Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The biosynthesis in Leydig cells of the C19 steroid testosterone from the C21 precursor progesterone requires the activities of the enzyme cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha)). Previous studies from this laboratory demonstrated that the de novo synthesis of the P450(17 alpha) protein and the accumulation of P450(17 alpha) mRNA in mouse Leydig cell cultures is absolutely dependent on cAMP stimulation. To investigate further the cAMP regulation of P450(17 alpha) expression in Leydig cells, the structural gene encoding P450(17 alpha) (Cyp17) was isolated from a mouse genomic library using a full-length mouse P450(17 alpha) cDNA. Two overlapping genomic clones were isolated and characterized by restriction mapping and partial sequencing. The two clones together contain the entire coding region and approximately 10 kilobases of 5'-flanking sequences of Cyp17. To identify regions necessary for cAMP-induced transcription, 5'-flanking regions of Cyp17 were fused with the chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into MA-10 tumor Leydig cells. Studies localized the cAMP-responsive region of the gene to a region between -346 and -245 basepairs relative to the transcription initiation site. Transient transfections of MA-10 cells with a construct consisting of the -346/-245 sequences fused to a heterologous promoter, thymidine kinase, and the CAT reporter gene demonstrated a marked increase in cAMP stimulation of CAT expression, providing additional evidence that the -346/-245 sequences of the Cyp17 5'-flanking region confer cAMP-induced expression of Cyp17. This cAMP-responsive region of mouse Cyp17 bears no apparent homology to the cAMP-responsive regions identified in the human and bovine Cyp17 genes.
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PMID:Isolation and characterization of the mouse P450 17 alpha-hydroxylase/C17-20-lyase gene (Cyp17): transcriptional regulation of the gene by cyclic adenosine 3',5'-monophosphate in MA-10 Leydig cells. 132 57

The molecular forms of estrogen receptor (ER) in estrogen-responsive mouse Leydig cell line (B-1) have been examined in relation to their biological activity. ER was predominantly recovered in the nuclear fraction upon homogenization even after cells were precultured in the absence of E2 and Phenol Red. This unoccupied nuclear ER (ERn) whose hormone binding ability was extremely thermostable could be extracted with 0.4 M KCl. This stability enabled us to determine hydrodynamic parameters in the ligand-free condition. The Stokes radius and sedimentation constant of this ERn in high salt condition were 5.5 nm and 6.0S, respectively, resulting in its molecular weight of 140,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ER labeled with [3H]tamoxifen aziridine gave a single band of 65,000 Da, indicating that this ERn had a oligomer structure similar to that of transformed nuclear ER complexed with estrogen in the putative target cells. Therefore, we further examined the possibility that this ERn in B-1 cells can activate estrogen-responsive genes without any aid from estrogen. Estrogen responsive element-thymidine kinase promoter-chloramphenicol acetyltransferase fusion gene (ERE-tk-CAT) was transfected into B-1 cells. CAT activity was enhanced only in cells stimulated with estrogen. It may be concluded from these results that transformed ERn can be formed in the absence of estrogen but that binding to estrogen may be required in order to exert its biological activity.
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PMID:Identification of unoccupied but transformed nuclear estrogen receptor in cultured mouse Leydig cell. 235 31

The Leydig insulin-like gene (Ley I-L), a member of the insulin-related gene family, is specifically expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. To determine the functional region of the mouse Ley I-L promoter and factors controlling the Ley I-L gene expression, we used 2.1 kb of the 5'-flanking region of the mouse Ley I-L gene to generate chimeric constructs with the chloramphenicol acetyltransferase gene (CAT). Transient transfections of MA10 Leydig cells, LTK- fibroblasts, and F9 embryonic cells by a series of 5'-deleted mouse Ley I-L promoter-CAT constructs revealed that the sequence between nucleotides -157 to +4 directs the transcription of the reporter gene in MA10 but not in LTK- and F9 cells, indicating that the determinants of Leydig cell-specific expression reside within this region. Deoxyribonuclease I (DNase I) footprint analysis revealed that the sequences designated SF-1/1, SF-1/2, and SF-1/3 within three DNase I-protected regions are homologous to the consensus binding site of the steroidogenic factor-1 (SF-1). Competition and antibody studies showed that the three SF-1-binding sites in the Ley I-L promoter have similar binding affinities for SF-1. Furthermore, transient transfections of MA10 cells with mutant reporter constructs, in which SF-1/1 or both SF-1/2 and SF-1/3 were deleted, demonstrated that all three SF-1-binding sites are required for SF-1-mediated stimulation of Ley I-L transcription. Cotransfection of an SF-1-containing expression vector together with a Ley I-L promoter-CAT construct into HeLa cells, which lack the endogenous SF-1 protein, resulted in CAT gene transcription, which indicated that SF-1 can transactivate the Ley I-L promoter. These data demonstrate an essential role of SF-1 in transcriptional activation of the Ley I-L promoter.
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PMID:Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1. 960 33