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     

Coding Sequence, Genomic Organization, Chromosomal Localization, and Expression Pattern of the Signalosome ComponentCops2:The Mouse Homologue ofDrosophila Alien

TheDrosophila aliengene is highly homologous to the human thyroid receptor interacting protein, TRIP15/COPS2, which is a component of the recently identified signalosome protein complex. We identified the mouse homologue ofDrosophila alienthrough homology searches of the EST database. We found that the mouse cDNA encodes a predicted 443-amino-acid protein, which migrates at 50 kDa. The gene for the mousealienhomologue, namedCops2,includes 12 coding exons spanning 30 kb of genomic DNA on the central portion of mouse chromosome 2. MouseCops2is widely expressed in embryonic, fetal, and adult tissues beginning as early as E7.5. MouseCops2cDNA hybridizes to two mRNA bands in all tissues at 2.3 and 4 kb, with an additional 1.9-kb band in liver. Immunostaining of native and epitope tagged proteins localized the mouseCops2protein in both the cytoplasm and the nucleus, with larger amounts in the nucleus in some cells.     

Genomic organization, chromosomal mapping, and analysis of the 5’ promoter region of the human MAdCAM-1 gene

 MAdCAM-1, the endothelial addressin cell adhesion molecule-1, interacts preferentially with the leukocyte β7 integrin LPAM-1 (α4β7), but also with L-selectin, and with VLA-4 (α4β1) on myeloid cells, and serves to direct leukocytes into mucosal and inflamed tissues. Overlapping cosmid and phage λ genomic clones were isolated, revealing that the human MAdCAM-1 gene contains five exons where the signal peptide, two Ig domains, and mucin domain are each encoded by separate exons. The transmembrane domain, cytoplasmic domain, and 3′ untranslated region are encoded together on exon 5. The mucin domain contains eight repeats in total that are subject to alternative splicing. Despite the absence of a human counterpart of the third IgA-homologous domain and lack of sequence conservation of the mucin domain, the genomic organizations of the human and mouse MAdCAM-1 genes are similar. An alternatively spliced MAdCAM-1 variant was identified that lacks exon 4 encoding the mucin domain, and may mediate leukocyte adhesion to LPAM-1 without adhesion to the alternate receptor, L-selectin. The MAdCAM-1 gene was located at p13.3 on chromosome 19, in close proximity to the ICAM-1 and ICAM-3 genes (p13.2-p13.3). PMA-inducible promotor activity was contained in a 700 base pair 5’ flanking fragment conserved with the mouse MAdCAM-1 gene including tandem NF-kB sites, and an Sp1 site; and in addition multiple potential AP2, Adh1 (ETF), PEA3, and Sp1 sites. In summary, the data establish that the previously reported human MAdCAM-1 cDNA does indeed encode the human homologue of mouse MAdCAM-1, despite gross dissimilarities in the MAdCAM-1 C-terminal structures. Received: 5 December 1996 / Revised: 2 January 1997     

Identification of alternatively spliced dab1 isoforms in zebrafish

We have investigated the genomic organization, the occurrence of alternative splicing and the differential expression of the zebrafish disabled1 (dab1) gene. Dab1 is a key effector of the Reelin pathway, which regulates neuronal migration during brain development in vertebrates. The coding region of the zebrafish dab1 gene spans over 600 kb of genomic DNA and is composed of 15 exons. Alternative splicing in a region enriched for tyrosine residues generates at least three different isoforms. These isoforms are developmentally regulated and show differential tissue expression. Comparison with mouse and human data shows an overall conservation of the genomic organization with different alternative splicing events generating species-specific isoforms. Because these alternative splicing events give rise to isoforms with different numbers of phosphorylateable tyrosines, we speculate that alternative splicing of the dab1 gene in zebrafish and in other vertebrates regulates the nature of the cellular response to the Reelin signal.Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5' untranslated regions.

epsilon BP (for epsilon binding protein) is a M(r) 31,000 S-type animal lectin that binds to IgE and has been identified as the homologue of Mac-2, a macrophage cell-surface marker, as well as the lectins RL-29, CBP35, and L-34. The protein is composed of two domains with the amino-terminal portion containing tandem repeats of nine amino acids and the carboxyl-terminal half containing consensus sequences shared by S-type animal lectins. We determined the genomic map in both rat and mouse and isolated overlapping genomic clones that contain the 5' two-thirds of the murine gene. The remaining portion of the gene was obtained by polymerase chain reaction (PCR) amplification of genomic murine DNA followed by subcloning into plasmid vectors. The epsilon BP gene is composed of six exons separated by five introns. The entire amino-terminal repetitive sequence is contained in exon III, and the carboxyl-terminal domain is encoded by the three succeeding exons (IV, V, VI). The latter three exons correspond well in size and share sequence homology with three exons coding for 14-kDa S-type lectins. The sequence in exon I offers an explanation for the generation of two mRNAs differing only in their 5' untranslated sequences, previously reported in Mac-2 cDNA clones. Using cDNA synthesis and PCR amplification, we determined that two alternative splice sites are used in many different types of cells. This alternative splicing results in different 5' untranslated regions of the murine epsilon BP mRNA.     

