The differentially spliced mouse tagL gene,homolog of tag7/PGRP gene family in mammals and Drosophila,can recognize Gram-positive and Gram-negative bacterial cell wall independently of T phage lysozyme homology domain

Tag7/PGRP, a recently characterized antimicrobial protein, is conserved from insects to mammals. Recently its involvement in Toll signalling in Drosophila was demonstrated. A number of genes representing a new family homologous to PGRP were identified in Drosophila and human. Here we describe a splicing pattern of the tagL gene, mouse member of tag7/PGRP family. Some of the identified splice variants lacked characteristics for the family T phage lysozyme homology domain (also known as PGRP domain). Accordingly to the predicted transmembrane domains, mouse TagL may be secreted as inducible proteins or retained on intracellular membranes. All detected splice variant isoforms of TagL bound Gram-positive, Gram-negative bacteria and peptidoglycan. This binding did not depend on the presence of T phage lysozyme homology domain but was associated with the C-terminal portion of the polypeptides. Thus, this variety of isoforms of a single gene may play a role in circulating bacteria recognition in mammals.     

Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule

BACKGROUND: Recent studies indicate that the innate component of immune defense plays an important role in the establishment of antigen-specific immune response. We have previously isolated a novel mouse gene tag7/PGRP that was shown to be involved in the innate component of the immune system, and its insect homologue is an upstream mediator of Toll signaling in Drosophila. METHODS: Transiently or stably genetically modified mouse tumor cell lines expressing Tag7 were used. Tumor growth rate and animal survival were analyzed. Possible effector cells involved in tumor suppression were detected immunohistochemically. RESULTS: Transfection of mammary gland adenocarcinoma cells with the tag7 cDNA did not alter their growth rate in vitro but diminished their tumorogenicity in vivo in syngeneic and immunodeficient animals. Increased incidence of apoptosis was registered in the modified tumors. Transient expression of Tag7 by mouse melanoma M3 cells elicited protective immunity against parental tumor cells. Immunohistochemical analysis revealed that tumors after immunization with the genetically modified cells were infiltrated with Mac1(+) cells, B220(+) cells, and NK cells. Using nude mice we observed rejection of modified cells, but did not detect memory formation. CONCLUSIONS: We can conclude that secretion of the Tag7 protein by genetically modified cells can induce mobilization of antigen-presenting cells and innate effectors. Memory mechanisms are mediated by T cell response. For the first time our results demonstrate that local secretion of Tag7-the molecule involved in innate immunity-may play an important role in the induction of effective antitumor response in mice.     

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.     

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and.ll introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFPtagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC- 1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.     

富亮氨酸重复超家族新成员LRRC4的克隆与在脑瘤中的表达分析

在染色体7q31-32多种肿瘤杂合性丢失（loss of heterozygosity,LOH）高频区,采用表达序列标签（expressed sequence tag,EST）介导的定位候选克隆策略获得了一个定位于人染色体7q31-32的新基因（GenBank 登录号: AF196976）.该基因编码653个氨基酸,蛋白质理论pI/m:6.58/72.7 ku.它包含七个典型的LRR、一个IgC2样结构域.此外,它还包含一个N端信号肽、一个C端跨膜区.其结构特征表明它是富亮氨酸重复（leucine-rich repeat,LRR）超家族的新成员.经过人类基因组命名委员会的同意,将该基因命名为LRRC4.此外,通过序列相似性匹配还获得了定位于小鼠6号染色体的LRRC4的同源基因（GenBank 登录号: AF290542）.RNA印迹和RT-PCR检测发现LRRC4在正常人脑组织相对特异表达,而在多种原发性脑瘤表达明显下调或缺失.综合考虑LRRC4基因的序列特征及表达谱,提示LRRC4基因可能在神经系统中发挥重要作用.     

Identification,genomic organization and mRNA expression of CRELD1, the founding member of a unique family of matricellular proteins

We have isolated and characterized a unique gene that encodes a highly conserved membrane bound extracellular protein that defines a new epidermal growth factor-related gene family. The CRELD1 (Cysteine-Rich with EGF-Like Domains 1) gene (previously known as cirrin) was cloned from a human chromosome 3 BAC. Mapping of the gene confirmed its position at chromosome 3p25.3. The gene is ubiquitously expressed in early development and later becomes more markedly expressed in the developing heart, limb buds, mandible and central nervous system. Expression persists in adulthood in most tissues. Sequence analysis suggests that this is a cell adhesion protein. The mouse orthologue was cloned and mapped to the syntenic region of mouse chromosome 6. Orthologues or homologues have also been identified for cow, Chinese hamster, Drosophila and Caenorhabditis elegans. The CRELD1 gene is deleted in the human cytogenetic disorder 3p- syndrome and is in the region of loss of heterozygosity for several types of cancer. A potential role for this protein in these disorders is discussed.     

