Molecular Cloning and Chromosomal Localization of the MouseGpr37Gene Encoding an Orphan G-Protein-Coupled Peptide Receptor Expressed in Brain and Testis

We report the cloning of the mouse ortholog of the humanGPR37gene, which encodes an orphan G-protein-coupled receptor highly expressed in brain tissues and homologous to neuropeptide-specific receptors ([20],Genomics 45:68–77;[45],Biochem. Biophys. Res. Commun. 233:559–567). The genomic organization of theGPR37gene is conserved in both mouse and human species with a single intron interrupting the receptor-coding sequence within the presumed third transmembrane domain. Comparative genetic mapping of theGPR37gene showed that it maps to a conserved chromosomal segment on proximal mouse chromosome 6 and human chromosome 7q31. The mouseGpr37gene contains an open reading frame coding for a 600-amino-acid protein 83% identical to the humanGPR37gene product. The predicted mouse GPR37 protein contains seven putative hydrophobic transmembrane domains, as well as a long (249 amino acid residues), arginine- and proline-rich amino-terminal extracellular domain, which is also a distinctive feature of the human GPR37 receptor. Northern blot analysis of mouse tissues withGpr37-specific probes revealed a main 3.8-kb mRNA and a much less abundant 8-kb mRNA, both expressed in the brain. A 3-kb mRNA is also expressed in the testis. Both the mouse and the humanGPR37genes may belong to a class of highly conserved mammalian genes encoding a novel type of G-protein-coupled receptor predominantly expressed in the brain.     

Homology between a 173-kb Region from Mouse Chromosome 10, Telomeric to the Ifng Locus, and Human Chromosome 12q15

We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow).     

Genomic organization and chromosomal localization of the murine epididymal retinoic acid-binding protein (mE-RABP) gene

The murine epididymal retinoic acid-binding protein (mE-RABP) is specifically synthesized in the mouse mid/distal caput epididymidis and secreted in the lumen. In this report, we have demonstrated by Southern blot analysis of genomic DNA that mE-RABP is encoded by a single-copy gene. A mouse 129/SvJ genomic bacterial artificial chromosome (BAC) library was screened using a cDNA encoding the minor form of mE-RABP. One positive BAC clone was characterized and sequenced to determine the nucleotide sequence of the entire mE-RABP gene. The molecular cloning of the mE-RABP gene completes the characterization of the 20.5-kDa–predicted preprotein leading to the minor and major forms of mE-RABP. Comparison of the DNA sequence of the promoter and coding regions with that of the rat epididymal secretory protein I (ESP I) gene showed that the mE-RABP gene is the orthologue of the ESP I gene that encodes a rat epididymal retinoic acid-binding protein. Several regulatory elements, including a putative androgen receptor binding site, “CACCC-boxes,” NF-1, Oct-1, and SP-1 recognition sites, are conserved in the proximal promoter. Analysis of the nucleotide sequence of the mE-RABP gene revealed the presence of seven exons and showed that the genomic organization is highly related to other genes encoding lipocalins. The mE-RABP gene was mapped by fluorescent in situ hybridization to the [A3-B] region of the murine chromosome 2. Our data, combined with that of others, suggest that the proximal segment of the mouse chromosome 2 may be a rich region for genes encoding lipocalins with a genomic organization highly related to the mE-RABP gene. Mol. Reprod. Dev. 50:387–395, 1998. © 1998 Wiley-Liss, Inc.     

Genomic characterization of the human and mouse protein tyrosine phosphatase-1B genes

PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20 q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3′ end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3′ end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitiative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5′ flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.     

Assignment of the Y4Receptor Gene (PPYR1) to Human Chromosome 10q11.2 and Mouse Chromosome 14

The human and mouse genes for the neuropeptide Y₄receptor have been isolated, sequenced, and shown to contain no introns within the coding region of the gene. Nonisotopicin situhybridization and interspecific mouse backcross mapping have localized the genes to human chromosome 10q11.2 and mouse chromosome 14. Five nucleotide variants, which do not alter the protein sequence, have been identified within the coding region of the human receptor gene. The human Y₄subtype is most closely related to the Y₁-receptor subtype (42%), suggesting that it evolved from an ancestral Y₁-like receptor via an RNA-mediated transpositional event.     

