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A novel member of the Krox family of proteins, designated Krox-25, was identified by screening clones from the cDNA libraries of a rat incisor and mouse embryo craniofacial tissue. Rat and mouse Krox-25 mRNAs are about 2.4 kb long, encoding 225 and 224 amino acids, respectively. Krox-25 consists of five zinc finger motifs homologous to the Drosophila Krüppel segmentation gene and also contains several consensus amino acid sequences for a protein kinase C binding domain. Northern blot analysis revealed an intense expression of Krox-25 mRNA in rat and mouse teeth, although it was expressed weakly in other tissues, including calvaria, brain, lung, thymus, kidney, and submandibular gland of mouse. In situ hybridization showed that Krox-25 mRNA began to be expressed weakly in the early odontogenic mesenchyme and primitive enamel epithelium located at the apical end of the rat incisor, and Krox-25 expression increased in the presecretory ameloblasts and became intense in the secretory ameloblasts. This expression was also similar to the results of immunohistochemistry and Western blot, especially the Krox-25 localization in the nuclei of enamel epithelial cells. These results suggest that Krox-25 plays an important role as a transcription factor for the cytodifferentiation and amelogenesis of enamel epithelium.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.

The human formylpeptide receptor (FPR) gene cluster has three members: FPR1 and FPRL1, which are expressed in neutrophils and monocytes and encode seven-transmembrane-domain chemotactic receptors specific for N-formylpeptides, and FPRL2, whose function is unknown. The FPRL1 receptor is also a lipoxin A4 receptor. Using probes for the three human genes we have cloned six distinct mouse genes, designated Fpr1 and Fpr-rs1 through Fpr-rs5, which form a cluster on chromosome 17 in a region of conserved synteny with human chromosome 19. Fpr1 encodes a functional receptor and is clearly the orthologue of FPR1. Both Fpr-rs1 and Fpr-rs2 have higher sequence homology to FPRL1 than to FPRL2; Fpr-rs1 is 97% identical in amino acid sequence to a previously reported cDNA that encodes a lipoxin A4 receptor, whereas the putative ligand for Fpr-rs2 is unknown. Fpr-rs3, Fpr-rs4, and Fpr-rs5 appear to lack human counterparts and are most similar in sequence to FPRL1. RNA for Fpr1, Fpr-rs1, and Fpr-rs2 is present in leukocytes, spleen, and lung, whereas RNA for Fpr-rs3 was detected only in skeletal muscle. We did not detect Fpr-rs4 or Fpr-rs5 RNA in any tissue tested. Moreover, Fpr-rs5 has a stop codon in the protein-coding region corresponding to transmembrane domain VI and may not encode a functional receptor. These results suggest that the FPR gene cluster has undergone differential expansion in mammals with FPRL2, Fpr-rs2, Fpr-rs3, Fpr-rs4, and Fpr-rs5 arising after divergence of human and mouse.

A genomic DNA fragment containing the rat epimorphin gene sequence was cloned from a rat DNA cosmid library using a mouse epimorphin cDNA probe. Within the cosmid insert, nine epimorphin exons were identified and sequenced. The predicted amino acid sequence of the rat epimorphin protein exhibited 96 and 86% identity with the mouse and human epimorphin proteins, respectively. Consistent with the developmentally related expression pattern of the mouse epimorphin gene, transcripts of the rat epimorphin gene were detected in 17-day postfertilization rat embryos. The gene, designated Epim, was assigned to rat chromosome 12 by somatic cell hybrid analysis and localized to 12q16 by fluorescence in situ hybridization. The mouse and human homologs of this gene were localized on mouse chromosome 5 and human chromosome 7 by linkage analysis and chromosomal in situ hybridization, respectively.

