Structural organization and chromosomal assignment of the gene encoding endothelin

Endothelin is a 21-amino acid vasoconstrictor synthesized and secreted by vascular endothelial cells. The human peptide is derived from a 212-amino acid precursor, preproendothelin. A nearly full length clone containing DNA complementary to human preproendothelin mRNA was isolated, and its nucleotide sequence was determined. Using this cDNA as a probe, the genomic organization of the human endothelin gene was determined and the promoter region delineated. The gene contains five exons and four intervening sequences. Nucleotide sequences encoding endothelin are contained within the second exon, and the third exon specifies a portion of preproendothelin that is homologous to endothelin. The second and third exons may represent descendants of a common progenitor exon. The 3'-untranslated portion of the gene contains a 250-base pair region that is highly conserved between human and porcine genomes and may have an important role in endothelin mRNA stability. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, the endothelin gene was assigned to human chromosome 6.     

cDNA cloning and chromosomal assignment of the endothelin 2 gene: vasoactive intestinal contractor peptide is rat endothelin 2.

Four members of the endothelin family of vasoactive and mitogenic peptides have been identified: human endothelins 1, 2, and 3 (ET1, ET2, and ET3, respectively) and mouse vasoactive intestinal contractor (VIC). To characterize the mRNA encoding ET2, a 192-bp fragment of the ET2 gene, amplified by the polymerase chain reaction from human genomic DNA, was used to screen cell lines and tissues for ET2 gene expression. ET2 mRNA was detected in a cell line (HTB119) derived from a human lung small cell carcinoma, and an ET2 cDNA was cloned from a cDNA library prepared from HTB119 mRNA. DNA prepared from human-mouse somatic hybrid cell lines was used to assign the gene encoding ET2 (EDN2) to the 1p21----1pter region of chromosome 1, demonstrating that EDN2 is not linked to genes encoding ET1 (EDN1; chromosome 6) and ET3 (EDN3; chromosome 20). Southern blot hybridization revealed a single gene in human and rat genomes that hybridized with the ET2 gene fragment, and the rat gene was cloned. The endothelin peptide encoded by the rat gene differed from ET2 at 1 of 21 residues and was identical to mouse VIC. We conclude that VIC is the mouse and rat analogue of the human ET2 gene.     

Mouse cathepsin F: cDNA cloning, genomic organization and chromosomal assignment of the gene

A murine cysteine protease of the papain family was identified by dbEST-database search. A 1.87kb full-length cDNA encoding a predicted polypeptide of 462 amino acids was sequenced. Since the encoded polypeptide shows more than 80% sequence identity with human cathepsin F, it is most likely that this cDNA represents the murine homologue of cathepsin F, and it was therefore named accordingly. Murine cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 20 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 228 amino acids and the domain of the mature protease comprising 214 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the 'catalytic triad'. Genomic clones covering the murine cathepsin F gene were isolated. The mouse cathepsin F gene consists of 14 exons and 13 introns and spans 5.8kb. Murine cathepsin F was mapped to chromosome 19, a region with synteny homology to a region of human chromosome 11 to which human cathepsin F has been mapped previously. Northern blot analysis of RNA from multiple tissues revealed a ubiquitous expression of cathepsin F in mouse and man.     

cDNA cloning and chromosomal mapping of the gene encoding adenylate kinase 2 from Drosophila melanogaster

As a step toward understanding of the role of adenylate kinase (AK) in energy metabolism, we analyzed this enzyme in Drosophila melanogaster. The enzyme activities of all three AK isozymes were determined in cell-free extracts of flies, and their proteins were detected by Western blot analysis using polyclonal antibodies against the mammalian isozymes. A cDNA encoding adenylate kinase was isolated from D. melanogaster cDNA library. The cDNA encodes a 240-amino acid protein, which shows high similarity to bovine, human and rat AK2, and hence was named DAK2. Preliminary subcellular fractionation analysis indicated that DAK2 is localized in both cytoplasm and mitochondria. In situ hybridization to salivary gland polytene chromosomes revealed that the Dak2 gene is located at 60B on the right arm of the second chromosome.     

Human liver type pyruvate kinase: cDNA cloning and chromosomal assignment

Pyruvate kinase (PK) has four isozymes (L,R,M1,M2) that are encoded mainly by two different genes. We isolated a cDNA clone from a Japanese adult liver lambda gt10 cDNA library by using a rat liver(L)-type PK cDNA probe. One positively hybridizing clone, hlPK-1, which contained a 1,049-base pair cDNA insert, was subjected to DNA sequence analysis. Comparisons of the sequence data with the rat PK cDNAs indicated that the cDNA encoded information for the carboxyl terminal 105 amino acids of a human L-type PK and a 3' untranslated region of 734 nucleotides. Furthermore, the karyotype analysis of several human-mouse hybrid cells and Southern blot analysis of DNAs of the hybrids with a hlPK-1 indicated that the human L-type PK gene is located on chromosome 1.     

