Characterization of the regulatory region of the human testis-specific form of the pyruvate dehydrogenase alpha-subunit (PDHA-2) gene.

The alpha-subunit of human pyruvate dehydrogenase (E(1)) is encoded by two separate genes. The gene located on chromosome X (PDHA-1) is expressed in somatic tissues, whereas the second gene (PDHA-2), located on chromosome 4, is expressed only in post-meiotic spermatogenic cells. A genomic fragment harboring the human gene encoding PDHA-2 has been isolated and approximately 800 nucleotides of the promoter region have been characterized. Functional studies of the promoter indicate the presence of both enhancer and repressor elements that are common to other genes that are only expressed in mature sperm.     

Characterization of the gene encoding human peroxisomal 3-oxoacyl-CoA thiolase (ACAA). No large DNA rearrangement in a thiolase-deficient patient.

We have characterized the gene encoding human peroxisomal 3-oxoacyl-CoA thiolase, an enzyme operative in the peroxisomal beta-oxidation system. We found one version of this gene (gene symbol ACAA) in the human genome, in contrast to the situation in rat where two versions have been described. The human gene shows a high structural similarity to the rat genes. It contains 12 exons and 11 introns and spans about 11 kb. We have determined the 5' end of the human thiolase mRNA by employing primer extension analysis and we have sequenced the region upstream of the gene. The putative promoter area displays some of the characteristics typical of promoters of other peroxisomal genes, in that it contains GC elements, but lacks TATA boxes. Finally, no large DNA rearrangement involving the thiolase gene could be observed in a patient suffering from pseudo-Zellweger syndrome (peroxisomal thiolase deficiency).     

Structure of the Hodgkin's lymphoma-associated human CD30 gene and the influence of a microsatellite region on its expression in CD30(+) cell lines.

The CD30 antigen is a member of the tumor necrosis factor receptor (TNFR) family which is overexpressed on the surface of the tumor cells of Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis. In this study the entire cd30 gene which is more than 24000 bp long and organized in eight exons was characterized by analyzing cosmid and phage lambda clones from human placental libraries with long-range polymerase chain reaction (PCR) and sequencing. Differences to other genes of the TNFR family were detected in the region encoding the extracellular domain of the cd30 gene. In nearly all other TNFR genes, the coding region of each cysteine-rich repeat is interrupted by one intron, i.e., the 3-4 cysteine-rich repeats of these receptors are encoded by at least 4-5 exons, whereas the six cysteine-rich repeats of the cd30 gene are encoded by two exons, i.e., each of these exons encode three cysteine-rich repeats. In addition, we also found a genetic polymorphism of tetranucleotide ATCC-repeats in the 5' part of the CD30 promoter. This region was amplified by PCR from seven CD30 overexpressing human lymphoid cell lines and five human tissues with an absent or very low CD30 expression. The amplification products showed length differences of more than 550 bp. The number of the ATCC-repeats was higher in CD30(+) cell lines than in normal tissues. Comparison of the individual PCR products in reporter gene assays revealed that the CD30 promoter activity increased with the length of this polymorphic region up to eightfold. The data suggest that the number of ATCC-repeats in the 5' region of the CD30 promoter modulates the regulation of CD30 expression.     

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.     

Human cellular retinol-binding protein gene organization and chromosomal location

The gene encoding the human cellular retinol-binding protein (CRBP) has been isolated from genomic libraries and its structure determined. Only one copy of the gene is present in the human genome. We have located the CRBP gene to segment 3p11-3qter on human chromosome 3 using hybridizations to mouse-human, rat-human and hamster-human cell hybrids. The gene harbors four exons encoding 24, 59, 33, and 16 amino acid residues respectively. The second intervening sequence alone occupies 19 kb of the 21 kb of the CRBP gene. The nucleotide sequence of the gene has been determined with the exception of the second intron. The positions of the introns agree with those in the rat CRBPII, the rat liver fatty-acid-binding protein and the mouse adipose P2 protein genes encoding molecules belonging to the same protein family as CRBP. In contrast to the other sequenced members of this family the promoter of the CRBP gene resembles those found in the 'housekeeping' genes in that it is (G + C)-rich, contains multiple copies of the CCGCCC sequence and lacks TATA box. A 9-bp homology containing the core sequence of the simian virus 40 enhancer repeat was found in the 5' upstream region. A genomic Southern blot probed with CRBP cDNA revealed hybridizing bands in restricted chicken and frog DNA.     

The gene for the beta subunit of bovine luteinizing hormone encodes a gonadotropin mRNA with an unusually short 5'-untranslated region

Both cDNA and genomic clones encoding the beta subunit of bovine luteinizing hormone (LH) have been isolated and characterized. The nucleotide sequence was determined for the entire gene and 776 base pairs of 5'-flanking sequence. The mRNA cap site and polyadenylation site were mapped by primer extension and S1 nuclease protection, respectively. The bovine LH beta spans less than 1.1 kilobase pairs and has three exons encoding a 550 nucleotide mRNA (excluding the poly(A) tail). Bovine LH beta is a single-copy gene, in contrast to human LH beta, which is a member of the LH/chorionic gonadotropin beta subunit multigene family. Comparison of the bovine LH beta gene with the human LH beta/chorionic gonadotropin gene family reveals a high degree of nucleotide sequence homology, both within the genes and in the 5'-flanking sequences. Despite this extensive sequence conservation, there is a major difference between the two species in the selection of a promoter site. As a result, the bovine LH beta gene produces an mRNA with an usually short 5'-untranslated region of only 6-11 nucleotides.     

Unusual ribosomal RNA gene organization in copepods of the genus Calanus

Ribosomal RNA genes in the nuclear genomes of eukaryotes are generally found in tandemly repeated units encoding 18 S, 5.8 S and 28 S rRNA (in that order). 5 S rRNA genes typically lie outside these units, most often in tandem clusters coding exclusively for 5 S rRNA. Inclusion of 5 S genes within the 18 S-5.8 S-28 S repeat unit is known only for certain protozoa and fungi. Here we report that, in the copepod Calanus finmarchicus, single 5 S genes are included within many or all of the 18 S-5.8 S-28 S repeat units. Sequence analyses of regions cloned from two of these repeat units show that they indeed include 5 S genes (which are distal to 28 S genes) and that these are transcribed from opposite strands.     

Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.

Filaggrin is an intermediate filament-associated protein which functions to aggregate keratin intermediate filaments in the stratum corneum of mammalian epidermis. It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. In this report, we describe the characterization of cosmid genomic clones containing the human profilaggrin gene coding for 11 complete filaggrin repeats of 324 amino acids each. At the amino- and carboxyl-terminal ends of human profilaggrin are leader and tail peptide sequences of 293 and 157 amino acids, respectively, which differ from filaggrin. The leader peptide is composed of two distinct domains: an 81-residue segment which shows significant homology to the S-100 family of EF hand-containing calcium-binding proteins, and a hydrophilic second domain of 212 residues. The gene is divided into three exons, with one intron (approximately 9.6 kilobase pairs) in the 5' noncoding region and a second one of 570 base pairs between the EF hands. The position of intron 2 is identical to that of other members of the S-100-like family. The presence of an S-100-like domain suggests that profilaggrin binds calcium and that the calcium binding domain is functionally significant in the formation of keratohyalin and/or the subsequent processing of profilaggrin to filaggrin, both of which may be calcium-dependent events.