A neural-specific activating subunit, p35, of cyclin-dependent kinase 5 (Cdk5) was recently reported to differ from other mammalian cyclins, suggesting a new type of regulatory subunit for Cdk activity. The mouse gene encoding p35, Cdk5r, was isolated from a mouse 129/SvJ genomic library, and the genomic structure of Cdk5r was characterized. The most notable features of Cdk5r are the absence of introns in the amino acid coding region and the high homology of amino acid sequence among species. The 5{prime}-flanking region of Cdk5r contained no canonical TATA or CAAT box but had several putative promoter elements, including Sp1, AP2, MRE, and NGFIA. The mouse Cdk5r transcript was detected only in the brain by Northern blot analysis. Mouse Cdk5r was mapped to a position on mouse chromosome 11. 14 refs., 2 figs.

A full-length cDNA encoding mouse annexin V (ANX5) was cloned, sequenced, and utilized for chromosomal mapping. The gene lies on mouse chromosome 3 in close linkage with the fibroblast growth factor 2 (basic) gene and is syntenic with other genes known to have orthologous counterparts on human chromosome 4q. The open reading frame encoded a protein of 319 amino acids (aa), with 92-96% identity to ANX5 in other species. Internal repeat 3 of mouse ANX5 exhibited the highest level of nonconservative aa replacements with respect to other annexin subfamilies, but the greatest sequence conservation among ANX5 species members. This region may thus contain features that distinguish ANX5 from other annexins in properties or function. Phylogenetic analysis and homology testing of ANX5 members indicated that the 34-kDa annexin from Torpedo marmorata may also belong to this subfamily. Comparison of nine species of ANX5 led to an estimation of the unit evolutionary mutation rate at 1% aa replacements every 8 million years, comparable to other annexins.

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5'-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5.

Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic α-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitos kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses.

The murine Wnt genes are implicated in the control of a variety of developmental processes. Using a PCR-based approach, we have isolated two novel members of the murine Wnt gene family, Wnt11 and Wnt12. These cDNAs display an amino acid sequence identity of between 38 and 49% with all other murine Wnts over the regions that we have isolated. In addition, two previously described Wnt genes, Wnt5a and Wnt7a, were detected in RT-PCR products. Interspecific crosses were used to demonstrate close linkage between Wnt12 and Wnt1 on Chromosome (Chr) 15. Wnt7a was mapped to mouse Chr 6, Wnt5a to the centromeric region of Chr 14, and Wnt11 to Chr7.

The 70,000-D heat shock protein (HSP70) gene family includes both heat-inducible and constitutively expressed genes. We have mapped five murine HSP70 genes to specific sites on three separate chromosomes. Southern blot analysis of Chinese hamster X mouse somatic cell DNAs was used to assign the gene for the 78,000-D glucose-regulated protein (Grp78) to Chromosome (Chr) 2, the mule germ cell-specific Hsp70-2 and Hsc70t genes to Chr 12 and Chr 17, respectively, and the heat-inducible Hsp70-3 gene also to Chr 17. Southern blot analysis of DNA from the progeny of two multilocus crosses confirmed the Grp78 location on Chr 2 and suggested the order: centromere-Vim-Abl-Grp78-Hc. Similar analysis also confirmed the initial Hsp70-2 assignment to Chr 12 with the order: Hsp70-2-Aat-Igh . The Hsp70-3 and Hsc70t genes on Chr 17, along with the heat-inducible Hsp70-1 gene, were further localized by Southern blot analysis of genomic clones to the H-2 histocompatibility region with the order: Hsp70-1-Hsp70-3-Hsc70t-Bat-6 (human G7a, valyl-tRNA synthetase).

