Molecular cloning, expression, and chromosomal localization of a human tubulointerstitial nephritis antigen

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix basement protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis (TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 kDa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human homologue of TIN-ag was cloned and its nucleotide sequence was determined (Accession No. AB022277; the DDBJ nucleotide sequence database). Deduced amino acid sequence (476 aa) exhibited the presence of a signal peptide (1-18 aa), cysteine residues termed follistatin module, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed approximately 85% homology with both rabbit and mouse TIN-ag, and also some ( approximately 40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracellular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression ( approximately 50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11.2-12 by fluorescence in situ hybridization. These results may provide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.     

Molecular cloning, structural characterization and chromosomal localization of human lipoyltransferase gene.

Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the lysine residue of the lipoate-dependent enzymes. We isolated human lipoyltransferase cDNA and genomic DNA. The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids. Predicted amino acid sequence of the protein shares 88 and 31% identity with bovine lipoyltransferase and Escherichia coli lipoate-protein ligase A, respectively. Northern blot analyses of poly(A)+ RNA indicated a major species of about 1.5 kb. mRNA levels of lipoyltransferase were highest in skeletal muscle and heart, showing good correlation with those of dihydrolipoamide acyltransferase subunits of pyruvate, 2-oxoglutarate and branched-chain 2-oxo acid dehydrogenase complexes and H-protein of the glycine cleavage system which accept lipoic acid as a prosthetic group. The human lipoyltransferase gene is a single copy gene composed of four exons and three introns spanning approximately 8 kb of genomic DNA. Some alternatively spliced mRNA species were found by 5'-RACE analysis, and the most abundant species lacks the third exon. The human lipoyltransferase gene was localized to chromosome band 2q11.2 by fluorescence in situ hybridization.     

Molecular cloning, tissue distribution, and chromosomal localization of MMEL2, a gene coding for a novel human member of the neutral endopeptidase-24.11 family

Members of the neutral endopeptidase (NEP, also known as MME for membrane metallo-endopeptidase in the Human Gene Nomenclature database) family play significant roles in pain perception, arterial pressure regulation, phosphate metabolism, and homeostasis. In this paper, we report the cloning of a new human member of the NEP family that we named MMEL2 for membrane metallo-endopeptidase-like 2. The MMEL2 protein has the structural characteristics of type II transmembrane proteins, although the presence of a furin-like cleavage site in the ectodomain suggests that it may be released into the medium following proteolytic cleavage. The MMEL2 protein contains the zinc-binding consensus sequence HEXXH and all the residues known to be essential for the enzymatic activity of other members of the family. The MMEL2 mRNA was detected predominantly in testis, but weak expression also was observed in brain, kidney, and heart. The human MMEL2 gene was mapped to 1p36 by fluorescence in situ hybridization. It will be important to test whether MMEL2 defects are associated with diseases such as hereditary motor sensory neuropathy 2A, Schwartz-Jampel-Aberfeld syndrome, or neuroblastoma, which all map to this locus.     

Molecular cloning, chromosomal mapping, and developmental expression of a novel protein tyrosine phosphatase-like gene

Protein tyrosine phosphatases (PTPs) mediate the dephosphorylation of phosphotyrosine. PTPs are known to be involved in many signal transduction pathways leading to cell growth, differentiation, and oncogenic transformation. We have cloned a new family of novel protein tyrosine phosphatase-like genes, the Ptpl (protein tyrosine phosphatase-like; proline instead of catalytic arginine) gene family. This gene family is composed of at least three members, and we describe here the developmental expression pattern and chromosomal location for one of these genes, Ptpla. In situ hybridization studies revealed that Ptpla expression was first detected at embryonic day 8.5 in muscle progenitors and later in differentiated muscle types: in the developing heart, throughout the liver and lungs, and in a number of neural crest derivatives including the dorsal root and trigeminal ganglia. Postnatally Ptpla was expressed in a number of adult tissues including cardiac and skeletal muscle, liver, testis, and kidney. The early expression pattern of this gene and its persistent expression in adult tissues suggest that it may have an important role in the development, differentiation, and maintenance of a number of different tissue types. The human homologue of Ptpla (PTPLA) was cloned and shown to map to 10p13-p14.     

