Cloning and characterization of human placental catechol-O-methyltransferase cDNA.
Catechol-O-methyltransferase (COMT) cDNA clones were isolated from a human placental cDNA library using synthetic oligonucleotides as probes. All four positive clones isolated contained an open reading frame, which potentially coded for a 24.4-kD polypeptide, presumably corresponding to the cytoplasmic form of the COMT (S-COMT). In addition to the S-COMT sequences, two of the clones carried extensions in the 5' end, which potentially coded for a 50-amino-acid peptide extending the S-COMT reading frame. This sequence contained a stretch of signal sequence-like hydrophobic amino acids in its amino terminus. The deduced human COMT polypeptide had 80% similarity with the previously characterized rat COMT. Expression of one of the cDNA clones in human K-562 cells resulted in cell clones with 3- to 10-fold increased COMT activity. Cell-free translation of transcripts synthesized in vitro from one of the short cDNAs yielded a 26-kD product, similar in size to human S-COMT. Translation of transcripts from one of the long cDNAs gave 30-kD and 26-kD polypeptides, suggesting translation initiation from two different AUG initiation codons. The 30-kD protein, but not the 25-kD protein, associated with microsomal membranes in translation lysates. A potential polyadenylation signal AATTAA was detected in the 3' ends of two of the clones 265 nucleotides downstream from the COMT translation termination codon. RNA blotting on human placental RNA revealed a 1.5-kb-long COMT-specific transcript. DNA analysis suggested that human, as well as rat, canine and monkey cells have one gene for COMT.
Lundström
K
K
Orion Corporation, Laboratory of Molecular Genetics, Helsinki, Finland.
Salminen
M
M
Jalanko
A
A
Savolainen
R
R
Ulmanen
I
I
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002394:Catechol O-Methyltransferase / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D014176:Protein Biosynthesis; D012313:RNA / Q000032:analysis; D051381:Rats; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D012313:RNA; D002394:Catechol O-Methyltransferase