A 13.5-kb genomic fragment of the mouse cystic fibrosis transmembrane conductance regulator (CFTR) gene was isolated from a C57BL/6J liver DNA library, using a human CFTR exon 10 probe. This region of the human gene includes the most common cystic fibrosis mutation (deletion of the Phe508 residue) in the first nucleotide binding domain of CFTR. Sequence analysis demonstrated 87% identity between the predicted mouse and the normal human CFTR exon 10 sequences, including conservation of the Phe508 residue. Northern analysis revealed that the mouse gene is expressed in intestine, lung, stomach, kidney, and salivary gland. In contrast to human CFTR, murine CFTR transcripts were not detectable by Northern analysis in the liver or pancreas. More sensitive PCR analysis, however, revealed that the mouse CFTR gene is weakly expressed in other tissues, including liver and pancreas. During development, mouse CFTR transcripts were observed as early as Embryonic Day 13. Southern analysis of mouse x Chinese hamster somatic cell hybrid DNAs mapped the mouse CFTR locus (Cftr) to Chromosome 6 (Chr 6). Subsequent typing of the progeny of an interspecies backcross revealed that Cftr is closely linked to the proto-oncogene c-met locus (Met) in the centromeric region of mouse Chr 6, consistent with the observation that there is a conserved chromosomal segment on human chromosome 7 and mouse Chr 6.

The gene for mouse transition protein 1 (mTP1) was isolated, sequenced, and chromosomally mapped. The nucleotide sequence of 1895 bp of a 6.4-kb mTP1 genomic subclone was determined to include 788 bp of 5′ flanking region, 564 bp of coding region including a 396-bp intron and a TAA stop codon, and 543 bp of 3′ flanking region. The mTP1 gene contains a B1 repeat sequence within the only intron of the gene. The transcriptional start site of the mTP1 mRNA was determined to be located 31 bases upstream of the ATG translational start codon. Southern blot analysis demonstrated the presence of sequences homologous to the mTP1 cDNA in the genomes of the rat, hamster, bull, boar, dog, horse, ram, human, and two marsupials (the American opossum and Monodelphis ), suggesting that the mTP1 gene sequence is widely conserved. The TP1 gene has been mapped by analysis of restriction fragment length variants (RFLV) in an interspecific backcross to a position 0.7 ± 0.4 cM telomeric of Mylf and 1.2 ± 0.5 cM centromeric of Vil on mouse chromosome 1.

The HSP86 gene family in BALB/c, AKR/J, C58/J, and NFS/N inbred mice comprises an intron-containing expressed gene and, depending on the strain, two to four other HSP86-related members that are apparently processed pseudogenes. The expressed gene locus, Hsp86-1, was identified by its sequence identity with the mouse HSP86 cDNA coding region together with the presence of an intron at the same position as in the homologous human gene. Hsp86-1 was mapped 11.6 cM from the immunoglobulin heavy chain gene IgH on Chromosome 12 using an intersubspecies backcross. Two of the other loci that were common to all inbred strains tested, designated Hsp86-ps1 and Hsp86-ps2, were mapped to positions on Chromosomes 11 and 3, respectively. An HSP86-related locus specific to NFS/N and C58/J mice, designated Hsp86-ps3, was mapped on Chromosome 9. Also, an HSP86-related locus that was unique to NFS/N mice, designated Hsp86-ps4, was mapped to Chromosome 4.

Glutamate dehydrogenase (GLUD) plays an important role in mammalian neuronal transmission. In human, GLUD is encoded by a small gene family. To determine whether defects in Glud genes are associated with known neurological mutations in the mouse and to contribute to the comparative mapping of homologous genes in man and mouse, the chromosomal location of genes reactive with a mouse brain GLUD cDNA were determined. Genomic Southern analysis of a well-characterized panel of Chinese hamster × mouse somatic cell hybrids identified two GLUD-reactive loci, one residing on mouse Chromosome 14 and the other on Chromosome 7. Progeny of an intersubspecies backcross were used to map one of these genes, Glud , proximal to Np-1 on Chromosome 14, but no restriction fragment polymorphisms could be identified for the second locus, Glud-2 .

