Molecular cloning and characterization of a chromosomal gene for human eosinophil peroxidase

Using human myeloperoxidase cDNA as a probe, a chromosomal gene related to myeloperoxidase was isolated from a human gene library. Comparison of the amino acid sequence deduced from the nucleotide sequence of the cloned gene with that of human eosinophil peroxidase purified from buffy coats has indicated that the isolated gene is the chromosomal gene for human eosinophil peroxidase. Like human myeloperoxidase gene, human eosinophil peroxidase gene consists of 12 exons and 11 introns spanning about 12 kilobases. The gene can code for a protein of 715 amino acids with a calculated Mr of 81,036. The heavy chain and the light chain of eosinophil peroxidase were located on the COOH and NH2 terminus of the protein, respectively. The coding sequences of eosinophil peroxidase and myeloperoxidase show homologies of 72.4% at the nucleotide and 69.8% at the amino acid level, while little homology was found in the 5'-flanking region. Northern hybridization and S1 mapping analysis of RNA from human leukemic cells have indicated that the eosinophil peroxidase gene is expressed in the eosinophilic subline of human HL-60 cells but not in the neutrophilic subline or in parental HL-60 cells.     

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     

Molecular cloning and chromosomal assignment of the human homolog of int-1, a mouse gene implicated in mammary tumorigenesis.

Viral mammary tumorigenesis in mice is frequently initiated by proviral activation of a highly conserved cellular gene called int-1. We have cloned the human homolog of this putative mammary oncogene and compared its structure to that of the mouse gene by heteroduplex analysis. The human int-1 gene was localized on chromosome 12 by use of somatic cell hybrids.     

Molecular cloning and chromosomal assignment of the porcine 54 and 56 kDa vacuolar H(+)-ATPase subunit gene (V-ATPase)

Vacuolar proton-translocating ATPases (V-ATPase) are multisubunit enzyme complexes located in the membranes of eukaryotic cells regulating cytoplasmic pH. So far, nothing is known about the genomic organization and chromosomal location of the various subunit genes in higher eukaryotes. Here we describe the isolation and analysis of a cDNA coding for the 54- and 56-kDa porcine V-ATPase subunit alpha and beta isoforms. We have determined the genomic structure of the V-ATPase subunit gene spanning at least 62 kb on Chromosome (Chr) 4q14-q16. It consists of 14 exons with sizes ranging from 54 bp to 346 bp, with a non-coding first exon and an alternatively spliced seventh exon leading to two isoforms. The 5′ end of the V-ATPase cDNA was isolated by RACE-PCR. The V-ATPase alpha isoform mRNA, lacking the seventh exon, has an open reading frame of 1395 nucleotides encoding a hydrophilic protein of 465 amino acids with a calculated molecular mass of 54.2 kDa and a pI of 7.8, whereas the beta isoform has a length of 1449 nucleotides encoding a protein of 483 amino acids with a calculated molecular mass of 55.8 kDa. Amino acid and DNA sequence comparison revealed that the porcine V-ATPase subunit exhibits a significant homology to the VMA13 subunit of Saccharomyces cerevisiae V-ATPase complex and V-ATPase subunit of Caenorhabditis elegans. Received: 14 May 1998 / Accepted: 20 October 1998     

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, 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.     

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, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.     

Molecular characterization and chromosomal assignment of the canine protein C gene

Protein C is a precursor to a serine protease present in the plasma that plays an important physiological role in the regulation of blood coagulation. Mutations in the human protein C gene have been linked to some cases of Morbus Perthes disease, a thrombophilic condition that results in aseptic necrosis of the femur head and neck. We have cloned the canine protein C gene to investigate whether Morbus Perthes disease in dogs is also caused by mutations within this gene. A genomic λFIXII clone was isolated, and 11,420 bp of DNA sequence were determined containing the complete protein C gene (Acc No. AJ001979). As in humans, the gene consists of nine exons with the translation start codon located in the second exon. The 1.7-kb mRNA contains a 1368-bp open reading frame coding for 456 amino acids. With the genomic protein C clone as a probe in a FISH experiment, the canine protein C gene was assigned to Chromosome (Chr) 19q21-q22. To search for possible mutations, we amplified genomic DNA from one healthy and 15 clinically and pathohistologically confirmed Morbus Perthes patients. Sequence analysis did not reveal any amino acid differences between the affected dogs and the normal control. Several nucleotide polymorphisms were detected, which however, did not result in an amino acid exchange. From these data we conclude that in contrast to human, canine Morbus Perthes disease is most likely not caused by mutations within the protein C gene. Received: 24 June 1998 / Accepted: 18 September 1998     

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 of a human Vent-like homeobox gene

We have isolated a previously unknown human homeobox-containing cDNA, VENT-like homeobox-2 (VENTX2), using PCR with a bone marrow cDNA library and primers designed from the VENTX1 (alias HPX42) homeobox sequence. Here we describe the molecular cloning, chromosomal localization to 10q26.3, and functional analysis of this gene. The 2.4-kb human VENTX2 cDNA encoded a protein with a predicted molecular weight of 28 kDa containing a homeodomain with 65% identity to the Xenopus laevis ventralizing gene Xvent2B. VENTX2 antisera detected a 28-kDa protein in cells transfected with a VENTX2 expression construct, in a human erythroleukemic cell line and in bone marrow samples obtained from patients in recovery phase after chemotherapy. The similarity of the homeodomains from VENTX2 and the X. laevis Vent gene family places them in the same homeodomain class. Consistent with this structural classification, overexpression of VENTX2 in zebrafish embryos led to anterior truncations and failure to form a notochord, which are characteristics of ventralization.     

Molecular cloning and structural analysis of human norepinephrine transporter gene（NETHG）

A cDNA molecule encoding a major part of the human Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene.A positive clone harbouring the whole gene was obtained from a human lymphocyte genomic library through utilizing the “genomic walking” technique.The clone,designated as phNET,harbours a DNA fragment of about 59 kd in length inserted into BamH I site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein.A single exon encodes a distinct transmembrane domain,except for transmembrane domain 10 and 11,which are encoded by part of two exons respectively,and exon 12,which encodes part of domain 11 and all of domain 12.These results imply that there is a close relationship between exon splicing of a gene and structureal domains of the protein,as is the case for the human γ－aminobutyric acid transporter(hGAT) and a number of other membrane proteins.