The human loricrin gene.

Loricrin is the major protein component of the cornified cell envelope of terminally differentiated mammalian epidermal (stratum corneum) cells. Using a specific human cDNA clone, we have isolated and characterized the human loricrin gene. We show that it has a very simple structure of a single intron of 1188 base pairs (bp) in the 5'-untranslated region; there are no introns in coding sequences. By use of rodent-human somatic cell hybrids, followed by in situ hybridization with a biotin-labeled genomic DNA clone, the single-copy gene maps to chromosome location 1q21. Polymerase chain reaction analyses of genomic DNAs from different individuals show that human loricrin consists of two allelic size variants, due to sequence variations in its second glycine loop domain, and these variants segregate in the human population by normal Mendelian mechanisms. Furthermore, there are multiple sequence variants within these two size class alleles due to various deletions of 12 bp (4 amino acids) in the major loop of this glycine loop domain. By use of a specific loricrin antibody, we show by immunogold electron microscopy that loricrin initially appears in the granular layer of human epidermis and forms composite keratohyalin granules with profilaggrin, but localizes to the cell periphery (cell envelope) of fully differentiated stratum corneum cells.     

CD14 is a member of the family of leucine-rich proteins and is encoded by a gene syntenic with multiple receptor genes.

We have isolated and characterized genomic and cDNA clones encoding the murine homolog of the human monocyte/granulocyte cell surface glycoprotein, CD14. As in man, the expression of murine CD14 is limited to the myeloid lineage. The murine and human CD14 genes are highly conserved in their intron-exon organization and nucleotide sequence. Their deduced protein sequences show 66% amino acid identity. In both mouse and man, the CD14 protein contains a repeating (10 times) leucine-rich motif (LXXLXLX) that is also found in a group of heterogeneous proteins from phylogenetically distant species. The CD14 gene has been mapped to mouse chromosome 18 which also contains at least five genes encoding receptors (Pdgfr, Adrb2r, li, Grl-1, Fms). Thus CD14 and the receptor genes form a conserved syntenic group localized on mouse chromosome 18 and human chromosome 5. The inclusion of CD14 in the family of leucine-rich proteins, its expression profile and the murine chromosomal localization support the hypothesis that CD14 may function as a receptor.     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

Organization and evolutionary relatedness of OR37 olfactory receptor genes in mouse and human

We report a comprehensive comparative analysis of human and mouse olfactory receptor (OR) genes encoding OR37 subtypes to determine the repertoire, chromosomal organization, and relatedness of these genes. Two OR37 clusters were found in both mouse (chromosome 4) and human (chromosome 9); with five genes in cluster I and three (mouse) and seven genes (human) in cluster II. The pronounced diversity of noncoding sequence regions in both genomic loci indicates a long-term coexistence of the two clusters and the genes within the clusters. In contrast, the coding regions, particularly of genes in cluster I, showed remarkably high sequence identity, a feature quite unique for OR genes. The conservation of only the coding sequences indicates that OR37 may be under negative selection pressure and suggests that the OR37 receptor family may be tuned to recognize distinct sets of signaling molecules. A comparison of mouse and human OR37 gene clusters revealed that genes in cluster I are highly related within each species whereas genes in cluster II are highly related across species. These data reflect a unique and complex evolutionary history of the OR37 family.     

Examination of sequence homology between human chromosome 20 and the mouse genome: intense conservation of many genomic elements

The conservation of genomic organization of mammalian species has been of interest for its usefulness in characterizing the genetics of traits and diseases and as one tool for examining evolution. The recent rough draft sequencing of the mouse and human genomes provides the opportunity for more detailed analyses. The current study examines the extent of homology between human chromosome 20 and the mouse genome by comparing putative coding and non-coding sequence to provide insight into organizational and sequence similarities between the species. The relative position of each of 460 putative coding orthologues was the same in both species, except for a single genomic segment rearrangement. The similarity extended to exon/intron structure, the size of introns, as well as strong evidence for the conservation of position of ancient LINE-1, LINE-2 and LTR repetitive sequence and the subtelomeric region of the long arm of human chromosome 20 and that of mouse chromosome 2. There was also evidence for conservation of a limited amount of non-coding single-copy sequence. Together these data provide additional insight into the extent of conservation of mammalian genomic organization and sequence.     