Intercellular adhesion molecule-1 (ICAM-1) is an integral membrane protein, a member of the immunoglobulin superfamily, and a ligand for LFA-1, a beta 2 leukocyte integrin. ICAM-1 has a tissue distribution similar to that of the major histocompatibility complex class II antigens and is likely to play a role in inflammatory responses. We have mapped this gene to proximal mouse chromosome 9 by using mouse-hamster somatic cell hybrids and an interspecies backcross. Since human ICAM-1 maps to chromosome 19, it joins the LDL receptor to establish a new conserved syntenic segment between human chromosome 19 and proximal mouse chromosome 9. Murine Icam-1 maps between Cbl-2 and the centromere in the same region as one of the susceptibility genes for insulin-dependent diabetes mellitus (Idd-2) that is postulated to play a role in immune function and inflammation leading to insulitis. The mapping of Icam-1 to the region known to contain the Idd-2 gene raises the question of whether the phenotypic differences attributed to the Idd-2 locus might be due to genetic variation in Icam-1.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.

Somatic cell hybrids, recombinant inbred (RI) strains, and progeny of an intersubspecific backcross were typed by Southern blot analysis to prepare a linkage map of mouse chromosome 10. The seven genetic markers in this map, four of which had not previously been positioned, include genes involved in oncogenesis (Gli, Myb, Tra-1), proviral integration (Emv-25), and immune responses (Ifg, Ifgr, Pfp). The linkage map spans much of the chromosome and covers a region of the mouse genome with few molecular markers. The gene order established here demonstrates that the genes for murine interferon-γ (Ifg) and its receptor (Ifgr) are at opposite ends of the chromosome and that Ifgr and the Myb oncogene are closely linked, a factor that may be related to their joint transcriptional enhancement in some plasmacytoid lymphosarcomas.

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mousehamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 ( Gad-1 ) and that an apparent pseudogene locus is on chromosome 10 ( Gad-1ps ). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.

The zona pellucida is a unique, oocyte-specific matrix that coats the surface of all mammalian eggs. Composed of three sulfated glycoproteins in the mouse (ZP1, ZP2, and ZP3), the zona pellucida facilitates early events in fertilization and protects the embryo during preimplantation development. Using DNA isolated from hamster-mouse somatic cell hybrids and from C 57 BL 6 J × Mus spretus interspecific backcross progeny, Zp -2 was located on chromosome 7, 11.3 ± 3.2 cM distal to Tyr , and Zp -3 was located on chromosome 5, 9.2 ± 2.9 cM distal to Gus .

CD45-AP is a recently identified phosphorylated protein that specifically associates with the leukocyte-specific transmembrane glycoprotein CD45. The gene for CD45-AP, Ptprcap (protein tyrosine phosphatase, receptor type c polypeptide associated protein), was mapped in mouse by typing the progeny of two multilocus crosses using the mouse CD45-AP cDNA as a Southern hybridization probe. The CD45-AP gene mapped to the centromeric region of Chr 19 proximal to the genes Fth, Cd5, and Pcna-rs. The gene for the human CD45-AP homologue, PTPRCAP, was localized to chromosome band 11q13.1-q13.3 by fluorescence in situ hybridization using human genomic CD45-AP DNA as a hybridization probe. The genetic mapping of the Ptprcap/PTPRCAP genes extends the previously defined synteny conservation of various genes that have been assigned to these regions of the mouse and the human chromosomes.

Oligodendrocyte-specific protein (OSP) is a recently described protein present only in myelin of the central nervous system. Several inherited disorders of myelin are caused by mutations in myelin genes but the etiology of many remain unknown. We mapped the location of the mouse OSP gene to the proximal region of chromosome 3 using two sets of multilocus crosses and to human chromosome 3 using somatic cell hybrids. Fine mapping with fluorescence in situ hybridization placed the OSP gene at human chromosome 3q26.2-q26.3. To date, there are no known inherited neurological disorders that localize to these regions.

We have mapped the mouse protein tyrosine phosphatase epsilon (PTP epsilon, gene symbol Ptpre) gene to the distal region of chromosome 7 by linkage analysis using two sets of multilocus genetic crosses. The human PTP epsilon gene (gene symbol PTPRE) was mapped to chromosome 10q26 by fluorescence in situ hybridization. We have previously documented the existence of two isoforms of PTP epsilon--a transmembranal, receptor-type isoform and a shorter, cytoplasmic one. Both isoforms have been suggested to arise from a single gene through the use of alternative promoters and 5' exons. The identification of a single PTP epsilon locus in both organisms is consistent with this suggestion.