Cloning, characterization, and tissue distribution of mouse phosphodiesterase 7A1

We have cloned a cDNA representing mouse phosphodiesterases (PDE) 7A1. The open reading frame encodes a protein of 482 amino acids with a predicted molecular mass of 55417. Like human PDE7A variants, mouse PDE7A1 and A2 are 5' splice variants from a common gene. The distinct N-terminal sequence of mouse PDE7A1 is highly homologous to the corresponding sequence of human PDE7A1 with a similarity of 98% but not to that of mouse PDE7A2 (with a similarity of 12%), and is more hydrophilic than that of mouse PDE7A2. Mouse PDE7A1 expressed in SF9 cells has been compared with human PDE7A1 under identical conditions. Mouse PDE7A1 has a Km for cAMP of 0.2 microM, an optimal pH of 7.5, an IC(50) value of 14 microM for 3-isobutyl-1-methylxanthine (IBMX), and is dependent on Mg(2+) for activity. All these characteristics are very similar to those of human PDE7A1. In mice, PDE7A1 is expressed in tissues of the immune system (lymph node, thymus, spleen, and blood leukocyte), testis, brain, kidney and lung but not in skeletal muscle, heart, embryo, or liver, while PDE7A2 is expressed in skeletal muscle, heart, embryo, and kidney, but not in the other tissues. This tissue distribution profile is very similar to that in humans, and hence suggests that PDE7A1 and 7A2 might play a similar role in different species.     

MDA-7/IL-24: novel cancer growth suppressing and apoptosis inducing cytokine

The melanoma differentiation-associated gene-7 (mda-7) was cloned by subtraction hybridization as a molecule whose expression is elevated in terminally differentiated human melanoma cells. Current information based on structural and sequence homology, has led to the recognition of MDA-7 as an IL-10 family cytokine member and its renaming as IL-24. Northern blot analysis revealed mda-7/IL-24 expression in human tissues associated with the immune system such as spleen, thymus, peripheral blood leukocytes and normal melanocytes. The MDA-7/IL-24 mouse counterpart, FISP, appears to be a Th2-specific protein and the rat counterpart, C49A/MOB-5, is associated with wound healing and is also induced as a consequence of ras-transformation. A notable property of MDA-7/IL-24 is its ability to induce apoptosis in a large spectrum of human cancer derived cell lines, in mouse xenografts and upon intratumoral injection in human tumors (phase I clinical trials). Various aspects of this intriguing molecule including its cytokine and anti-tumoral effects are described and discussed.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.     

Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form

The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.     

Cloning and genomic organization of the mouse gene slc23a1 encoding a vitamin C transporter.

Vitamin C is known to exist in particularly high concentrations in brain tissue, and its free radical scavenging function is thought to represent a major antioxidative defense system. We have cloned, sequenced and analyzed the genomic structure of a mouse sodium-dependent vitamin C transporter gene, Slc23a1 (also known as Svct2). The mouse Slc23a1 cDNA is 6.4 kb long and was cloned directly from a mouse brain RNA preparation. Hybridization screening of a mouse genomic BAC library identified BAC 53L21 which contains at least the entire coding sequence of the mouse Slc23a1 gene. Determination of the exon-intron structure of the gene revealed 17 exons ranging from 58 bp to 4407 bp extending over 50 kb of the mouse genome, with the translation start codon located in exon 3. Its 1944 nucleotide open reading frame encodes a polypeptide of 647 aa, which is highly similar to rat and human orthologs. The mouse gene was assigned to chromosome 2qG2 by fluorescence in situ hybridization analysis. Expression of this gene was demonstrated in a wide range of tissues, with especially high levels in brain. Neurodegenerative diseases with an established role for oxidative stress in the cytoplasm may therefore be conditions of SLC23A1 dysfunction. Key words: gene structure; Vitamin C; transporter; oxidative stress     

Cloning and expression of sterol Delta 14-reductase from bovine liver.