Molecular cloning and chromosomal localization of a novel Drosophila protein phosphatase

A 1.0 kilobase cDNA coding for the complete amino acid sequence of a putative protein phosphatase (314 amino acid residues, molecular mass 36 kDa) has been isolated from a Drosophila head cDNA library. The cDNA hybridises to a single site on the right arm of the second chromosome at cytological position 55A1-3. The deduced sequence of the protein, designated protein phosphatase-Y, is homologous to the catalytic subunits of Drosophila and rabbit protein phosphatase-1 alpha (64 and 59% identity, respectively) and rabbit protein phosphatase-2A (39% identity). These and other comparisons demonstrate that this novel enzyme is not the Drosophila counterpart of mammalian protein phosphatases 1, 2A, 2B, 2C or X.     

Molecular cloning,expression and chromosomal localization of a human gene encoding a 33 kDa putative metallopeptidase (PRSM1)

The zincins are a superfamily of structurally-related Zn²⁺-binding metallopeptidases which play a major role in a wide range of biological processes including pattern formation, growth factor activation and extracellular matrix synthesis and degradation. In this paper we report the identification and complete primary structure of a novel 33 kDa protein which contains the zinc-binding HEXXH motif found in the zincin superfamily. We have named this novel protein PRSM1 (PRoteaSe, Metallo, number 1). The gene was identified by the immunoscreening of a human placental cDNA library using polyclonal antibodies raised to the 70 kDa human matrix metalloendopeptidase, type III procollagen N-proteinase [Halila, R. and Peltonen, L. (1986) Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum. Biochem. J. 239, 47–52]. The protein is found in placenta and cultured osteosarcoma cells. PRSM1 could share sequence homology with the type III procollagen N-proteinase. The prsml gene is represented once in the human genome and is localized on chromosome 16 (q24.3).     

Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase.

Murine macrophages have previously been shown to secrete a zinc-dependent proteinase that can degrade elastin. In this report, we identify murine macrophage elastase (MME) cDNA and show that it is a distinct member of the metalloproteinase gene family. Small amounts of MME were purified to homogeneity, and N-terminal amino acid sequence was obtained. This sequence was used to obtain a partial cDNA clone by the polymerase chain reaction; a cDNA library derived from a mouse macrophage-like cell line (P388D1) was screened with this probe. A full-length MME cDNA spanning approximately 1.8 kilobases contained an open reading frame of 1386 base pairs; the predicted molecular mass of the MME proenzyme is 53 kDa. The gene encoding MME is represented only once in the mouse genome and is located on chromosome 9. Despite a size that is similar to other metalloproteinases, MME is distinct, sharing only 33-48% amino acid homology with other metalloproteinases. In contrast to other metalloenzymes, MME appears to be rapidly processed to an active truncated form (N-terminal and C-terminal cleavage). We expressed recombinant MME in Escherichia coli and demonstrated that it has significant elastolytic activity that is specifically inhibited by the tissue inhibitor of metalloproteinases. MME is therefore a true metalloproteinase that may be involved in tissue injury and remodeling.     

人类Connexin基因家族新成员-Cx44基因的克隆和特性分析

用绵羊和牛的Cx44基因(connexin 44 protein gene)序列对NCBI数据库进行Blast检索,得到一个相似性很高的人DNA序列(Human Genome Bank:AL138688),用GENSCAN程序分析AL138688,推测AL138688中包含一个编码区由1个外显子构成的基因——Cx44基因。人Cx44基因的开放阅读框为1320bp,推测编码435个氨基酸。用PROMOTORSCAN程序分析了其启动子。人Cx44基因与绵羊Cx44基因在1320bp有84.75％的一致性,其表达的蛋白有83％的一致性。用Map View将人的Cx44基因定位于13号染色体。     

Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.     

Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor.

The sequencing of endopeptidase-generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2-terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h-pPBS11, is 831 nucleotides and contains a full-length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.     