The chromosomal localizations of Scnn1b and Scnn1g, genes corresponding to the {beta}- and {gamma}-subunits, respectively, of an epithelial non-voltage-gated amiloride-sensitive sodium channel, were determined by analyses of two sets of multilocus crosses using probes generated by polymerase chain reaction and a mouse kidney cortical collecting tubule cell line (M1). Scnn1b and Scnn1g were determined to be closely linked on distal mouse chromosome 7, showing no recombination with Zp2, whereas the gene for the {alpha}-subunit, Scnn1a, was confirmed to map to distal mouse chromosome 6. 26 refs., 1 fig.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and RANTES are members of the beta chemokine family of leukocyte chemoattractants. We have previously cloned three mouse genes by cross-hybridization with the human MIP-1 alpha/RANTES receptor gene CMKBR1. One of the mouse genes, Scya3r, encodes a functional MIP-1 alpha receptor. The functions of the other two, Scya3r-rs1 and Scya3r-rs2, are not known. We have now mapped Scya3r, Scya3r-rs1, and Scya3r-rs2 to chromosome 9, in a region of conserved synteny with the location of CMKBR1. Thus, like chemokine genes and alpha chemokine receptor genes, this group of beta chemokine receptor genes arose by tandem duplication.

The protein-tyrosine kinase gene Itk is expressed preferentially in T lymphoid cells of the mouse and is induced by IL-2. A related gene, Btk, is expressed in the murine B lymphoid and myeloid lineages. Because mutations in Btk and the corresponding human gene are associated with X-linked immunodeficiency syndromes, it was of interest to map Itk and its human counterpart. By Southern blot analysis of DNA from the progeny of two multilocus crosses, murine Itk was mapped to Chromosome 11. By fluorescence in situ hybridization, human ITK was mapped to 5q32-q33. Murine Itk and its human homologue lie within regions of conserved synteny that include several growth factor and growth factor receptor genes. This region in humans is frequently deleted in the myelodysplastic syndrome, suggesting possible involvement of ITK in this disorder.

Using mouse probes specific to cytochrome c S and to cytochrome c T , the single-copy genes encoding these two proteins have been mapped to paralogous chromosomal regions by analysis of restriction fragment length variants in interspecific crosses. The gene for cytochrome c S , Cycs , maps to a position between Tcrb and Cbl-1 on proximal mouse Chromosome 6, and the gene for cytochrome c T , Cyct , maps between Gad-1 and Sfpi-1 on mouse Chromosome 2.

We have isolated a unique gene from a mouse JB6 epidermal cell cDNA expression library, termed FKBPRP, that codes for a protein having domains that share between 37 and 44% amino acid sequence identity and 60% similarity with members of the family of FK506-binding proteins. The FKBPRP protein has three repeats contained within its sequence that share between 48 and 58% identity to each other. We have localized the FKBPRP gene to mouse Chromosome 11, and crosses of different murine strains provided the gene order centromere-FKBPRP-Int-4-Pkca-Es-3.

A ratbrain cDNA probe for the gene encoding the α2 subunit of the dihydropyridine-sensitive L-type calcium channel was used as a hybridization probe for the Southern blot analysis of Chinese hamster × mouse somatic cell hybrids and the progeny of an intersubspecies backcross. This gene, termed Cchl2a, was mapped near the centromeric end of the Chromosome 5 linkage group with gene order: centromere-Pgy-1-Cchl2a-Il-6-Pgm-1.

The gene Blk , which encodes a novel tyrosine kinase expressed preferentially in B-lymphoid cells, was mapped by Southern blot analysis of DNA from the progeny of an intersubspecific backcross. Blk maps to the proximal region of chromosome 14 with the gene order centromere- (Np-1, Tcra)-Blk-sys-Es-10 .

