Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit

G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.     

Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in beta cells of the pancreatic islets

A novel immunoglobulin superfamily (Igsf) protein gene was discovered by computational analysis of human draft genomic DNA, and multiple cDNA clones were obtained. The protein encoded by this gene contains five Ig domains, one transmembrane domain, and an intracellular domain. It has significant similarity with several known Igsf proteins, including Drosophila RST (irregular chiasm C-roughest) protein and mammalian KIRREL (kin of irregular chiasm C-roughest), NEPH1, and NPHS1 (nephrin) proteins. All these proteins have multiple Ig domains, possess properties of cell adhesion molecules, and play important roles in organ development. RT-PCR and Northern blot results indicate this gene is predominantly expressed in pancreas, and public sequence databases indicate there is also expression in the nervous system. We have named this gene Kirrel2 (kin of irregular chiasm-like 2), to reflect its similarity to irregular chiasm C-roughest and Kirrel. Four splice forms of Kirrel2 were observed, including two that we cloned from pancreas mRNA as well as two GenBank entries, one from the brain and one from a retinoblastoma cell line. A partial cDNA clone of the mouse orthologue was obtained by RT-PCR from mouse brain, and the inferred protein sequence has 85% sequence identity to the human protein. Immunohistochemical staining results indicate that the KIRREL2 protein is conserved from rodents to primates, and it is highly expressed in pancreatic islets. RT-PCR results on mouse pancreatic cell lines indicate that expression in the pancreas is restricted to beta cells. Thus, KIRREL2 protein is a beta-cell-expressed Ig domain protein and may be involved in pancreas development or beta cell function.     

小鼠脂联素受体2基因编码区的克隆及序列分析

构建小鼠脂联素受体2(mAdipoR2)基因cDNA克隆,并进行序列及基因结构分析,为进一步研究小鼠AdipoR2的表达和生物学活性奠定基础。用RT—PCR方法扩增mAdipoR2基因cDNA,获得的片段连接至pGEM-T载体,转化JM109大肠杆菌,经酶切鉴定后,进行序列测定。利用因特网生物信息学资源分析mAdipoR2基因序列。结果成功地构建了mAdipoR2基因cDNA克隆,其序列与GenBank登录序列一致。通过与小鼠基因组序列比对,分析了mAdipoR2基因结构,其编码序列由7个外显子组成,编码1个含7个跨膜区的膜蛋白,但该受体不属于G蛋白偶联受体家族(GPCRs)。mAdipoR2与人及大鼠蛋白的同源性分别为91％和95％。     

Cloning, characterization and primary function study of a novel gene, Cymg1, related to family 2 cystatins

Cystatins are cysteine proteinase inhibitors. We found two expression sequence tags (ESTs), CA463109 and AV042522, from a mouse testis library using Digital differential display (DDD). By electrical hybridization, a novel gene, Cymg1 (GenBank accession No. AY600990), which has a full length of 0.78kb, and contains four exons and three introns, was cloned from a mouse testis cDNA library. The gene is located in the 2G3 area of chromosome 2. The full cDNA encompasses the entire open reading frame, encoding 141 amino acid residues. The protein has a cysteine protease inhibitor domain that is related to the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the CRES subfamily, which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract. CYMG1 has a 44% (48/108) identity with mouse CRES and 30% (42/140) identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 is specifically expressed in adult mouse testes. Cell location studies showed that the GFP-tagged CYMG1 protein was localized in the cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium, spermatocytes, round spermatids, elongating spermatids and spermatozoa. RT-PCR results also showed that Cymg1 was expressed in mouse testes and spermatogonium. The Cymg1 expression level varied in different developmental stages: it was low 1 week postpartum, steadily increased 2 to 5 weeks postpartum, and was highest 7 weeks postpartum. The expression level at 5 weeks postpartum was maintained during 13 to 57 weeks postpartum. The Cymg1 expression level in the testes over different developmental stages correlates with the mouse spermatogenesis and sexual maturation process. All these indicate that Cymg1 might play an important role in mouse spermatogenesis and sexual maturation.     

The mouse lysyl oxidase-like 2 gene (mLOXL2) maps to chromosome 14 and is highly expressed in skin, lung and thymus.

The predicted amino acid sequence derived from a mouse expressed sequence tag (EST) contig contained two domains that are highly conserved among members of the lysyl oxidase gene family: a copper binding-site with four histidines and a catalytic domain that includes a tryptophan residue. This new cDNA sequence showed the highest level of sequence homology with the human loxl2 cDNA and suggested that it encoded the mouse equivalent of hLOXL2. The mLOXL2 gene was mapped to chromosome 14 by radiation hybrid analysis. The mLOXL2 locus was tightly linked with a LOD score over 9 to the marker D14Mit32. The mLOXL2 gene is expressed as a 4-kb mRNA in almost all tissues analyzed, with highest levels of mRNA in skin, lung and thymus.     

Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1

 Human membrane cofactor protein (MCP, CD46) is widely distributed and is one of the plasma membrane complement inhibitors. We isolated cDNA clones encoding genetic homologues of human MCP from a rat testis cDNA library. Northern blot analysis indicated that rat MCP is preferentially expressed in testis, similar to what is found with guinea pig MCP. We identified several different cDNAs, which were presumably generated by alternative splicing from a single-copy gene. The most prevalent isoform corresponded to the Ser/Thr/Pro-rich C type of human MCP. Mouse MCP cDNA was cloned by polymerase chain reaction based on the nucleotide sequence of rat MCP. The deduced amino acid sequence showed 77.8% identity to rat MCP. Mouse MCP was also preferentially expressed in testis. Unique expression in testis in rat and mouse as well as guinea pig suggests that MCPs in these species not only act as complement regulatory proteins but may also have more specialized functions in fertilization or reproduction. Genetic mapping by linkage analysis indicated that the mouse Mcp gene is located on distal chromosome 1, closely linked to the complement receptor 2 (Cr2) gene. Received: 24 February 1998 / Revised: 11 May 1998     

The mouse one P-domain (pS2) and two P-domain (mSP) genes exhibit distinct patterns of expression

We have previously shown that the human pS2 gene, which codes for a secreted peptide of 60 amino acids, is expressed in a number of human carcinomas, including carcinomas of the breast, the pancreas, and the large bowel. Strong pS2 gene expression was also observed in the normal gastric mucosa and in the regenerative tissues surrounding ulcerous lesions of the gastrointestinal tract. A number of pS2 similar peptides, designated as P-domain peptides, have been described, notably the porcine (PSP), murine (mSP), and human (hSP) spasmolytic polypeptides, which correspond to duplicated pS2 proteins. We have now cloned a mouse homolog of the human pS2 cDNA to dispose of an animal model to study the pS2 protein function, which remains unknown at the present time. We show that the mouse putative pS2 protein sequence and the physiological pattern of expression of the mouse pS2 gene are well conserved. The mouse pS2 gene is highly expressed in the stomach mucosa cells, whereas no pS2 gene expression could be detected in the mouse mammary gland, even during postnatal development processes dependent on growth factors or hormones. Using in situ hybridization, we show that although coexpressed in the fundus, the antrum and the antrum-pyloric regions of the stomach, the mouse pS2 and mSP genes exhibit distinct and complementary cellular patterns of expression.     

Mouse cPLA2gamma, a novel oocyte and early embryo-abundant phospholipase A2 gamma-like protein, is targeted to the nuclear envelope during germinal vesicle breakdown

This report documents the characterization of a novel mouse oocyte protein which was originally identified by microsequence analysis of a 67.8 kDa protein spot (pI 5.7) on a Coomassie-stained two-dimensional (2D) gel of murine egg proteins. Tandem mass spectroscopic analysis of the peptides obtained from the cored protein yielded sequences that appeared to match only ovary, egg, and preimplantation embryo cDNAs. We then cloned the novel gene by RACE-PCR, and analysis of the deduced cDNA sequence found that this maternal product was ∼56% identical to human cytosolic phospholipase A2γ (cPLA2γ). Based on this sequence homology, we named the molecule mouse cytosolic phospholipase A2γ (cPLA2γ). As with human cPLA2γ, mouse cPLA2γ contains a lipase consensus sequence and lacks the calcium binding domain that is found in other PLA2 proteins. However, mouse cPLA2γ is different from human cPLA2γ in that mouse cPLA2γ expression is restricted to the ovary and that the protein does not contain the myristoylation and prenylation lipid-anchoring motifs that are present in human cPLA2γ. Within oocytes, mouse cPLA2γ localizes mainly to the oocyte cortex and to the nucleoplasm. Interestingly, during germinal vesicle breakdown, mouse cPLA2γ aggregates dynamically relocate from the oocyte cortex to the nuclear envelope, suggesting a possible role for this putative egg-restricted phospholipase A2γ in membrane remodeling. Furthermore, mouse cPLA2γ protein continues to be expressed in the embryo until the 4-8-cell stage of development, suggesting that mouse cPLA2γ may function as a previously uncharacterized maternal effect gene.     

Organization and structure of the mouse interleukin-2 gene.

We have cloned a chromosomal DNA segment which covers the entire sequence for the murine interleukin-2 gene and analysed the structure of the gene. The coding regions are separated into four blocks by three introns each of which is located similarly to the corresponding human gene. The exon sequences can be aligned perfectly with the previously cloned cDNA sequence. Of particular interests is the presence of sequences within the 5'-flanking region which are highly conserved between mouse and man. The conserved region which spans more than 400 base pairs may play a role in the regulation of IL-2 gene expression.