Biosynthesis of cholesterol represents one of the fundamental cellular metabolic processes. Sterol Delta 14-reductase (Delta 14-SR) is a microsomal enzyme involved in the conversion of lanosterol to cholesterol in mammals. Amino-acid sequence analysis of a 38-kDa protein purified from bovine liver in our laboratory revealed > 90% similarity with a human sterol reductase, SR-1, encoded by the TM7SF2 gene, and with the C-terminal domain of human lamin B receptor. A cDNA encoding the 38-kDa protein, similar to human TM7SF2, was identified by analysis of a bovine expressed sequence tag (EST) database. The cDNA was synthesized by RT-PCR, cloned, and sequenced. The cDNA encodes a 418 amino-acid polypeptide with nine predicted transmembrane domains. The deduced amino-acid sequence exhibits high similarity with Delta 14-SR from yeasts, fungi, and plants (55-59%), suggesting that the bovine cDNA encodes Delta 14-SR. Northern blot analysis of bovine tissues showed high expression of mRNA in liver and brain. The polypeptide encoded by the cloned cDNA was expressed in COS-7 cells. Immunofluorescence analysis of transfected cells revealed a distribution of the protein throughout the ER. COS-7 cells expressing the protein exhibited Delta 14-SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes Delta 14-SR and provide evidence that the human TM7SF2 gene encodes Delta 14-SR.     

Isolation of a chicken gene that confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses.

We used a genetic strategy to isolate the chicken gene believed to encode the receptor for subgroup A avian leukosis and sarcoma viruses (ALSV-A). Chicken genomic DNA was transfected into monkey COS-7 cells, and two independent primary transfectants susceptible to ALSV-A infection were identified by using ALSV-A vectors containing a hygromycin B resistance gene. A second round of transfection and selection in mouse BALB/3T3 fibroblasts again led to isolation of a transfectant susceptible to infection by ALSV-A. Plasmid DNA sequences linked to chicken DNA during the primary transfection segregated with chicken DNA in the secondary transfectant and served as a molecular tag to clone the gene conferring susceptibility. Expression of the cloned gene in mouse BALB/3T3 cells conferred susceptibility to infection by ALSV-A but not by ALSV-B. Therefore the cloned gene most probably represents the tv-a locus, the genetically defined receptor gene for ALSV-A.     

A novel zebrafish kelchlike gene klhl and its human ortholog KLHL display conserved expression patterns in skeletal and cardiac muscles

In this study, a novel gene, kelchlike (klhl) was identified in zebrafish by whole-mount in situ hybridization screen for important genes involved in embryogenesis. A full-length klhl cDNA was cloned and characterized. We found that klhl was a member of the kelch-repeat superfamily, containing two evolutionary conserved domains--broad-complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, and kelch motif. Database mining revealed the presence of putative orthologs of klhl in human, mouse, rat, and pufferfish. klhl was determined to map to zebrafish linkage group (LG) 13 and was found to be syntenic with the proposed orthologs of klhl in human, mouse, and rat. In an effort to elucidate the function of klhl, klhl expression was investigated by Northern blot analysis and in situ hybridization. klhl is specifically expressed in the fast skeletal and cardiac muscle. Northern blot analyses show that the human ortholog, KLHL, is also specifically expressed in the skeletal muscles and heart. In silico analyses of rat expressed sequence tag (EST) clones corresponding to rat Klhl ortholog also indicate that its expression is also restricted to rat muscle tissues, suggesting a conserved role of klhl in vertebrates. The expression pattern of klhl, as well as the presence of the kelch repeats indicates a possible role for Klhl in the organization of striated muscle cytoarchitecture.     

