Rat glycine methyltransferase. Complete amino acid sequence deduced from a cDNA clone and characterization of the genomic DNA.
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.
Ogawa
H
H
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Konishi
K
K
Takata
Y
Y
Nakashima
H
H
Fujioka
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004251:DNA Transposable Elements; D005796:Genes; D050938:Glycine N-Methyltransferase; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D051381:Rats; D014158:Transcription, Genetic
D000596:Amino Acids; D004251:DNA Transposable Elements; D004247:DNA; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase; C494512:Gnmt protein, rat