Molecular cloning and characterization of <Emphasis Type="Italic">Izumo1</Emphasis> gene from sheep and cashmere goat reveal alternative splicing

We cloned the cDNA and genomic DNA encoding for Izumo1 of cashmere goat (Capra hircus) and sheep (Ovis aries). Analysis of 4.6 kb Izumo1 genomic sequences in sheep and goat revealed a canonical open reading frame (ORF) of 963 bp spliced by eight exons. Sheep and goat Izumo1 genes share >99% identity at both DNA and protein levels and are also highly homologous to the orthologues in cattle, mouse, rat and human. Extensive cloning and analysis of Izumo1 cDNA revealed three (del 69, del 182 and del 217) and two (del 69 and ins 30) alternative splicing isoforms in goat and sheep, respectively. All of the isoforms are derived from splicing at typical GT-AG sites leading to partial or complete truncation of the immunoglobulin (Ig)-like domain. Bioinformatics analysis showed that caprine and ovine Izumo1 proteins share similar structure with their murine orthologue. There are a signal peptide at the N-terminus (1–22 aa), a transmembrane domain at the C-terminus (302–319 aa), and an extracellular Ig-like region in the middle (161–252 aa) with a putative N-linked glycosylation site (N²⁰⁵-N-S). Alignment of Izumo1 protein sequences among 15 mammalian species displayed several highly conserved regions, including LDC and YRC motifs with cysteine residues for potential disulfide bridge formation, CPNKCG motif upstream of the Ig-like domain, GLTDYSFYRVW motif upstream of the putative N-linked glycosylation site, and a number of scattered cysteine residues. These distinctive features are very informative to pinpoint the important gene motifs and functions. The C-terminal regions, however, are more variable across species. Izumo1 cDNA sequences of goat, sheep, and cow were found to be largely homologous, and the molecular phylogenetic analysis is consistent with their morphological taxonomy. This implies the Izumo1 gene evolves from the same ancestor, and the mechanism of sperm–egg fusion in mammals may be under the same principle in which Izumo1 plays an important role.     

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.     

The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).     

Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse

We have recently cloned a cDNA encoding mitochondrial porin in Drosophila melanogaster and shown its chromosomal localization (Messina et al., FEBS Lett. (1996) 384, 9–13). Such cDNA was used as a probe for screening a genomic library. We thus cloned and sequenced a 4494-bp genomic region which contained the whole gene for the mitochondrial porin or VDAC. It was found that this D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3′ untranslated sequence (3′-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5′-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5′-UTR sequence, demonstrated to be transcribed by 5′-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2–1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites.     

Isolation of the Mouse Homologue of BRCA1 and Genetic Mapping to Mouse Chromosome 11

The BRCA1 gene is in large part responsible for hereditary human breast and ovarian cancer. Here we report the isolation of the murineBrca1homologue cDNA clones. In addition, we identified genomic P1 clones that contain most, if not all, of the mouseBrca1locus. DNA sequence analysis revealed that the mouse and human coding regions are 75% identical at the nucleotide level while the predicted amino acid identity is only 58%. A DNA sequence variant in theBrca1locus was identified and used to map this gene on a (Mus m. musculusCzech II × C57BL/KsJ)F1 × C57BL/KsJ intersubspecific backcross to distal mouse chromosome 11. The mapping of this gene to a region highly syntenic with human chromosome 17, coupled with Southern and Northern analyses, confirms that we isolated the murineBrca1homologue rather than a related RING finger gene. The isolation of the mouseBrca1homologue will facilitate the creation of mouse models for germline BRCA1 defects.     

DNA Sequence, Chromosomal Localization, and Tissue Expression of the Mouse Proteasome SubunitLmp10(Psmb10) Gene

Proteasomes are nonlysosomal multicatalytic proteases involved in antigen processing. Three of the 10 mammalian proteasome β subunits (LMP2, LMP7, and LMP10) are induced by IFN-γ. Two of these (LMP2 and LMP7) are encoded in the major histocompatibility complex of both human (chromosome 6) and mouse (chromosome 17). However, the human homologue ofLmp10, MECL1,is found on chromosome 16. Here we show that in mice,Lmp10is a single-copy gene localized to chromosome 8, in a region of conserved synteny with human chromosome 16. Sequencing of a 129/SvJ strain genomic clone revealed that the gene has eight exons spanning 2.3 kb. Characterization of a full-length mouse cDNA clone indicates thatLmp10encodes a protein of 273 amino acids with a calculated molecular weight of 29 kDa and an isoelectric point of 6.86. Northern analysis ofLmp2, Lmp7,andLmp10showed expression in heart, liver, thymus, lung, and spleen, but not in brain, kidney, skeletal muscle, or testis.     

Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22 kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA^Tyr is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation. Received: 11 August 1997 / Accepted: 2 February 1998