Proximal Region Of Mouse Chromosome Research Articles

Fine Mapping and Candidate Gene Search of Quantitative Trait Loci for Growth and Obesity Using Mouse Intersubspecific Subcongenic Intercrosses and Exome Sequencing

Although growth and body composition traits are quantitative traits of medical and agricultural importance, the genetic and molecular basis of those traits remains elusive. Our previous genome-wide quantitative trait locus (QTL) analyses in an intersubspecific backcross population between C57BL/6JJcl (B6) and wild Mus musculus castaneus mice revealed a major growth QTL (named Pbwg1) on a proximal region of mouse chromosome 2. Using the B6.Cg-Pbwg1 intersubspecific congenic strain created, we revealed 12 closely linked QTLs for body weight and body composition traits on an approximately 44.1-Mb wild-derived congenic region. In this study, we narrowed down genomic regions harboring three (Pbwg1.12, Pbwg1.3 and Pbwg1.5) of the 12 linked QTLs and searched for possible candidate genes for the QTLs. By phenotypic analyses of F2 intercross populations between B6 and each of four B6.Cg-Pbwg1 subcongenic strains with overlapping and non-overlapping introgressed regions, we physically defined Pbwg1.12 affecting body weight to a 3.8-Mb interval (61.5–65.3 Mb) on chromosome 2. We fine-mapped Pbwg1.3 for body length to an 8.0-Mb interval (57.3–65.3) and Pbwg1.5 for abdominal white fat weight to a 2.1-Mb interval (59.4–61.5). The wild-derived allele at Pbwg1.12 and Pbwg1.3 uniquely increased body weight and length despite the fact that the wild mouse has a smaller body size than that of B6, whereas it decreased fat weight at Pbwg1.5. Exome sequencing and candidate gene prioritization suggested that Gcg and Grb14 are putative candidate genes for Pbwg1.12 and that Ly75 and Itgb6 are putative candidate genes for Pbwg1.5. These genes had nonsynonymous SNPs, but the SNPs were predicted to be not harmful to protein functions. These results provide information helpful to identify wild-derived quantitative trait genes causing enhanced growth and resistance to obesity.

Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis

Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1(c.204+1G>A), an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.

Short-Limbed Dwarfism: slw Is a New Allele of Npr2 Causing Chondrodysplasia

Short-limbed dwarfism (SLW) is a new mutant mouse characterized by a dwarf phenotype with markedly short body, limbs, and tail. In the present study, we investigated the skeletal phenotypes of the SLW mouse and determined the chromosomal localization to identify the gene responsible for the phenotypes (slw). Skeletal preparations stained with alcian blue and alizarin red revealed that longitudinal growth of the extremities of the affected (slw/slw) mice was significantly reduced in comparison with that of normal mice, whereas the positions and numbers of skeletal elements were normal. Histological examination of tibial growth plates of the affected mice showed that the numbers of proliferating and hypertrophic chondrocytes were obviously diminished. These phenotypes resembled to those of human chondrodysplasias caused by defective chondrocyte proliferation and differentiation. We mapped the slw locus on an 11.7-cM interval of the proximal region of mouse chromosome 4 by linkage analysis. Furthermore, allelism test using Npr2(cn) locus, a mutant allele of Npr2 gene encoding a natriuretic peptide receptor B, revealed that slw locus is an allele of the Npr2 gene. These results suggest that the dwarf phenotype of the SLW mouse is caused by the disturbed endochondral ossification, and a mutation in the Npr2 gene is expected to be responsible for the phenotypes of the SLW mouse.

Defining the breakpoints of the quakingviable mouse mutation reveals a duplication from a Parkin intron

The quakingviable (qkv) mutant mouse shows a recessive neurological phenotype that includes central nervous system (CNS) dysmyelination, seizures, and tremor associated with voluntary movement. The molecular defect of qkv has been previously reported to be a spontaneous approximately 1 megabase (Mb) deletion in the proximal region of mouse chromosome 17 that occurred in the DBA mouse strain more than four decades ago. The mutation has recently been shown to affect three genes in the region: Quaking (qk), Parkin-coregulated gene (Pacrg), and Parkin. Here we determine the exact deletion breakpoints and demonstrate that the mutation is not just comprised of a approximately 1.1 Mb deletion, but also harbors a small 163 bp duplication fragment between the deletion breakpoints. Although the distal deletion breakpoint is within the fifth intron of the mouse Parkin gene, the duplicated sequence is derived from the sixth Parkin intron and shows positive transcriptional activity on a reporter gene in vitro. This complexity provides insight into a well-studied neurological mutant and may have a role in affecting the phenotype observed.

