TY - JOUR
T1 - Human indolethylamine N-methyltransferase
T2 - cDNA cloning and expression, gene cloning, and chromosomal localization
AU - Thompson, Michael A.
AU - Moon, Eunpyo
AU - Kim, Ung Jin
AU - Xu, Jingping
AU - Siciliano, Michael J.
AU - Weinshilboum, Richard M.
N1 - Funding Information:
Supported in part by NIH Grants RO1 GM28157 (R.M.W.), RO1 GM35720 (R.M.W.) and RO1 CA34936 (M.J.S.) as well as DOE Grant DEFC03-97ER62299 (U.J.K.). Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. (human INMT cDNA) AF128846 and AF128847 and (human INMT gene) AF128848. 1To whom correspondence and reprint requests should be addressed. Telephone: (507) 284-2246. Fax: (507) 284-9111. E-mail: weinshilboum.richard@mayo.edu.
PY - 1999/11/1
Y1 - 1999/11/1
N2 - Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K(m) value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon- intron splice junctions conformed to the 'GT-AG' rule, and no canonical TATA or CAAT sequences were present within the 5'-flanking region of the gene. Human INMT mapped to chromosome 7p15.2-p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.
AB - Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K(m) value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon- intron splice junctions conformed to the 'GT-AG' rule, and no canonical TATA or CAAT sequences were present within the 5'-flanking region of the gene. Human INMT mapped to chromosome 7p15.2-p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.
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U2 - 10.1006/geno.1999.5960
DO - 10.1006/geno.1999.5960
M3 - Article
C2 - 10552930
AN - SCOPUS:0032757201
SN - 0888-7543
VL - 61
SP - 285
EP - 297
JO - Genomics
JF - Genomics
IS - 3
ER -