Molecular cloning, characterization, and chromosomal localization of the mouse homologue of CD84, a member of the CD2 family of cell surface molecules

 CD84 is a member of the immunoglobulin gene superfamily (IgSF) with two Ig-like domains expressed primarily on B lymphocytes and macrophages. Here we describe the cloning of the mouse homologue of human CD84. Mouse CD84 cDNA clones were isolated from a macrophage library. The nucleotide sequence of mouse CD84 was shown to include an open reading frame encoding a putative 329 amino acid protein composed of a 21 amino acid leader peptide, two extracellular immunoglobulin (Ig)-like domains, a hydrophobic transmembrane region, and an 87 amino acid cytoplasmic domain. Mouse CD84 shares 57.3% amino acid sequence identity (88.7%, considering conservative amino acid substitutions) with the human homologue. Chromosome localization studies mapped the mouse CD84 gene to distal chromosome 1 adjacent to the gene for Ly-9, placing it close to the region where other members of the CD2 IgSF (CD48 and 2B4) have been mapped. Northern blot analysis revealed that the expression of mouse CD84 was predominantly restricted to hematopoietic tissues. Two species of mRNA of 3.6 kilobases (kb) and 1.5 kb were observed. The finding that the pattern of expression was restricted to the hematopoietic system and the conserved sequence of the mouse CD84 homologue suggests that the function of the CD84 glycoprotein may be similar in humans and mice. Received: 1 July 1998 / Revised: 31 August 1998     

Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe11), KE3 (probe7), KE2 (probe5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the 2(XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb 3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.     

Genomic organization and characterization of human PEX2 encoding a 35-kDa peroxisomal membrane protein

Peroxins are proteins involved in peroxisome biogenesis and are encoded by PEX genes. The human PEX2 gene encodes a 35-kDa peroxisomal integral membrane protein which is a member of the zinc finger protein family. Mutations in the PEX2 gene are the primary defect in a subset of patients with Zellweger syndrome and related peroxisome biogenesis disorders. The role of zinc finger proteins in peroxisome assembly and function is poorly understood. Here we report the cloning and characterisation of the human PEX2 structural gene. PEX2 was assigned to human chromosome 8q13-q21 and its murine homologue to mouse chromosome 3. The gene is approximately 17.5 kb in length, and contains four exons. The entire coding sequence is included in one exon, exon 4. The 5'-flanking region has features of housekeeping genes (GC enrichment, two Sp1 sites) and tissue-specific, inducible genes (two CCAAT boxes). In more than 1.5 kb of 5'-flanking sequences we did not identify consensus peroxisomal proliferator responsive elements (PPRE).     

A Human Homologue (BICD1) of theDrosophila Bicaudal-DGene

We previously isolated a cDNA fragment homologous to theDrosophila Bicaudal-Dgene (Bic-D) using a hybridization selection procedure with cosmids derived from the short arm of human chromosome 12. A PCR-mediated cDNA cloning strategy was applied to obtain the coding sequence of the human homologue (BICD1) and to generate a partial mouse (Bicdh1) cDNA. TheDrosophila Bicaudal-Dgene encodes a coiled coil protein, characterized by five α-helix domains and a leucine zipper motif, that forms part of the cytoskeleton and mediates the correct sorting of mRNAs for oocyte- and axis-determining factors during oogenesis. Analysis of the predicted amino acid sequence of theBICD1cDNA clones indicates that the sequence similarity is essentially limited to the amphipatic helices and the leucine zipper, but the conserved order of these domains suggests a similar function of the protein in mammalians. A database search further indicates the existence of a second human homologue on chromosome arm 9q and aCaenorhabditis eleganshomologue. Northern blot analysis indicates that both the human and the murine homologues produce an mRNA species of 9.5 kb expressed in brain, heart, and skeletal muscle and during mouse embryonic development. The conserved structural characteristics of theBICD1protein and its expression in muscle and especially brain suggest thatBICD1is a component of a cytoskeleton-based mRNA sorting mechanism conserved during evolution.     

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.     