Identification of Fetuin-B as a member of a cystatin-like gene family on mouse chromosome 16 with tumor suppressor activity

Studies of mouse models for multistage carcinogenesis have led to the identification of a susceptibility locus for skin tumor development (Skts9) in the proximal region of mouse chromosome 16. This chromosome region shows a loss of heterozygosity or an allelic imbalance in mouse skin and pancreatic islet carcinoma, and has been associated with angiogenesis. The microsatellite marker D16Mit2, which has the strongest linkage to skin tumor susceptibility, was used to screen a bacterial artificial chromosome (BAC) library, leading to the identification of the histidine-rich glycoprotein (Hrg) and Fetuin-B as the most tightly linked genes. These genes are members of a cystatin-like superfamily that includes the neighboring genes Kng and Ahsg/Fetuin. Overexpression of Fetuin-B in skin squamous carcinoma cells led to suppression of tumor growth in nude mice. The neighboring genes Kng and Ahsg also have potential roles in angiogenesis and (or) tumor development, and several genes in this locus may be candidates for the Skts9 gene.

Biliary atresia.

Biliary atresia.

Ohx is a homeobox-encoding gene preferentially expressed in mature oocytes

There is abundant evidence to indicate that the homeobox genes are developmentally important. We used the NCBI dbEST databases of early mouse embryos to identify novel homeobox-containing sequence tags. Ohx ( oocyte-specific homeobo x gene) was one of several genes identified by in silico cloning. The full-length Ohx cDNA was cloned and its genomic organization was characterized. The Ohx gene spans 1.6 kb, encodes three exons and maps to the proximal region of mouse chromosome 7. Reverse transcriptase polymerase chain reaction analyses show that Ohx is preferentially expressed in one- and two-cell embryonic stages, as well as in the ovary. Whole mount in situ hybridization analysis demonstrates that the Ohx mRNA was exclusively localized to the oocytes of the mature ovary. Ohx is one of the few homeobox-encoding genes preferentially expressed during mammalian oogenesis.

Murine NFX.1: isolation and characterization of its messenger RNA, mapping of its chromosomal location and assessment of its developmental expression.

We have previously isolated (by expression cloning) a human cDNA, termed NFX.1, encoding a nucleic acid-binding protein that interacts with the conserved X1 box cis-element first discovered in class II major histocompatibility complex (MHC) genes. Functional studies involving expression of NFX.1 and assessment of expression from class II reporter constructs and endogenous class II MHC genes indicated that the factor could repress transcription of class II MHC genes. Subsequent studies have extended the biological significance of the factor, indicating that it plays an important role in neuronal development. Indeed, the reiterated RING finger motifs in the central domain of the polypeptide strongly suggest that NF-XI is a probable E3 ubiquitin protein ligase, indicating that the protein may have multiple activities. Here we report the cloning of the mouse homologue of the human NfX.1 cDNA: m-Nfx.1. Comparison of the deduced primary sequence of mouse and human NFX.1 proteins shows very high homology and confirms that m-NFX.1 contains the conserved cysteine-rich DNA-binding motif first described in human NFX.1 (95% homology). Expression of MHC class II genes is substantially reduced following expression of m-NFX.1, which confirms that we have isolated the functional murine homologue of human NfX.1 cDNA. Further evidence comes from the mapping of m-Nfx.1 gene to the proximal region of mouse chromosome 4, a region syntenic to the location of human Nfx.1 (short arm of chromosome 9). Expression profiling shows that m-NFX.1 is expressed ubiquitously in both adult tissues and during development, supporting the hypothesis that it may have yet-undescribed roles in distinct biological processes.

Identification of a novel isoform of Murr1 transcript, U2mu, which is transcribed from the portions of two closely located but oppositely oriented genes.

Here we identified a novel transcript in mouse that is transcribed from the portions of two independent genes, U2af1-rs1 and Murr1, and we designated it U2mu. The U2af1-rs1 gene is located in the intron and transcribed in the opposite direction from the Murr1 gene on the proximal region of mouse chromosome 11. The U2mu cDNA sequence is derived from three genomic regions--an intron of the Murr1 gene, an antisense sequence of U2af1-rs1 gene, and the last exon of Murr1 gene--in the order of 5' to 3'. The U2mu transcript of 2.8 kb is expressed ubiquitously in adult mice. It is transcribed biallelically, and is not imprinted, in neonatal and adult mice.

Random monoallelic expression of three genes clustered within 60 kb of mouse t complex genomic DNA.