The Heat-Shock Gene hsp83 of Drosophila auraria: Genomic Organization, Nucleotide Sequence, and Long Antiparallel Coupled ORFs (LAC ORFs)

The genomic organization of the hsp83 gene of Drosophila auraria, a far-eastern endemic species belonging to the montium subgroup of the melanogaster species group, is presented here. Based on in situ hybridization on polytene chromosomes, cDNA and genomic clone mapping, nucleotide sequencing, and genomic Southern analysis, hsp83 is shown to be present as a single-copy gene at locus 64B on the 3L chromosome arm in D. auraria. This gene is organized into two exons separated by a 929-bp intron. The first exon represents the mRNA leader sequence and is not translated, while the coding region, having a length of 2,151 bp, is solely included in the second exon. Nucleotide sequence comparisons of D. auraria hsp83 with homologous sequences from other organisms show high conservation of the coding region (88–92% identity) in the genus Drosophila, in addition to the conserved genomic organization of two-exons–one-intron, of comparable size and arrangement. A phylogenetic tree based on the protein sequences of homologous genes from representative organisms is in accord with the accredited phylogenetic position of D. auraria. In the hsp83 gene region, a second case of long antiparallel coupled open reading frames (LAC ORFs) for this species was found. The antiparallel to the hsp83 gene ORF is 1,554 bases long, while the two ORFs overlap has a size of 1,548 bp. The anti-hsp83 ORF does not show significant homology to any known gene sequences. In addition, no similar LAC ORF structures were found in homologous gene regions of other organisms. Received: 18 April 1997 / Accepted: 1 August 1997     

The haemochromatosis candidate gene HFE (HLA-H) of man and mouse is located in syntenic regions within the histone gene cluster

The HFE (HLA-H) gene is a strong candidate gene for hereditary haemochromatosis and was localized on the short arm of chromosome 6 to 6p21.3-p22. In addition, the sequence of the homologous mouse and rat cDNA and a partial sequence from the mouse gene have been reported recently. In this report, we describe the location of the human and the mouse HFE (HLA-H) gene within the histone gene clusters on the human chromosome 6 and the mouse chromosome 13. Both the human and the murine gene were located on syntenic regions within the histone gene clusters in the vicinity of the histone H1t gene. The genomic sequence of the human HFE (HLA-H) gene and the 3′ portion of the homologous mouse gene were determined. Comparison of the genomic sequences from man and mouse and the cDNA sequence from rat shows significant similarities, also beyond the transcribed region of the mouse gene. J. Cell. Biochem. 69:117–126, 1998. © 1998 Wiley-Liss, Inc.     

Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4.

We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124-126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of approximately 52 kb. The first intron is approximately 16 kb in length and is followed by an unusually large exon (approximately 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromosome 16, which is the orthologous region of human chromosome 3q29 encompassing the large membrane-anchored mucin MUC4. Contigs analysis of the Human Genome Project did not reveal any other mucin on chromosome 3q29 and, interestingly, our analysis allowed the determination of the genomic organization of the human MUC4 and showed that its exon/intron structure is identical to that of the mouse gene we cloned. Furthermore, the human MUC4 shares considerable homologies with the mouse gene. Based on these data, we concluded that we isolated the mouse ortholog of MUC4 we propose as Muc4. Expression studies showed that Muc4 is ubiquitous like SMC and MUC4, with highest levels of expression in trachea and intestinal tract.     

Structure and Chromosomal Localization of the Human Homeobox Gene Prox 1

The genomic organization and nucleotide sequence of the human homeobox gene Prox 1 as well as its chromosomal localization have been determined. This gene spans more than 40 kb, consists of at least 5 exons, and encodes an 83-kDa protein. It shows 89% identity with the chicken sequence at the nucleotide level in the coding region, while the human and chicken proteins are 94% identical. Among the embryonic tissues analyzed (lens, brain, lung, liver, and kidney), the human Prox 1 gene is most actively expressed in the developing lens, similar to the expression pattern of the chicken Prox 1 gene. The Prox 1 gene was mapped to human chromosome 1q32.2–q32.3.