Cloning, chromosome mapping and functional characterization of a human homologue of murine gtse-1 (B99) gene

Murine Gtse-1 (G(2) and S phase expressed protein), previously named B99, is a wt-p53 inducible gene that encodes a microtubule-localized protein which is able to induce G(2)/M phase accumulation when ectopically expressed. Here we report the cloning and characterization of a new cDNA (GTSE-1) encoding a human homologue of the mouse Gtse-1 protein. Chromosome mapping of mouse and human genes assigned Gtse-1 to chromosome 15 and GTSE-1 to chromosome 22q13.2-q13.3 in a region with conserved synteny to that where Gtse-1 mapped. Analysis of the genomic structure revealed that GTSE-1 contains at least 11 exons and 10 introns, spanning approximately 33kb of genomic DNA. Similar to murine Gtse-1, the product of GTSE-1 localized to the microtubules, was able to delay G(2)/M progression when ectopically expressed and was cell cycle regulated. Taken together, these results indicate GTSE-1 as the human functional homologue of murine Gtse-1.     

Cloning and chromosomal mapping of the human p53-related KET gene to Chromosome 3q27 and its murine homolog Ket to mouse Chromosome 16

KET is a member of the newly discovered family of proteins that is related to the tumor suppressor p53. Here we describe the molecular cloning of a human cDNA of 4846 bp encoding a protein of 680 amino acids. The human KET protein shares 98% identity with the previously characterized rat homolog. The remarkably high degree of conservation lends support to the notion that KET proteins have important basic functions in development and differentiation. Using the GeneBridge 4 radiation hybrid panel, we have mapped KET to human Chromosome (Chr) 3q27. KET is located between the somatostatin gene SST (proximal) and the apolipoprotein D gene APOD (distal) in a region of conserved synteny to mouse Chr 16. This chromosomal region is deleted in early stages of tumorigenesis of mouse islet cell carcinomas and contains the hitherto unidentified Loh2 gene, a putative suppressor of angiogenesis. The murine homolog Ket was mapped in an interspecific backcross panel and falls into this region of loss of heterozygosity. From our mapping data we infer that KET might act as a tumor suppressor and is considered as a candidate for Loh2. Received: 30 April 1998 / Accepted: 17 July 1998     

Murine sex-limited protein (Slp) antigenic sites in human complement component C4

Human complement component C4 is encoded by two HLA-linked loci, A and B. In the mouse, the H-2 region contains structural genes for two serum proteins that react with antibodies to human C4, but one of these proteins (Slp) has no C4 hemolytic activity. Because the product of C4-A locus in man has low hemolytic activity, a previous report suggests it may be the homologue of murine Slp. We show here that Slp antigenic determinants are found in human C4. However, they are expressed in the products of both loci A and B, that is, C4A and C4B, since both proteins were specifically immunoprecipitated by the IgG fraction of alloantisera to mouse Slp. Therefore, Slp-associated structural features are preserved in evolution, although they do not seem to be relevant to the hemolytic properties of C4.     

The Mouse VLA-2 Homologue Supports Collagen and Laminin Adhesion but Not Virus Binding

Human VLA-2 (α₂β₁) mediates cellular adhesion to collagen and laminin and cell attachment by the human pathogen echovirus 1. We report here the cloning, sequencing and functional expression of the mouse VLA-2 α subunit homologue. This integrin subunit is closely related to its human counterpart, with 84% amino acid identity between the human and murine proteins. Conserved structural features include an identical number of amino acids, the presence of an I domain, and identity in the number and position of N-linked glycosylation sites and putative divalent cation binding regions. Murine and human α₂ show 30% amino acid divergence within the cytoplasmic tail, a difference that can be detected with antisera directed against the C-terminal peptides. Functionally, mouse α₂ was capable of mediating cell attachment to collagen and laminin, and responded to both intra- and extracellular signals with changes in its ligand affinity. In contrast, unlike its human homologue, mouse α₂ did not promote binding of echovirus 1. Comparison of the primary structure of the homologues leads us to predict that echovirus 1 may bind in the region of the first two thirds of the human α₂ I domain, where the sequences are most divergent, whereas more conserved flanking regions, and the conserved terminal one third of the I domain, may be involved in adhesion to collagen and laminin.     

