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.     

Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

Imprinted methylation profiles for proximal mouse Chromosomes 11 and 7 as revealed by methylation-sensitive representational difference analysis

Proximal mouse Chromosome (Chr) 11 shares regions of orthology with the candidate gene region for the imprinting growth disorder Silver-Russell syndrome (SRS) on human Chr 7p. It has previously been shown that mice with two maternal or two paternal copies (duplications, Dp) of proximal Chr 11 exhibit reciprocal growth phenotypes. Those with two paternal copies show fetal and placental overgrowth, while those with two maternal copies are growth retarded. The growth retardation observed in the latter is reminiscent of the intrauterine growth restriction (IUGR) observed in SRS patients with maternal uniparental disomy for Chr 7 (mUPD7). We have carried out a methylation-sensitive representational difference analysis (Me-RDA) screen to look for regions of differential methylation (DMRs) associated with imprinted genes. For these experiments, we have used mouse embryos with uniparental duplications of Chrs 11 and 7 proximal to the breakpoint of the reciprocal translocation T(7;11)40Ad. Two previously known imprinted loci associated with paternal allele hypomethylation were recovered on proximal mouse Chr 11, U2af1-rs1 and Meg1/Grb10. These two genes map 15 cM apart, so it seems likely that they are within separate imprinted domains that do not contain additional DMRs. The known imprinted gene Peg3, located on mouse proximal Chr 7, was also detected in our screen. The finding that Peg3 was differentially methylated in embryos with uniparental inheritance of proximal Chr 7 confirms that Peg3 is located proximal to the breakpoint of T40Ad in G-band 7A2. Because GRB10 has previously been reported to be a candidate gene for SRS, we analysed 22 patients for epimutations of the GRB10 differentially methylated region that could lead to the altered expression of this gene. No such mutations were found.     

Mouse genes encoding DNA topoisomerase I

We have initiated a genetic analysis of the physiologically important enzyme type I DNA topoisomerase in mouse. The exon-intron structures of the 5 part and the 3 part of the active gene, Top-1, were determined and shown to be quite similar to those of the previously determined human gene TOP1. The active mouse gene was mapped to the distal Chromosome (Chr) 2. In addition, the mouse genome contains one truncated processed topoisomerase-I-related pseudogene (retroposon), Top-1ps, on Chr 16. The Top-1ps locus, together with the immunoglobulin-lambda-light-chain locus, defines and additional conserved linkage group common to murine Chr 16 and human Chr 22, the site of the human pseudogene TOP1P2. The mapping data suggest that the pseudogene was established before mammalian radiation. Structural features, shared by the mouse and the human pseudogene, support this possibility.     

Identification and genetic mapping of the murine gene and 20 related sequences encoding chromosomal protein HMG-17

HMG-17 is an abundant, nonhistone chromosomal protein that binds preferentially to nucleosomal core particles of mammalian chromatin. The human gene for HMG-17 has been localized to Chromosome (Chr) 1p, but the murine gene has not been previously mapped. Here we identify the murine functional gene, Hmg17, from among more than 25 related sequences (probably processed pseudogenes) and show that it is located on mouse Chr 4, in a region known to have conserved linkage relationships with human Chr 1p. We also report the map locations of 20 additional Hmg17-related sequences on mouse Chrs 1, 2, 3, 5, 7, 8, 9, 13, 15, 16, 17, 18, and X. The multiple, dispersed members of the Hmg17 multigene family can be detected efficiently with a single cDNA probe and provide useful markers for genetic mapping studies in mice.     

Trypanosoma cruzi: TcRAB7 protein is localized at the Golgi apparatus in epimastigotes

In mammalian cells, the Rab7 protein is a key element of late endocytic membrane traffic. Several results suggest that it is involved in the transport from early to late endosome or from late endosome to lysosome. We have previously characterized a Rab7 gene homologue (TcRAB7) in Trypanosoma cruzi. Now, using an affinity-purified antibody specific to TcRAB7 protein we have determined that it is localized at the Golgi apparatus of the parasite. Our results indicate that the T. cruzi Rab7 homologue may function in a different route than its counterparts in mammalian cells.     