We have recently characterized a novel mammalian gene family, encoding membrane glycoproteins with four trans-membrane domains. This gene family includes the previously studied PMP22, which is involved in the Charcot-Marie-Tooth neuropathy, and three novel genes: TMP, XMP, and YMP (HGMW-approved symbols EMP1, EMP2 and EMP3, respectively). The Tmp (tumor-associated membrane protein) gene was isolated from a c-myc induced mouse brain tumor and is expressed in several highly proliferative cell types. We have now isolated cDNAs of the mouse Xmp and Ymp genes and determined the chromosomal localization of mouse Tmp, Xmp, and Ymp. Tmp was mapped to mouse chromosome 6, Xmp was mapped to chromosome 16, and Ymp was mapped to chromosome 7. Tmp and Ymp map to paralogous chromosomal regions, whereas Xmp maps to a chromosomal region that is putatively paralogous to a region on chromosome 11, to which Pmp22 was previously mapped. These data suggest that this family of membrane glycoproteins evolved as a result of chromosomal duplications.

N-RAP is a nebulin-related actin-binding protein found at the myotendon junction in skeletal muscle and at the intercalated disks in cardiac muscle. We mapped the NRAP gene to mouse chromosome 19 using interspecific crosses and to human chromosome 10 using radiation hybrid panels. Comparative analysis of the mouse and human genomes indicates that the NRAP gene is located in regions of conserved synteny between the two species.

We have cloned and characterized the organization of the mouse orphan nuclear receptor Nurr1 gene. The Nurr1 gene is approximately 7 kb long, contains eight exons and seven introns, and mapped to mouse chromosome 2. Although the exon/intron structure of Nurr1 is nearly identical to that of Nur77, Nurr1 possesses an additional untranslated exon. Primer extension was used to identify two major transcription initiation sites mapped 37 nucleotides apart in the first untranslated exon. Functional studies of chimeric Nurr1–luciferase reporter genes delineated the promoter region and underscored the importance of the +1 transcription start site. Sequence analysis of the 5′ flanking region surrounding +1 revealed several possible response elements such as a hexanucleotide glucocorticoid binding site, a cAMP-response element, a CArG box, and two c-Jun-binding sites. These data help to explain the different response characteristics of two closely related early response genes, Nurr1 and Nur77.

Chromogranin A, chromogranin B, and secretogranin II, members of the chromogranin/secretogranin secretory protein family, are overexpressed in some human hereditary maladies and may have arisen, in part, from common ancestor genes. To understand better the mammalian chromosomal dispersion of this gene family and to facilitate studies of these genes in human illnesses and their animal models, we positioned the locus of each member in the rat, mouse, and human genomes. Our results indicate that each locus lies in a region of locally syntenic chromosomal homology across the three species.

The genes encoding the alpha 1 and beta subunits of voltage-sensitive calcium channel were mapped in the mouse by analysis of the progeny of two multilocus crosses. The alpha 1, beta 2, and beta 4 subunit genes, termed Cchna1, Cchb2, and Cchb4, are located at different sites on proximal Chr 2, while the beta 3 subunit gene Cchb3 maps to Chr 15 near Wnt1. These results together with previous mapping data indicate that the calcium channel genes are dispersed in the mouse genome, unlike the sodium channel genes, which are clustered.

The Int6 gene is a common insertion site for the mouse mammary tumor virus (MMTV) in mouse mammary tumors. We have determined that this gene is located centromeric of the Myc protooncogene on mouse chromosome 15. In the mouse genome there are several other Int6-reactive restriction fragments that are located on mouse chromosomes 6, 11, 14, 17, and 18. Nucleotide sequence analysis of four of six of these additional Int6 fragments showed that they contain processed Int6 pseudogenes. Comparisons between the Int6 genes of the inbred mouse laboratory strains and the wild mouse species Mus spretus and Mus mus musculus indicate that some pseudogenes were present before divergence of these species and others were acquired since their separation.

