Salminen M, Lundström K, Tilgmann C, Savolainen R, Kalkkinen N, Ulmanen I
Orion Corporation, Laboratory of Molecular Genetics, Helsinki, Finland.
Gene. 1990 Sep 14;93(2):241-7. doi: 10.1016/0378-1119(90)90231-f.
The coding sequence of rat liver catechol-O-methyl-transferase (COMT; EC 2.1.1.6) was determined from rat cDNA and genomic libraries were screened with DNA probes and specific antiserum. The open reading frame consisted of 663 nucleotides coding for a 221-amino acid (aa) polypeptide with a deduced Mr of 24,747. No obvious hydrophobic signal sequence, membrane-spanning domains, or potential N-glycosylation sites were found in this sequence. The identity of the clone and the accuracy of the sequence was verified by direct aa sequencing of the tryptic peptides derived from the purified rat liver enzyme. Primer extension analysis showed that the transcription start point of the rat liver COMT mRNA was 450 bp upstream from the translation start codon. A putative polyadenylation signal (ATTAAA) was found in the 3'-noncoding region. The predicted size of the COMT transcript was 1.8-2.0 kb, which could be confirmed from Northern hybridization analyses of the isolated rat liver mRNA. One polypeptide of 25 kDa, could be immunoprecipitated with anti-COMT antibody from in vitro translation of rat liver mRNA. Employing the DNA blot analysis only one COMT-encoding gene was found in the rat genome.
从大鼠cDNA中确定了大鼠肝脏儿茶酚-O-甲基转移酶（COMT；EC 2.1.1.6）的编码序列，并用DNA探针和特异性抗血清筛选了基因组文库。开放阅读框由663个核苷酸组成，编码一个221个氨基酸（aa）的多肽，推导的Mr为24747。在该序列中未发现明显的疏水信号序列、跨膜结构域或潜在的N-糖基化位点。通过对纯化的大鼠肝脏酶衍生的胰蛋白酶肽进行直接氨基酸测序，验证了克隆的身份和序列的准确性。引物延伸分析表明，大鼠肝脏COMT mRNA的转录起始点在翻译起始密码子上游450 bp处。在3'-非编码区发现了一个推定的聚腺苷酸化信号（ATTAAA）。预测的COMT转录本大小为1.8 - 2.0 kb，这可以通过对分离的大鼠肝脏mRNA的Northern杂交分析得到证实。用抗COMT抗体从大鼠肝脏mRNA的体外翻译产物中可免疫沉淀出一条25 kDa的多肽。采用DNA印迹分析，在大鼠基因组中仅发现一个编码COMT的基因。