Synteny conservation of the Huntington's disease gene and surrounding loci on mouse Chromosome 5

The mouse homologs of the Huntington's disease (HD) gene and 17 other human Chromosome (Chr) 4 loci (including six previously unmapped) were localized by use of an interspecific cross. All loci mapped in a continuous linkage group on mouse Chr 5, distal to En2 and Il6, whose human counterparts are located on Chr y. The relative order of the loci on human Chr 4 and mouse Chr 5 was maintained, except for a break between D5H4S115E and Idua/rd, with relocation of the latter to the opposite end of the map. The mouse HD homolog (Hdh) mapped within a cluster of seven genes that were completely linked in our data set. In human these loci span a1.8 Mb stretch of human 4p 16.3 that has been entirely cloned. To date, there is no phenotypic correspondence between human and mouse mutations mapping to this region of synteny conservation.     

Characterization and chromosomal mapping of a mouse ortholog of the late-infantile ceroid-lipofuscinosis gene CLN2

Late-infantile ceroid-lipofuscinosis (CLN2) is an autosomal recessively inherited, neurodegenerative disease in humans. The CLN2 locus has been mapped to Chromosome (Chr) 11p15, and its sequence and genomic organization have recently been reported. In the present study, the cDNA sequence, exon/intron organization, and chromosomal localization of a mouse ortholog of the CLN2 gene are described. The mouse cDNA contains an open reading frame that predicts a protein product of 562 amino acids. The mouse and human coding regions are 86% and 88% identical at the nucleic acid and amino acid levels, respectively. One less codon appears in the mouse cDNA when compared with the human ortholog. The mouse gene (Cln2) spans more than 6 kb and consists of 13 exons separated by introns ranging in size from 111 to 1259 bp. Length polymorphism in an (AC)_n microsatellite in intron 3 of the mouse Cln2 gene was used to perform segregation analysis with The Jackson Laboratory DNA Panel Mapping Resource. On the basis of this analysis, the Cln2 gene was localized to a region of mouse Chr 7 that corresponds to human Chr 11p15. Characterization of the mouse Cln2 gene will facilitate generation of a mouse model for late-infantile ceroid-lipofuscinosis by gene targeting and identification of functionally important regions of the Cln2 protein. Received: 25 May 1999 / Accepted: 22 July 1999     

The mouse Chromosome 7 distal imprinting domain maps to G-bands F4/F5

Mouse Chromosome (Chr) 7 distal to band F3 on the physical map is known to be subject to imprinting, maternal duplication (MatDp) of the region leading to a late embryonic lethality, while paternal duplication (PatDp) causes death in utero before 11.5 dpc. Using a new mouse reciprocal translocation T(7;11)65H to produce MatDp for distal Chr 7, we have mapped the region subject to imprinting more precisely to bands 7F4/F5 on the cytogenetic map. Fluorescence in situ hybridization (FISH) studies on mitotic and meiotic chromosomes of a T65H heterozygote show that the imprinted gene Igf2 is located in the same region. This was confirmed by the finding that embryos with MatDp of bands 7F4/F5 did not express Igf2. We suggest that other members of the imprinted domain containing Igf2, namely Mash2, H19, Ins2, and p57 ^K1P2 , are also located in 7F4/F5 and that some or all of these genes may be responsible for the two imprinting lethalities seen with MatDp and PatDp for this region. Received: 13 October 1996 / Accepted: 8 December 1996     

Chromosomal assignment of 11 loci in the rat by mouse-rat somatic hybrids and linkage

Eleven rat genes have been assigned to rat chromosomes by use of mouse × rat somatic hybrids and/or use of linkage to known chromosome markers. Among them, the genes for the inducible nitric oxide synthase (Nos2) and for a vasoactive intestinal peptide receptor (Vipr) are potential candidates for genetic regulation of blood pressure and were localized to rat Chromosomes (Chrs) 10 and 8 respectively. Genes for gastric H,K-ATPase alpha subunit (Atp4a). Class I alcohol dehydrogenase (Adh), and aldolase C (Aldoc) were localized to Chrs 1, 2, and 10 respectively, and thus provide more DNA markers for genetic mapping of quantitative trait loci for blood pressure on those chromosomes. Genes for alkaline phosphatase (Alp1) and cardiac AE-3 Cl^-/HCO₃ ^- exchanger (Ae3) were both localized to Chr 9. Genes for glutamate dehydrogenase (Glud) and gastric H,K-ATPase beta subunit (Atp4b) were localized to Chr 16. The ornithine decarboxylase (Odc) gene and ornithine decarboxylase pseudogene (Odcp) were localized to Chrs 6 and 11 respectively.     