The single functional mouse gene for MIF (macrophage migration inhibitory factor) has been cloned from a P1 library, and its exon/intron structure determined and shown to resemble that of the human gene. The gene was mapped to chromosome 10 using two multilocus crosses between laboratory strains and either Mus musculus musculus or Mus spretus. Nine additional loci containing related sequences, apparently all processed pseudogenes, were also mapped to chromosomes 1, 2, 3, 7, 8, 9, 12, 17, and 19. While most of these pseudogenes were found in inbred mice and M. spretus, some are species specific. This suggests that there have been active phases of pseudogene formation in Mus both before and after the separation of musculus and spretus. The human genome contains no pseudogenes; we assigned the human gene to chromosome 19, consistent with the location of mouse and human functional genes for MIF in a region of conserved linkage.

beta-1,4-N-Acetylgalactosaminyltransferase (EC 2.4.1.92; GalNAc-T) is a glycosyltransferase involved in the synthesis of gangliosides GM2 and GD2 as well as glycolipid GA2. We have isolated and sequenced the mouse Gal-NAc-T cDNA, studied GalNAc-T mRNA expression in adult tissues and in embryos, and determined the chromosomal location of the GalNAc-T gene, Ggm2. In comparison with the human cDNA, the mouse sequence was 83 and 87% identical at the nucleic acid and amino acid levels, respectively. The GalNAc-T transcript was most abundantly expressed in brain, liver, lung, spleen, and testis among the eight adult tissues examined. Relatively high levels of expression were seen early in mouse development (7-day embryos) compared to later times (11, 15, and 17 days). The Ggm2 gene was mapped to a distal position on mouse chromosome 10 that is homologous to a portion of human chromosome 12.

Uroporphyrinogen-III synthase (URO-S; EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 × 10 6 recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5′ and 3′ untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli . In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy.

The phenol-preferring sulfotransferases aryl sulfo-transferase IV and N-hydroxyarylamine sulfotransferase catalyze sulfate conjugation of N-hydroxy-2-acetylaminofluorene, a metabolite capable of causing hepatocarcinogenesis in rats. We utilized published cDNA sequences of these sulfotransferases to type the progeny of two multilocus crosses and determined that the genes, aryl sulfotransferase (Stp) and N-hydroxyarylamine sulfotransferase (Stp2), map to positions on mouse chromosomes 7 and 17. 19 refs., 1 fig.

Productive infection with HIV-1, the virus responsible for AIDS, requires the involvement of host cell factors for completion of the replicative cycle, but the identification of these factors and elucidation of their specific functions has been difficult. A human cDNA, TRBP, was recently cloned and characterized as a positive regulator of gene expression that binds to the TAR region of the HIV-1 genome. Here we demonstrate that this factor is encoded by a gene, TARBP2, that maps to human chromosome 12 and mouse chromosome 15, and we also identify and map one human pseudogene (TARBP2P) and two mouse TRBP-related sequences (Tarbp2-rs1, Tarbp2-rs2). The map location of the expressed gene identifies it as a candidate for the previously identified factor encoded on human chromosome 12 that has been shown to be important for expression of HIV-1 genes. Western blotting indicates that despite high sequence conservation in human and mouse, the TARBP2 protein differs in apparent size in primate and rodent cells.

The GM2 activator protein forms a substrate-complex with GM2 ganglioside, which enables degradation of the ganglioside by beta-hexosaminidase A. Mutations in the human GM2 activator protein gene (GM2A) result in the GM2 gangliosidosis AB variant, a severe neurological disease. We have isolated and sequenced a mouse GM2 activator protein (Gm2a) cDNA with complete protein coding and 3' untranslated regions. Expression of the Gm2a transcript (approximately 2.3 kb) was apparent in all tissues examined and was most abundant in kidney and testis. The Gm2a gene was mapped to a region on mouse chromosome (Chr) 11 that is homologous with a segment of human chromosome 5 containing the orthologous human gene. In addition, a Gm2a-related sequence (Gm2a-rs1) was mapped to mouse Chr 5.

