus of glycine methyltransferase from rat liver is blocked. A hexapeptide containing the blocked amino-terminal residue was obtained from a tryptic digest of the purified enzyme and its amino acid sequence was determined to be Ac-Val-Asp-Ser-Val-Tyr-Arg by Edman degradation and fast-atom-bombardment mass spectrometry after fragmentation with Staphylococcus aureus protease V8. A full-length cDNA clone for the enzyme was isolated from a lambda gt11 rat liver cDNA library using the previously obtained pGMT A56 cDNA [Ogawa, H., Gomi, T., Horii, T., Ogawa, H. & Fujioka, M. (1984) Biochem. Biophys. Res. Commun. 124, 44-50] as a probe. The amino acid sequence deduced from the nucleotide sequence contained both amino- and carboxyl-terminal sequences. The predicted amino acid composition and molecular mass were also in agreement with the published data obtained with the purified protein. Five clones for the glycine methyltransferase gene were isolated from a Charon 4A library containing EcoRI digest of rat liver DNA by in situ plaque hybridization. All clones had inserts of 6500 base pairs, consistent with the size of EcoRI genomic DNA fragment determined by Southern blot hybridization. Sequence analysis of a 5400-bp fragment of the insert DNA lacking a 1100-bp 5' region and comparison of the sequence with that of the cDNA showed that the insert DNA entirely encoded glycine methyltransferase and the gene consisted of six exons and five introns. S1 nuclease protection 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, CAAT and GC sequences, and the complementary sequence to the enhancer core element, were located upstream of the transcription initiation site.
大鼠肝脏甘氨酸甲基转移酶的氨基末端被封闭。从纯化酶的胰蛋白酶消化物中获得了一个包含被封闭氨基末端残基的六肽，经金黄色葡萄球菌蛋白酶V8切割后，通过埃德曼降解法和快原子轰击质谱法确定其氨基酸序列为Ac-Val-Asp-Ser-Val-Tyr-Arg。使用先前获得的pGMT A56 cDNA [小川浩、古美、堀井哲、小川浩和藤冈正(1984年)，《生物化学与生物物理学研究通讯》，124卷，44 - 50页]作为探针，从λgt11大鼠肝脏cDNA文库中分离出该酶的全长cDNA克隆。从核苷酸序列推导的氨基酸序列包含氨基末端和羧基末端序列。预测的氨基酸组成和分子量也与纯化蛋白的已发表数据一致。通过原位菌斑杂交从包含大鼠肝脏DNA的EcoRI消化物的Charon 4A文库中分离出五个甘氨酸甲基转移酶基因克隆。所有克隆的插入片段均为6500个碱基对，与通过Southern印迹杂交确定的EcoRI基因组DNA片段大小一致。对插入DNA的一个缺少1100 bp 5'区域的5400 bp片段进行序列分析，并将该序列与cDNA序列进行比较，结果表明插入DNA完全编码甘氨酸甲基转移酶，该基因由六个外显子和五个内含子组成。S1核酸酶保护图谱分析和引物延伸分析使我们推测，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、CAAT和GC序列以及增强子核心元件的互补序列位于转录起始位点的上游。