Structure of the murine E-selectin ligand 1 (ESL-1) gene and assignment to Chromosome 8

A 150-kDa glycoprotein designated in the mouse as E-selectin ligand-1 (ESL-1; gene symbol Selel) was first isolated based on its ability to function as a ligand for E-selectin. The gene appears equivalent to that for membrane glycoprotein MG160 encoded in the human by the locus for Golgi apparatus protein 1 (GLG1). ESL-1 is also highly homologous to the chicken cysteine-rich fibroblast growth factor receptor (CFR). We describe the genomic structure and chromosomal localization of the Selel locus. The gene is encoded by 27 exons and extends over approximately 75 kb. It maps to murine Chromosome (Chr) 8 in a region homologous to human Chr 16q where the GLG1 locus maps, further indicating that Selel and GLG1 are mouse and human equivalents of the same gene. Received: 21 April 1999 / Accepted: 12 July 1999     

Genomic structure, mapping, and expression analysis of the mammalian Lunatic, Manic, and Radical fringe genes

The three members of the mammalian fringe gene family, Manic fringe (Mfng), Radical fringe (Rfng), and Lunatic fringe (Lfng), were identified on the basis of their similarity to Drosophila fringe (fng) and their participation in the evolutionarily conserved Notch receptor signaling pathway. Fringe genes encode pioneer secretory proteins with weak similarity to glycosyltransferases. Both expression patterns and functional studies support an important role for Fringe genes in patterning during embryonic development and an association with cellular transformation. We have now further characterized the expression and determined the chromosomal localization and genomic structure of the mouse Mfng, Rfng, and Lfng genes; the genomic structure and conceptual open reading frame of the human RFNG gene; and the refined chromosomal localization of the three human fringe genes. The mouse Fringe genes are expressed in the embryo and in adult tissues. The mouse and human Fringe family members map to three different chromosomes in regions of conserved synteny: Mfng maps to mouse Chr 15, and MFNG maps to human Chr 22q13.1 in the region of two cancer-associated loci; Lfng maps to mouse Chr 5, and LFNG maps to human Chr 7p22; Rfng maps to mouse Chr 11, and RFNG maps to human Chr 17q25 in the minimal region for a familial psoriasis susceptibility locus. Characterization of the genomic loci of the Fringe gene family members reveals a conserved genomic organization of 8 exons. Comparative analysis of mammalian Fringe genomic organization suggests that the first exon is evolutionarily labile and that the Fringe genes have a genomic structure distinct from those of previously characterized glycosyltransferases. Received: 19 February 1999 / Accepted: 22 February 1999     

Genetic mapping near the myd locus on mouse Chromosome 8

Myodystrophy (myd), an autosomal recessive mutation of the mouse characterized by progressive weakness and dystrophic muscle histology, maps to the central portion of Chromosome (Chr) 8 (Lane et al. J. Hered 67, 135, 1976). This portion of Chr 8 contains the genes for a mitochondrial uncoupling protein (Ucp) and kallikrein (Kal3), which map to distal 4q in the human, providing evidence for a segment of homology. Characteristics of the myd phenotype coupled with this homology suggest that myd may be a mouse homolog of facioscapulohumeral muscular dystrophy (FSHD), which maps to human 4q35. We have confirmed and expanded the region of mouse 8-human 4 homology by generating a map of Chr 8 in an interspecific backcross of C57BL/6J and a partially inbred strain derived from M. spretus. The map is comprised of the genes for Ucp, coagulation factor XI (Cf11), and chloride channel 5 (Clc5), all of which have homologs on distal human 4q, 15 microsatellite loci, and the membrane cofactor protein pseudogene (Mcp-ps). To place myd in the genetic map, 75 affected progeny from an intersubspecific backcross of animals heterozygous for myd with Mus musculus castaneus were genotyped with Chr 8 microsatellite loci. The mutation maps between D8Mit30 and D8Mit75, an interval that is flanked by genes with human homologs at distal 4q. These results are consistent with the possibility that myd is the mouse homolog of FSHD.     

Genetic and physical mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) in chicken

The chicken natural resistance-associated macrophage protein 1 (NRAMP1) gene has been mapped by linkage analysis by use of a reference panel to develop the chicken molecular genetic linkage map and by fluorescence in situ hybridization. The chicken homolog of the murine Nramp1 gene was mapped to a linkage group located on Chromosome (Chr) 7q13, which includes three genes (CD28, NDUSF1, and EF1B) that have previously been mapped either to mouse Chr 1 or to human Chr 2q. Physical mapping by pulsed-field gel electrophoresis revealed that NRAMP1 is tightly linked to the villin gene and that the genomic organization (gene order and presence of CpG islands) of the chromosomal region carrying NRAMP1 is well conserved between the chicken and mammalian genomes. The regions on mouse Chr 1, human Chr 2q, and chicken Chr 7q that encompass NRAMP1 represent large conserved chromosomal segments between the mammalian and avian genomes. The chromosome mapping of the chicken NRAMP1 gene is a first step in determining its possible role in differential susceptibility to salmonellosis in this species.     

