Cloning and characterization of mouse homolog of the CXC chemokine receptor CXCR1

CXCR2/IL-8RB was the only receptor previously reported in mice for ELR+ CXC chemokines, whereas the receptors for these chemokines in human include both CXCR1 and CXCR2. In this study, we cloned the full length cDNA of the mouse CXCR1 (mCXCR1) gene. The deduced amino acid of mCXCR1 was 77% and 58% identical to the rat and human CXCR1, respectively. RT-PCR and Northern blot analysis showed that mCXCR1 mRNA was expressed in lung, spleen, thymus, peripheral blood leukocytes, as well as in the isolated neutrophils. In a mouse respiratory inflammation model induced by lipopolysaccharide, a large number of neutrophils infiltrated into the lung and, meanwhile, the mCXCR1 expression was significantly increased in the recruited neutrophils, suggesting that mCXCR1 may mediate the recruitment of neutrophils to the inflammation site under certain infections.     

Cloning and characterization of a secY homolog from Chlamydia trachomatis

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc-α ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50 195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SceY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.     

Cloning and characterization of mouse Dhm2 cDNA,a functional homolog of budding yeast SEP1

We have isolated mouse Dhm2 cDNAs encoding a homolog of budding yeast SEP1, whose product is involved in many cellular processes including meiosis, cellular senescence, and telomere maintenance. The putative Dhm2 protein (Dhm2p), which consists of 1687 amino acids and whose molecular weight is 191 400, matches the size of Sep1p and shares extensive homology with Sep1p especially in their N-terminal regions. A multicopy plasmid containing of the Dhm2 cDNA complements the slow growth phenotype, sporulation defect, and DNA recombination defect caused by the sep1 mutation in yeast, indicating that Dhm2 is a functional homolog of SEP1. Since Dhm1, another SEP1 homolog we reported previously, only partially compensates for the sep1 mutation, we conclude that Dhm2 is a true homolog of SEP1. Northern analysis revealed that 5.8 kb mRNA corresponding to Dhm2 open reading frame is produced highly in testis. These results strongly suggest that Dhm2p participates in gametogenesis in mouse.     

Cloning and characterization of a novel human RNA binding protein gene PNO1.

Present work reported the cloning and characterization of a human novel RNA binding gene Partner of NOB1 (PNO1), with a length of 1637bp and a putative open reading frame of 759 bp, isolated from human kidney. It is composed of seven exons and is localized on chromosome 2p14. Western blot showed that the molecular weight of PNO1 is about 35kDa. RT-PCR results in 16 human tissues indicated that PNO1 is expressed mainly in liver, lung, spleen and kidney, slightly in thymus, testis, ovary, respectively, but not in heart, brain, skeletal muscle, placenta, pancreas, prostate, small intestine, colon and peripheral blood leukocytes. GFP fusion expression in mammalian cells exhibited its localization in the nucleus, especially in nucleoli. Subcellular localization of thirteen GFP fusion PNO1 deletion proteins showed that the region of 92-230 aa is solely responsible for its nucleolar retention, and KH domain alone is not sufficient for nucleolar retention. The PNO1 family shows significant conservation in both eukaryotes and prokaryotes.     

Cloning and characterization of a novel human homolog* of mouse U26, a putative PQQ-dependent AAS dehydrogenase

Mouse U26 has been defined as a 2-aminoadipic 6-semialdehyde dehydrogenase. It was speculated to be a PQQ-dependent AAS dehydrogenase due to the research of demonstrating PQQ as a new B vitamin. We isolated a novel human cDNA from the human fetal brain cDNA library we constructed. Its deduced protein was most related to mouse U26. Thus, we termed it human U26. This putative protein contains an AMP-binding domain, a Phosphopantetheine-binding domain and six PQQ-binding motifs. Human U26 mRNA is ubiquitously expressed in adult tissues and is highly expressed in colon adenocarcinoma (CX-1) and colon adenocarcinoma (GI-112) cell lines. Further study should be made to clarify the precise function of human U26.The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY314787.     

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.     

Cloning and characterization of bovine insulin-like growth factor binding protein-3 (bIGFBP-3).

We report for the first time the isolation of a cDNA encoding the complete amino acid sequence for bovine growth hormone-dependent insulin-like growth factor binding protein-3 (bIGFBP-3). The deduced amino acid sequence from the cDNA revealed a mature polypeptide consisting of 264 amino acids and a 27 amino acid putative signal peptide. The amino acid sequence is over 80% homologous with human IGFBP-3 with complete conservation of the 18 cysteine residues and the 3 Asn-linked glycosylation sites. Between the two species there are 44 amino acid substitutions. Northern analysis of the bIGFBP-3 mRNA in bovine tissue revealed a single mRNA species of 1.65 kilobases.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

Identification and characterization of an SPO11 homolog in the mouse

The SPO11/TOPVIA family includes proteins from archaebacteria and eukaryotes. The protein member from the archaebacterium Sulfulobus shibatae is the catalytic subunit of TopoVI DNA topoisomerase. In Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, SPO11 is required for meiotic recombination, suggesting a conserved mechanism for the initiation step of this process. Indeed, S. cerevisiae SPO11 has been shown to be directly involved in the formation of meiotic DNA double-strand breaks that initiate meiotic recombination. Here, we report the identification of a Mus musculus Spo11 cDNA, which encodes a protein closely related to all members of the SPO11/TOPVIA family. cDNAs resulting from alternative splicing were detected, suggesting that there are potential variants of the mouse SPO11 protein. By RNA-blotting analysis, expression of the mouse Spo11 gene was detected only in the testis, in agreement with its predicted function in the initiation of meiotic recombination. We mapped the mouse Spo11 gene to chromosome 2, band H2–H4. Received: 11 August 1999; in revised form; 11 October 1999 / Accepted: 13 October 1999