Exon-intron organization of the human 5-HT3A receptor gene

The gene structure of the human 5-HT3A receptor gene was analyzed by exon to exon polymerase chain reaction and subsequent sequencing. The results were confirmed by restriction analysis and genomic Southern blotting. The coding region of the human gene was found to be split by eight introns at identical positions as in the murine 5-HT3A receptor gene. All exon-intron boundaries exhibited fully conserved splice donor and acceptor consensus sequences. The alternative splice acceptor in intron eight of the murine gene was not found in the human counterpart. The length of particular introns differs markedly from the murine gene. With the exception of intron 5, all human introns are longer than their murine counterparts. From the start to the stop codon the human gene stretches over about 14.5 kb. The human exon sequences confirm one of three published human 5-HT3A receptor cDNA sequences. Knowledge of the gene structure, including 1.9 kb of the 5' noncoding region, all introns and the exon-intron boundaries of the human 5-HT3A receptor gene should facilitate investigation of its potential role in psychiatric disorders.     

Identification of a new human CYP2A gene fragment with close linkage to CYP2GP1.

Human genomic libraries were screened to identify CYP2G-related cytochrome P450 genes. A genomic fragment comprising exons 7 through 9 of CYP2GP1 and exons 6 through 9 of a previously unidentified CYP2A gene, designated CYP2A7P2, was isolated from an EMBL3 library; the two genes were arranged in outward opposite directions with about 8 kbp of intervening sequence. The same structure was also detected in a bacteriophage P1 clone, which contained a full-length CYP2GP1 gene, exons 6 through 9 of CYP2A7P2, and the CYP2B7 gene. However, additional CYP2A-related exons as well as other CYP2A genes, CYP2A7P1, CYP2B6, CYP2F1, and CYP2GP2 were not detected. These results indicate that CYP2A7P2 is located near CYP2B7 in the middle of the CYP2A-2B-2F gene cluster on chromosome 19. Furthermore, an analysis of CYP2A sequence alignment suggests that CYP2A7P2 may be derived from the same ancestral gene that gave rise to CYP2A7P1, which was corrupted by a large insertion at intron 5.     

人细胞色素P450 3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18重组酶的异源表达与活性

目的重组表达人细胞色素P450(CYP)3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,为CYP3A4代谢活性的体外研究提供单一酶源。方法应用杆状病毒表达系统构建含有上述各CYP3A4突变体基因序列的重组病毒,将其连同含人源还原型烟酰胺腺嘌呤二核苷磷酸-P450氧化还原酶(POR)和细胞色素b5基因的重组病毒共同感染昆虫草地夜蛾细胞Sf9得到CYP3A4突变体与POR和细胞色素b5共表达的重组蛋白,分别以高效液相色谱法和荧光分析法测定各重组酶对睾酮和7-甲氧基-4-三氟甲基香豆素的代谢活性。结果在mRNA分子水平上验证了CYP3A4突变体CYP3A4*3,CYP3A4*4,CYP3A4*5和CYP3A4*18基因在Sf9细胞中的转录。感染了各重组病毒的Sf9细胞裂解液对睾酮和7-苄氧基-4-三氟甲基香豆素有明显代谢。结论应用杆状病毒-昆虫细胞表达系统在体外成功表达了具有催化活性的人CYP3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,其中CYP3A4.5活性显著低于野生型蛋白,CYP3A4.18活性显著高于野生型蛋白,而CYP3A4.3和CYP3A4.4与野生型蛋白活性近似。     

CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.     

Genetic polymorphisms of the CYP3A4, CYP3A5, CYP3A7 and CYP1A2 among the Jordanian population

Cytochromes P450 (CYP450) plays an extremely vital role in oxidation, reduction, and peroxidation of numerous endogenous and exogenous compounds, like drugs and procarcinogens. Mainly, expression occurs in the liver, in varying polymorphic forms. Therefore, proposed as biomarkers of susceptibility to carcinogenicity and toxicity. The objective of this study was to find the allelic frequencies of CYP3A5*2,*3,*4,*5,*6,*7, CYP3A4*1B, CYP3A7*1C and CYP1A2*1C, *1D, *1E, *1F enzymes in Jordanians, and to compare them with other ethnic groups. We used polymerase chain reaction-restriction fragment length (PCR-RFLP) to genotype alleles, and we calculated frequencies using Hardy Weinberg's equation (HWE). Allelic frequencies results were: CYP3A5*2 (0.2%), CYP3A5*3 (86.6%), CYP3A5*6 (1.7%), CYP*3A5*4,*5*7 not detected, CYP3A4*1B (11.7%), CYP3A7*1C (1.7%). Finally 6.5%, 18.2%, 6.0%, 67.3% were the results of CYP1A2*1C, 1D, 1E and 1F, respectively. In conclusion, genotyping method and results of this study can be adopted or used in pharmacotherapy, toxicity and carcinogenic studies in Jordan.     

PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides

OCP are xenobiotics which display various toxic effects on animal and human health. One of their effects is to bind and activate estrogen receptor alpha (ERalpha). We have previously studied the down-regulation of induced CYP1A1 (cytochrome P450) expression by this class of molecules in mammary carcinoma cells and shown the importance of ERalpha in this process. However, an alternative mechanism was suggested by those experiments in hepatoma cells. In this study, we have performed Northern blot and transient transfection assays in various cell lines and shown that OCP activate human pregnane X receptor (PXR) and subsequent CYP3A4 mRNA expression. This effect is mediated by the distal xenobiotic responsive element modulator of the promoter. The induction of CYP3A4 by OCP was dose-dependent within the 1-10 microM range. The data suggest that chronic exposure to OCP could alter a major metabolite pathway in human liver and putatively modify the pharmacokinetics of drugs and pollutants.