Opioid receptors are the membrane proteins that mediate the pain-relieving effect of opioid drugs, such as morphine and fentanyl as well as endogenous opioid peptides enkephalins and endorphins. Using cDNAs for the μ and the κ opioid receptors, we mapped the chromosomal locations of their genes in mouse. Multilocus cross analysis located the μ receptor gene Oprm on Chr 10 and the κ receptor gene Oprk1 on Chr 1. Both genes are near centromere, with no markers more centromeric. These data indicate that the two opioid receptors are different gene products, ruling out the possibility that they may be differential splicing products from the same gene.

GABAA receptors are a heterogeneous group of heterooligomeric ligand-gated anion channels that represent the main means of synaptic inhibition in neuronal tissue. At least 14 distinct GABAA subunit proteins representing 5 different classes have been identified. Using rat brain cDNAs for the GABAA receptor beta 1-, alpha 2-, and alpha 4-subunits, we have assigned the corresponding murine genes Gabrb-1, Gabra-2, and Gabra-4 to specific chromosomes with somatic cell hybrids, and we have positioned these genes by analysis of the progeny of three genetic crosses. Gabrb-1 and Gabra-2 are tightly linked on mouse Chromosome 5 between the markers Pgm-1 and Kit, and Gabra-4 maps to proximal mouse Chromosome 7 in apparent proximity to the previously mapped Gabrb-3. The Gabrb-1, Gabra-2 pair of genes on Chr 5 maps near the mouse "circling mutation" pi (pirouette).

Cchl1a3 encodes the dihydropyridine-sensitive calcium channel alpha 1 subunit isoform predominantly expressed in skeletal muscle. mdg (muscular dysgenesis) has previously been implicated as a mutant allele of this gene. Hybridization of a rat brain cDNA probe for Cchl1a3 to Southern blots of DNAs from a panel of Chinese hamster x mouse somatic cell hybrids suggested that this gene maps to mouse Chromosome 1. Analysis of the progeny of an inbred strain cross-positioned Cchl1a3 1.3 cM proximal to the Pep-3 locus on Chr 1.

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.

The alpha 3, alpha 5, and beta 4 genes (human gene symbols CHRNA3, CHRNA5, and CHRNB4 respectively; mouse gene symbols Acra-3, Acra-5, and Acrb-4, respectively) are members of the nicotinic acetylcholine receptor gene family and are clustered within a 68-kb segment of the rat genome (Boulter et al., 1990, J. Biol. Chem. 265:4472). By somatic cell hybrid analysis, three cDNAs corresponding to these genes were used to map the homologous loci to human chromosome 15 and to mouse chromosome 9. Linkage analysis using CEPH pedigrees showed that the CHRNA5 gene was closely linked to the following chromosome 15 loci: D15S46, D15S52, D15S28, D15S34, and D15S35. Using interspecies crosses in mice, the Acra-5 gene was found closely linked to the Mpi-1 locus. The mapping of these members of a neurotransmitter receptor gene family may facilitate the identification of relationships between the neurotransmitter receptors and murine or human phenotypes.

Interphotoreceptor retinoid-binding protein (IRBP) is a large glycoprotein known to bind retinoids and found primarily in the interphotoreceptor matrix of the retina between the retinal pigment epithelium and the photoreceptor cells. It is thought to transport retinoids between the retinal pigment epithelium and the photoreceptors, a critical role in the visual process. We have used a 900-bp bovine IRBP cDNA fragment to map the corresponding gene, Rbp-3, to mouse chromosome 14 with somatic cell hybrids and have positioned the gene near Np-1 (nucleoside phosphorylase-1) by analysis of the progeny of an intersubspecific backcross. In the human genome, NP maps to human chromosome 14 and RBP3 to human chromosome 10. Thus, these two genes span the putative site of a chromosomal translocation which contributed to divergent karyotype evolution of man and mouse.