Mammals achieve gene dosage control by (1) random X-chromosome inactivation in females, (2) parental origin-specific imprinting of selected autosomal genes, and (3) random autosomal inactivation. Genes belonging to the third category of epigenetic phenomenon are just now emerging, with only six identified so far. Here we report three additional genes, Nubp2, Igfals, and Jsap1, that show 50%-methylated CpG sites by Southern blot analyses and primarily monoallelic expression in single-cell allele-specific RT-PCR analysis of bone marrow stromal cells and hepatocytes. Furthermore, we show that, in contrast to X inactivation, alleles can switch between active and inactive states during the formation of daughter cells. These three genes are the first in their category to exist as a tight cluster, in the proximal region of mouse chromosome 17, providing a thus far unique example of a region of autosomal random monoallelic expression.

Physical Mapping of Male Fertility and Meiotic Drive Quantitative Trait Loci in the Mouse t Complex Using Chromosome Deficiencies

The t complex spans 20 cM of the proximal region of mouse chromosome 17. A variant form, the t haplotype (t), exists at significant frequencies in wild mouse populations and is characterized by the presence of inversions that suppress recombination with wild-type (+) chromosomes. Transmission ratio distortion and sterility are associated with t and affect males only. It is hypothesized that these phenomena are caused by trans-acting distorter/sterility factors that interact with a responder locus (Tcr(t)) and that the distorter and sterility factors are the same because homozygosity of the distorters causes male sterility. One factor, Tcd1, was previously shown to be amorphic using a chromosome deletion. To overcome limitations imposed by recombination suppression, we used a series of deletions within the t complex in trans to t chromosomes to characterize the Tcd1 region. We find that the distorter activity of Tcd1 is distinct from a linked sterility factor, originally called tcs1. YACs mapped with respect to deletion breakpoints localize tcs1 to a 1.1-Mb interval flanked by D17Aus9 and Tctex1. We present evidence for the existence of multiple proximal t complex regions that exhibit distorter activity. These studies demonstrate the utility of chromosome deletions for complex trait analysis.

A transgenic insertional inner ear mutation on mouse chromosome 1.

To clone and characterize the integration site of an insertional inner ear mutation, produced in one of fourteen transgenic mouse lines. The insertion of the transgene led to a mutation in a gene(s) necessary for normal development of the vestibular labyrinth. Molecular genetic analysis of a transgene integration site. Molecular cloning, Southern and northern blotting, DNA sequencing and genetic database searching were the methods employed. The integration of the transgene resulted in a dominantly inherited waltzing phenotype and in degeneration of the pars superior. During development, inner ear fluid homeostasis was disrupted. The integration consisted of the insertion of a single copy of the transgene. Flanking DNA was cloned, and mapping indicated that the genomic DNA on either side of the transgene was not contiguous in the wild-type mouse. Localization of unique markers from the two flanks indicated that both were in the proximal region of mouse chromosome 1. However, in the wild-type mouse the markers were separated by 6.3 cM, indicating a sizable rearrangement. Analysis of the mutant DNA indicated that the entire region between the markers was neither deleted nor simply inverted. These results are consistent with a complex rearrangement, including at least four breakpoints and spanning at least 6.3 cM, resulting from the integration of the transgene. This genomic rearrangement disrupted the function of one or more genes critical to the maintenance of fluid homeostasis during development and the normal morphogenesis of the pars superior.

Two Novel Mouse Genes—Nubp2, Mapped to the t-Complex on Chromosome 17, and Nubp1, Mapped to Chromosome 16— Establish a New Gene Family of Nucleotide-Binding Proteins in Eukaryotes

Two novel mouse genes and one novel human gene that define distinctive eukaryotic nucleotide-binding proteins (NUBP) and are related to the mrp gene of prokaryotes are characterized. Phylogenetic analyses of the genes, encoding a short form (Nubp2) and a long form (Nubp1) of NUBP, clearly establish them as a new NUBP/MRP gene family that is well conserved throughout phylogeny. In addition to conserved ATP/GTP-binding motifs A (P-loop) and A′, members of this family share at least two highly conserved sequence motifs, NUBP/MRP motifs α and β. Only one type of NUBP/MRP gene has been observed thus far in prokaryotes, but there are two types in eukaryotes. One group includes mouse Nubp1, human NBP, yeast NBP35, and Caenorhabditis elegans F10G8.6 and is characterized by a unique N-terminal sequence with four cysteine residues that is lacking in the other group, which includes mouse Nubp2, human NUBP2, and yeast YIA3w. Northern blot analyses of the two mouse genes show distinctive patterns consistent with this classification. Mouse Nubp2 is mapped to the t-complex region of mouse Chromosome 17, whereas Nubp1 is mapped to the proximal region of mouse Chromosome 16. Interestingly, both regions are syntenic with human chromosome 16p13.1–p13.3, suggesting that a chromosomal breakage between Nubp2 and Nubp1 probably occurred during the evolution of mouse chromosomes.

