Structural characterization of the chicken SF-1/Ad4BP gene

SF-1/Ad4BP was identified as a master regulator controlling steroidogenic P-450 genes and belongs to the steroid hormone receptor superfamily. It is expressed in the adrenal cortex, gonads, and pituitary gonadotroph. Targeted disruption of the mouse SF-1/Ad4BP gene showed that it plays a critical role in the development of the steroidogenic tissues and pituitary gonadotroph. We have recently cloned the chicken SF-1/Ad4BP cDNA and have now cloned the chicken SF-1/Ad4BP gene and analyzed its promoter activity. This gene consists of seven exons as well as mammalian counterparts and spans about 15 kb. In mice, the gene encodes another protein, ELP, but we could not find the open reading frame of ELP in the chicken SF-1/Ad4BP gene. The promoter of this gene included five putative cis elements (E, CCAAT, GC and TATA boxes and a GA-rich element), although no TATA box has been found in mammalian counterparts. The E and CCAAT boxes moderately affected promoter activity and the GA-rich element and TATA box were essential for the expression of the chicken SF-1/Ad4BP gene.     

Gene structure of human cytochrome P-450(SCC), cholesterol desmolase

Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

The chimeric gene linked to glucocorticoid-suppressible hyperaldosteronism encodes a fused P-450 protein possessing aldosterone synthase activity.

Glucocorticoid-suppressible hyperaldosteronism (GSH) is one variety of primary aldosteronism with hypertension and is inherited in an autosomal dominant mode. A recent report has indicated that GSH is caused by a gene duplication arising from unequal crossing over between the two genes, CYP11B1 and CYP11B2, encoding P-450(11 beta) and P-450C18, respectively (Lifton et al. Nature (1992) 355, 262-265). The nucleotide sequence analysis in the present study has demonstrated that unequal crossing over in the chimeric gene formed by the gene duplication occurs within the region from the 3'-portion of exon 4 through the 5'-portion of intron 4 in Australian GSH patients. Namely, the chimeric gene encodes a fused P-450 protein consisting of the amino-terminal side of P-450(11 beta) (encoded by exons 1-4 of CYP11B1) and the carboxyl-terminal side of P-450C18 (encoded by exons 5-9 of CYP11B2). When a cDNA corresponding to the chimeric gene is transfected into COS-7 cells, the fused P-450 protein expressed in the mitochondria exhibits steroid 18-hydroxylase or aldosterone synthase activity. These results provide the molecular genetic basis for the characteristic biochemical phenotype of GSH patients.     

Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.     

Genomic organization and characterization of human PEX2 encoding a 35-kDa peroxisomal membrane protein

Peroxins are proteins involved in peroxisome biogenesis and are encoded by PEX genes. The human PEX2 gene encodes a 35-kDa peroxisomal integral membrane protein which is a member of the zinc finger protein family. Mutations in the PEX2 gene are the primary defect in a subset of patients with Zellweger syndrome and related peroxisome biogenesis disorders. The role of zinc finger proteins in peroxisome assembly and function is poorly understood. Here we report the cloning and characterisation of the human PEX2 structural gene. PEX2 was assigned to human chromosome 8q13-q21 and its murine homologue to mouse chromosome 3. The gene is approximately 17.5 kb in length, and contains four exons. The entire coding sequence is included in one exon, exon 4. The 5'-flanking region has features of housekeeping genes (GC enrichment, two Sp1 sites) and tissue-specific, inducible genes (two CCAAT boxes). In more than 1.5 kb of 5'-flanking sequences we did not identify consensus peroxisomal proliferator responsive elements (PPRE).     

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.