Cloning, Genomic Structure, and Expression of Mouse Ring Finger Protein GeneZnf179

ZNF179,a RING finger protein encoding gene, has been mapped within the critical deletion region for Smith–Magenis syndrome (SMS), a disorder characterized by mental retardation and multiple congenital anomalies associated with del(17)(p11.2). Here we report the cloning ofZnf179,the mouse homologue ofZNF179,and characterization of its gene structure. The 3028-bp cDNA has a 1.9-kb open reading frame that contains a RING finger domain at its N-terminus and an alanine-rich and glycine-rich domain at its C-terminus.Znf179genomic sequence includes 15 introns and spans about 10 kb on mouse chromosome 11, which maintains conserved synteny with human 17p. Northern analysis indicates thatZnf179is predominantly expressed in brain and testis. Although contained within the SMS common deletion interval, FISH experiments show thatZNF179is not deleted in two SMS patients with smaller deletions.     

PLAC1, an Xq26 Gene with Placenta-Specific Expression

A novel human X-linked gene shows placenta-specific expression and has been named PLAC1. The gene maps 65 kb telomeric to HPRT at Xq26 and has been completely sequenced at the cDNA and genomic levels. The mouse orthologue Plac1 maps to the syntenically equivalent region of the mouse X chromosome. In situ hybridization studies with the antisense mRNA during mouse embryogenesis detect Plac1 expression from 7.5 dpc (days postcoitum) to 14.5 dpc in ectoplacental cone, giant cells, and labyrinthine trophoblasts. The putative human and murine PLAC1 proteins are 60% identical and 77% homologous. Both include a signal peptide and a peptide sequence also found in an interaction domain of the ZP3 (zona pellucida 3) protein. These results make PLAC1 a marker for placental development, with a possible role in the establishment of the mother–fetus interface.     

Cloning, Structure, and Chromosome Localization of the Mouse Glutaryl-CoA Dehydrogenase Gene

Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, an inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains an open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdh was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped.     

YAC Contigs of theRab1andwobbler(wr) Spinal Muscular Atrophy Gene Region on Proximal Mouse Chromosome 11 and of the Homologous Region on Human Chromosome 2p

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized thewobblerspinal atrophy genewrto proximal mouse Chr 11, tightly linked toRab1,a gene coding for a small GTP-binding protein, andGlns-ps1,an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of theRab1region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescencein situhybridization (FISH), and sequence-tagged site (STS) isolation and mapping.Rab1andGlns-ps1were found to be only 200 kb apart. A potential CpG island near a methylatedNarI site and a trapped exon,ETG1.1,were found between these loci, and a new STS,AHY1.1,was found over 250 kb fromRab1.Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising theRAB1locus,AHY1.1,and the human homologue ofETG1.1,indicating a high degree of conservation of this region in the two species. We mappedAHY1.1and thus humanRAB1on Chr 2p13.4–p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the geneLMGMD2Bfor a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13–p16. The conservation between the mouseRab1and humanRAB1regions will be helpful in identifying candidate genes for thewobblerspinal muscular atrophy and in clarifying a possible relationship betweenwrandLMGMD2B.     

A Cluster of Keratin-Associated Proteins on Mouse Chromosome 10 in the Region of Conserved Linkage with Human Chromosome 21

A gene cluster of three to five high-cysteine keratin-associated proteins (KAPs) has been identified on mouse Chromosome 10 (MMU10) in the region of conserved linkage with human chromosome 21 (HSA21). One of these genes,Krtap12-1,has been sequenced in its entirety and shown to be an intronless gene encoding a predicted 130-amino-acid protein.Krtap12-1is most closely related to two previously identified KAP4 genes, but variation in sequence and cysteine content suggests that it represents a new KAP family.Krtap12-1is expressed in the skin of a 3-day-old mouse. The corresponding region of HSA21, betweenITGB2(integrin β2) andPFKL(the liver isoform of phosphofructokinase), has proven refractory to cloning, and thus mapping of this region at high resolution has been problematic. Based on the KAP gene cluster position in mouse, evidence has been found for an orthologous human KAP cluster on HSA21q22.3, reinforcing the observation that comparative genomics can play an essential and practical role in determining mammalian genome organization.