Molecular cloning, expression analysis, and chromosomal localization of human syntaxin 8 (STX8)

We report the cloning of a cDNA encoding human syntaxin 8 (STX8), using the regulator (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) as a bait to screen a human fetal lung cDNA library by the yeast two-hybrid system. This gene was found broadly transcribed and its mRNA size is about 1.3 kb. The STX8 gene maps to chromosomal band 17p12 and it encodes a 236-amino-acid protein. Syntaxin 8 contains in its C-terminal half a coiled-coil domain found highly conserved in the t-SNARE (SNAP receptor on target membrane) superfamily of proteins, which are involved in vesicular trafficking and docking. In syntaxin 8, a C-terminal hydrophobic domain may constitute a transmembrane anchor. It was recently shown that CFTR-mediated chloride currents can be regulated by syntaxin 1A, a t-SNARE family member, through direct protein-protein interaction. This raises the possibility that syntaxin 8 may also be involved in such regulations.     

A novel gene member of the human glycophorin A and B gene family. Molecular cloning and expression

A new gene closely related to the glycophorin A (GPA) and glycophorin B (GPB) genes has been identified in the normal human genome as well as in that of persons with known alterations of GPA and/or GPB expression. This gene, called glycophorin E (GPE), is transcribed into a 0.6-kb message which encodes a 78-amino-acid protein with a putative leader peptide of 19 residues. The first 26 amino acids of the mature protein are identical to those of M-type glycophorin A (GPA), but the C-terminal domain (residues 27-59) differs significantly from those of glycophorins A and B (GPA and GPB). The GPE gene consists of four exons distributed over 30 kb of DNA, and its nucleotide sequence is homologous to those of the GPA and GPB genes in the 5' region, up to exon 3. Because of branch and splice site mutations, the GPE gene contains a large intron sequence partially used as exons in GPA and GPB genes. Compared to its counterpart in the GPB gene, exon 3 of the GPE gene contains several point mutations, an insertion of 24 bp, and a stop codon which shortens the reading frame. Downstream from exon 3, the GPE and the GPB sequences are virtually identical and include the same Alu repeats. Thus, it is likely that the GPE and GPB genes have evolved by a similar mechanism. From the analysis of the GPA, GPB and GPE genes in glycophorin variants [En(a-), S-s-U- and Mk], it is proposed that the three genes are organized in tandem on chromosome 4. Deletion events within this region may remove one or two structural gene(s) and may generate new hybrid structures in which the promoter region of one gene is positioned upstream from the body of another gene of the same family. This model of gene organization provides a basis with which to explain the diversity of the glycophorin gene family.     

Molecular cloning and chromosomal localization of the Bombyx Sex-lethal gene.

We cloned Bm-Sxl, an orthologue of the Drosophila melanogaster Sex-lethal (Sxl) gene from embryos of Bombyx mori. The full-length cDNAs were of 2 sizes, 1528 and 1339 bp, and were named Bm-Sxl-L and Bm-Sxl-S, respectively. Bm-Sxl-L consists of 8 exons and spans more than 20 kb of genomic DNA. The open reading frame (ORF) codes for a protein 336 amino acids in length. Bm-Sxl-S is a splice variant that lacks the second exon. This creates a new translation start 138 nucleotides downstream and an ORF that codes for 46 amino acids fewer at the N-terminus. Linkage analysis using an F2 panel mapped Bm-Sxl to linkage group 16 at 69.8 cM. We isolated 2 BACs that include the Bm-Sxl gene. With BAC-FISH we located Bm-Sxl cytogenetically on the chromosome corresponding to linkage group 16 (LG16) at position >68.8 cM.     

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.     

Molecular cloning and chromosomal mapping of a novel five-span transmembrane protein gene, M83

In an attempt to identify novel transmembrane molecules expressed on hematopoietic cells, we identified a novel transmembrane protein gene, M83. Cloning of the full-length cDNAs of human and mouse M83 revealed that M83 encodes a type I transmembrane protein with a region containing five hydrophobic segments within the C-terminal part of the protein, suggesting that M83 is a five-span transmembrane molecule. The M83 protein was expressed on the cell surface as a glycosylated protein with a molecular mass of 84 kDa. The M83 gene was localized to human chromosome 16p13.3, mouse chromosome 17B1, and rat chromosome 10q12.3 distal. In human, M83 mRNA was highly expressed in placenta, pancreas, and lymphohematopoietic tissues including peripheral blood, spleen, and bone marrow. Among hematopoietic cells, it was highly expressed in resting T lymphocytes and was downregulated by cell activation, suggestive of its biological role related to the T cell resting status.