Identification of CD7 as a cognate of the human K12 (SECTM1) protein

CD7 is a 40-kDa protein found primarily on T, NK, and pre-B cells; the function of the CD7 protein in the immune system is largely unknown. The K12 (SECTM1) protein was originally identified by its location just upstream of the CD7 locus. The K12 gene encodes a transmembrane protein of unknown function. In order to clone a K12-binding protein, we generated a soluble version of the human K12 protein by fusing its extracellular domain to the Fc portion of human IgG(1). Flow cytometry experiments showed that the K12-Fc fusion protein bound at high levels to both human T and NK cells. Precipitation experiments using K12-Fc on (35)S-radiolabeled NK cells lysates indicated that the K12 cognate was an approximately 40-kDa protein. A human peripheral blood T cell cDNA expression library was screened with the K12-Fc protein, and two independent, positive cDNA clones were identified and sequenced. Both cDNAs encoded the same protein, which was CD7. Thus, K12 and CD7 are cognate proteins that are located next to each other on human chromosome 17q25. Additionally, we have cloned the gene encoding the mouse homologue of K12, shown that it maps near the mouse CD7 gene on chromosome 11, and established that the mouse K12 protein binds to mouse, but not human, CD7. Mouse K12-Fc inhibited in a dose-dependent manner concanavalin A-induced proliferation, but not anti-TcRalpha/beta induced proliferation, of mouse lymph node T cells. Human K12-Fc stimulated the up-regulation of CD25, CD54, and CD69 on human NK cells in vitro.     

Natural antisense mRNAs to hyaluronan synthase 2 inhibit hyaluronan biosynthesis and cell proliferation

We report the identification of a natural antisense mRNA of hyaluronan synthase 2 that we have chosen to designate as HASNT (for HA synthase 2 antisense) in human and mouse. HASNT is transcribed from the opposite strand of the HAS2 gene locus and is represented by several independent expressed sequence tags in human. Portions of the mouse Hasnt gene were identified through an exon-trapping approach. Sequence conservation is extremely low between human and mouse HASNT, and it is not clear whether these mRNAs contain functional open reading frames. HASNT has an alternate splice site in both human and mouse. This splice site is located at an identical position within the gene in both species and results in mRNAs of two different lengths. In each species, the antisense portion of the HASNT gene is complementary to the first exon of HAS2, which represents the 5'-untranslated region. To study the biological activity of HASNT, two human expressed sequence tag clones, representing long and short HASNT splice variants, were cloned into a tetracycline-inducible vector and were stably transfected into human osteosarcoma U2-OS Tet-on cells. The long and short HASNT-expressing cells had a reduction in HAS2 mRNA levels up to 94 and 86%, respectively, whereas hyaluronan biosynthesis was inhibited by 40 and 37%, respectively. Cell proliferation was reduced throughout the time frame of the experiment. Exogenous high molecular mass hyaluronan failed to rescue the suppressed cell proliferation, whereas adenoviral-mediated overexpression of hyaluronan synthase 3, which stimulated endogenous hyaluronan biosynthesis, was able to rescue. Collectively, our data suggest that natural antisense mRNAs of HAS2 are able to regulate HAS2 mRNA levels and hyaluronan biosynthesis in a cell culture model system and may have an important and novel regulatory role in the control of HAS2, HA biosynthesis, and HA-dependent cell functions in vivo.     

Characterization of mouse muc6 and evidence of conservation of the gel-forming mucin gene cluster between human and mouse

Using degenerate primers designed from conserved cysteine-rich domains of gel-forming mucins, we cloned two new mouse mucin cDNAs. Blast searching showed that they belong to the same new gene assigned to chromosome 7 band F5. This gene is clustered with the three secreted large gel-forming mucins Muc2, Muc5ac, and Muc5b in a region that exhibits synteny with human chromosome 11p15. Computer analysis and sequence alignments with mucin genes predict that the new gene is composed of 33 exons and spans 30 kb from the initiation ATG codon to the Stop codon. Sequence similarities, domain organization of the deduced peptide, and expression analysis allow us to conclude that this newly cloned mouse gene is Muc6, i.e., the mouse ortholog of human MUC6. Like those of their human homologs, the genomic order and arrangement of the four mucins within the cluster of mucin genes are conserved.     

Cloning of human immunoglobulin mu gene and comparison with mouse mu gene

We have cloned a 12 kb DNA segment containing human mu gene and its flanking sequence from human fetal liver DNA library using mouse mu gene as a probe. Partial nucleotide sequence determination shows that the cloned DNA contains the sequence encoding human mu chain. This is the first constant region gene of the human heavy chain that is cloned. We have compared human and mouse mu genes by heteroduplex analysis and Southern blot hybridization. The results clearly show that not only the sequence encoding the CH4 domain but also the 5'-flanking (S mu) sequence is conserved between human and mouse mu genes, suggesting that the nucleotide sequence in the S mu region has an important biological function, presumably a recognition signal for the class switch recombinant as proposed previously.