Isolation and characterization of a UDP–glucuronosyltransferase (UGT1A01) cloned from female rhesus monkey
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Materials and methods
Materials. RNeasy, PolyFect transfection reagent, and other molecular biology products were obtained through Qiagen (Valencia, CA). M-MLV Reverse Transcriptase (RNase H minus) and the pGEM-T Easy vector were purchased from Promega (Madison, WI). The E-gels, tris–acetate NuPAGE gels, Dulbecco's modified eagle's media (DMEM), heat-inactivated fetal bovine serum, Hepes, geneticin (G418), and the pcDNA3.1 vector were obtained through Invitrogen (Carlsbad, CA). AmpliTAQ Gold was purchased through
Results
Detection and isolation of novel UGTs from rhesus monkey liver cDNA were performed using primers for human UGT isoforms based on the high degree of homology between primates. Screening of various primer combinations has led to the detection of a 1645-bp PCR product using the human UGT1A1 primers. This cDNA was sequenced and data analysis confirmed existence of a 1599-bp open reading frame encoding a 533-amino acid translational product (Fig. 1). The deduced amino acid sequence revealed greater
Discussion
This paper describes the isolation and characterization of female rhesus UGT1A01, an ortholog of human UGT1A1 and cynomolgus monkey UGTlA01 [32] and the first UGT to be sequenced and characterized from this species. The amplified UGT1A01 cDNA encodes for a functional full-length enzyme of 533 amino acids possessing UGT characteristics such as an ER-directing signal peptide (residues 1–27), a transmembrane-spanning domain (residues 490–508), a UDPGA-binding domain (residues 354–402), and
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