Genomic cloning and chromosomal assignment of rat regucalcin gene

The gene for a Ca²⁺-binding protein regucalcin was cloned from a rat genomic library which was constructed in FIX II by screening with radiolabeled probe (complementary DNA of rat liver regucalcin). Positive clone had 19.9 kb insert of size and contained four exons of the gene coding for a rat regucalcin. These exons included the partial coding sequence (61.2% of open reading frame) and the entire 3-untranslated region of the gene. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. The sequence analysis of the clone showed that the identifier sequence and two simple repeated sequences exist in the intron of the gene. Moreover, chromosomal location of the rat regucalcin gene was determined by direct R-banding fluorescencein situ hybridization (FISH) method with the 19.9 kb clone containing four exons. The regucalcin gene was localized on rat chromosome Xq11.1–12 proximal end.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases with the following accession number D31662     

cDNA cloning, genomic structure and chromosomal localization of the human BUP-1 gene encoding beta-ureidopropionase.

A full-length cDNA clone encoding human beta-ureidopropionase was isolated. A 1152-nucleotide open reading frame which corresponds to a protein of 384 amino acids with a calculated molecular weight of 43? omitted?158 Da, surrounded by a 5'-untranslated region of 61 nucleotides and a 3'-untranslated region of 277 nucleotides was identified. The protein showed 91% similarity with the translation product of the rat beta-ureidopropionase cDNA. Expression of the human cDNA in an Escherichia coli and eukaryotic COS-7 expression system revealed a very high beta-ureidopropionase enzymatic activity, thus confirming the identity of the cDNA. Since human EST libraries from brain, liver, kidney and heart contained partial beta-ureidopropionase cDNAs, the enzyme seems to be expressed in these tissues, in agreement with the expression profile of this enzyme in rat. Using the human cDNA as a probe a genomic P1 clone could be isolated containing the complete human beta-ureidopropionase gene. The gene consist of 11 exons spanning approximately 20 kB of genomic DNA. Fluorescence in situ hydridization localized the human beta-ureidopropionase gene to 22q11.2.     

Gene encoding the mouse sulphamidase: cDNA cloning, structure, and chromosomal mapping

Sulphamidase is an exoglycosidase involved in the degradation of heparan sulfate. Lack of sulphamidase activity leads to the lysosomal storage disorder Mucopolysaccharidosis type IIIA (Sanfilippo type A OMIM No. 252900). At present there are no naturally occurring small animal models of this disease that could be of fundamental importance to study the pathophysiology of the disease and to try therapeutic strategies. Cloning of the mouse gene is an important step to create a mouse model for this common mucopolysaccharidosis. We have isolated and sequenced the gene encoding mouse sulphamidase. Comparison of the deduced amino acid sequences of human and mouse sulphamidase showed 88% identity and 93% similarity. The exon-intron structure of the gene has been determined with the mouse 10-kb gene divided in 8 exons. The mouse sulphamidase gene (Sgsh) was mapped to the distal end of Chromosome (Chr) 11, in a region that is homologous with a segment of human Chr 17 containing the orthologous human gene. Received: 26 July 1999 / Accepted: 3 February 2000     

Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

Platelets metabolize arachidonic acid to thromboxane A2, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide-derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human--hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and its gene regulation.     

Molecular cloning and chromosomal assignment of a human perforin (PFP) gene

Human perforin cDNA was isolated and the complete nucleotide sequence of the gene determined. The deduced amino acid sequence of human perforin showed 68.4% similarity to that of mouse perforin. RNA blot analysis of the human perforin gene revealed that the gene product is expressed preferentially in killer-type cells among cell lines tested, and in large granular lymphocytes among the peripheral blood mononuclear cells. In situ hybridization analysis with a human perforin cDNA probe revealed that the human perforin (PFP) gene is located on chromosome17q11-21. The nucleotide sequence data reported in this paper have been submitted to the GeBank nucleotide sequence database and have been assigned the accession number M28393.     

Human indolethylamine N-methyltransferase: cDNA cloning and expression, gene cloning, and chromosomal localization.

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K(m) value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon-intron splice junctions conformed to the "GT-AG" rule, and no canonical TATA or CAAT sequences were present within the 5'-flanking region of the gene. Human INMT mapped to chromosome 7p15.2-p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma

Two overlapping clones, covering the entire coding sequence of human M2-type pyruvate kinase (PK) cDNA, were isolated and sequenced. Nucleotide sequencing results showed that they contained the 109-bp 5'-untranslated region, the 1593-bp coding region and the 585-bp 3'-untranslated region. Nucleotide sequence homology was 90% and 69% with rat M2-type and L-type PK cDNA, respectively. In situ hybridization using the human M2-type PK cDNA probe disclosed that the gene for M2-type PK is located at band q22 on chromosome 15. Northern blot analysis with RNA from human hepatoma demonstrated that M2-type PK was predominantly expressed in hepatoma cells, whereas L-type PK was preferentially expressed in the non-tumor portion of the liver.