Genomic organization and mapping of the human and mouse neuronal β2-nicotinic acetylcholine receptor genes

As a first step in determining whether there are polymorphisms in the nicotinic acetylcholine receptor (nAChR) genes that are associated with nicotine addiction, we isolated genomic clones of the β2-nAChR genes from human and mouse BAC libraries. Although cDNA sequences were available for the human gene, only the promoter sequence had been reported for the mouse gene. We determined the genomic structures by sequencing 12 kb of the human gene and over 7 kb of the mouse gene. While the sizes of exons in the mouse and human genes are the same, the introns differ in size. Both promoters have a high GC content (60–80%) proximal to the AUG and share a neural-restrictive silencer element (NRSE), but overall sequence identity is only 72%. Using a 6-Mb YAC contig of Chr 1, we mapped the human β2-nAChR gene, CHRNB2, to 1q21.3 with the order of markers cen, FLG, IVL, LOR, CHRNB2, tel. The mouse gene, Acrb2, had previously been mapped to Chr 3 in a region orthologous to human Chr 1. We refined mapping of the mouse gene and other markers on a radiation hybrid panel of Chr 3 and found the order cen, Acrb2, Lor, Iv1, Flg, tel. Our results indicate that this cluster of markers on human Chr 1 is inverted with respect to its orientation on the chromosome compared with markers in the orthologous region of mouse Chr 3. Received: 26 January 1999 / Accepted: 10 May 1999     

The Mouse Glutathione PeroxidaseGpx2Gene Maps to Chromosome 12; Its PseudogeneGpx2-psMaps to Chromosome 7

TheGPX2gene codes for GSHPx-GI, a glutathione peroxidase whose mRNA is readily detectable in the gastrointestinal tract. AlthoughGPX2is a single gene in humans, there are two genes in the mouse genome with homology toGPX2.By analyzing a panel of mouse interspecies DNA from the Jackson Laboratory's backcross resource, we have chromosomally mapped these two genes. One was mapped to the central region of mouse chromosome 12 betweenD12Mit4andD12Mit5,nearfosandTgfb3.This region is homologous to human 14q24.1, where humanGPX2has been mapped, and most likely represents the functional mouseGpx2gene. The otherGpx2-like gene was mapped to mouse chromosome 7 betweenPcsk3andHbb.We have isolated the latter gene from a P1 phage library. Its pseudogene nature is revealed by the sequence analysis: (a) it is intronless; (b) it has a single nucleotide deletion in the coding region; and (c) it has a poly(A) tail at its 3′-untranslated region.     

Genomic structure and chromosomal localization of the mouse gene Punc

The mouse gene Punc encodes a member of the immunoglobulin superfamily of cell surface proteins. It is highly expressed in the developing embryo in nervous system and limb buds. At mid-gestation, however, expression levels of Punc decrease sharply. To allow investigation of such a regulatory mechanism, the genomic locus encompassing the Punc gene was cloned, characterized, and mapped. Fluorescent in situ hybridization was used to determine the chromosomal location of the Punc gene of mouse and human. Mouse Punc maps to Chromosome (Chr) 9 in the region D-E1, whereas the human PUNC gene is localized to Chr 15 at 15q22.3-23, a region known to be syntenic to mouse 9D-E1. The human PUNC gene therefore maps close to a genetic locus that is linked to Bardet-Biedl Syndrome, an autosomal recessive human disorder. Confirmation for the location of human PUNC was obtained through sequence relationships between mouse Punc cDNA, human PUNC cDNA, genomic sequence upstream of the murine Punc gene, and human STS markers that had been previously mapped on Chr 15. The STS sequence WI-14920 is in fact derived from the 3′-untranslated region of the human PUNC gene. WI-14920 had been placed at 228cR from the top of the Chr 15 linkage group, which provided positional information for the human PUNC gene at high resolution. Thus, this study identifies PUNC as the gene corresponding to a previously anonymous marker and serves as a basis to investigate its role in genetic disorders. Received: 8 July 1998 / Accepted: 14 October 1998