Identification and characterization of G90, a novel mouse RNA that lacks an extensive open reading frame.

We describe the cloning and characterization of the murine G90 gene, identified by subtractive hybridization based on the differential presence of its transcript in large and small intestine. The full-length cDNA and genomic sequences were cloned and found to produce a 1.5kb transcript that is polyadenylated but has no open reading frame larger than 249bp. The G90 gene was mapped to the proximal region of mouse chromosome 6. Expression analysis by Northern blotting showed that G90 is transcribed at very high levels in the small intestine and at lower levels in large intestine, testis and kidney of the mouse. In situ hybridization analysis on sections of small and large intestine and testis showed that G90 transcripts are present only in post-mitotic cells.

Isolation and Characterization of Mouse Homologue for the Human Epilepsy Gene,EPM2A

Mutations in the novel gene,EPM2A,have been shown recently to cause the progressive myoclonus epilepsy of Lafora type.EPM2Ais predicted to encode a putative protein-tyrosine phosphatase but its specific role in normal brain function and in the Lafora disease is not known. As a first step towards understanding the cellular function ofEPM2Ain an animal model, we have isolated cDNA clones for mouseEPM2Aand analyzed its expression. Sequence analyses of the mouse cDNA clones revealed a complete ORF that supports the 5′ coding sequence predicted for humanEPM2Afrom the genomic sequence. When compared toEPM2A,the mouse homologue, namedEpm2a,shows 86% identity at the nucleotide level and 88% identity and 93% similarity at the amino acid level. Similar to the human counterpart,Epm2ashowed ubiquitous expression in Northern with a major transcript size of 3.5 kb. We have mapped theEpm2ato the proximal region of mouse chromosome 10 which is the syntenic region for human chromosome band, 6q24. Our results suggest thatEPM2Ais highly conserved in mammals and might have a conserved function.

Genomic structure and chromosome mapping of the genes encoding clathrin-associated adaptor medium chains μ1A (Ap1m1) and μ1B (Ap1m2)

The protein μ1B is a member of the medium chain family of the clathrin-associated adaptor complex and is expressed exclusively in epithelial cells. We determined the genomic structure of previously cloned murine genes for μ1B (Ap1m2) and its closely related homolog, μ1A (Ap1m1). Comparison of their genomic structures revealed that the positions of introns are identical between these two genes, except for the insertion of an additional intron in Ap1m1 (intron 4). By contrast, these structures are different from that of the more distantly related Ap2m1 gene encoding μ2. Taken together with the similarity of amino acid sequences among these genes, the data presented in this study suggest that Ap1m1/2 and Ap2m1 diverged long before the separation of Ap1m1 and Ap1m2, which most likely resulted from a relatively recent gene duplication. We also mapped AP1M2 to human chromosome 19p13.2 and Ap1m2 to the proximal region of mouse chromosome 9. The results are consistent with the fact that these regions are syntenic.

Structure of the gene encoding the ubiquitin-conjugating enzyme Ubcm4, characterization of its promoter, and chromosomal location.

Ubiquitin-conjugating enzymes (E2 or Ubc) play a key role in the post-translational modification of proteins by ubiquitylation. They are encoded by a large family of genes that are closely related to each other. In this paper we present the first complete structural analysis, including the promoter and the chromosomal location, of a member of this family, the mouse Ubcm4 gene. At the genomic level the Ubcm4 gene spans approx. 50kb and is composed of four exons. Only about 1% of the total gene codes for amino acids. The four different Ubcm4 specific RNAs encode the same protein and differ only in the length of the 3' untranslated region. The polyadenylation signals used by the four different RNAs are all within the 3' terminal exon. At the 5' end of the gene, multiple transcriptional start sites were mapped within a region of 25bp. The region proximal to the initiation sites does not contain a TATA box and is not GC-rich. Transient chloramphenicol acetyltransferase assays, however, showed that this region can promote the expression of a reporter gene and that 15bp upstream of the first initiation site were sufficient for basal expression. The Ubcm4 gene was mapped by interspecific backcross analysis to the proximal region of mouse chromosome 16.

Assignment of Mouse Cardiac Two-Pore Background K+Channel Gene (Kcnk4) to the Proximal Region of Mouse Chromosome 5

Assignment of Mouse Cardiac Two-Pore Background K+Channel Gene (Kcnk4) to the Proximal Region of Mouse Chromosome 5

Genomic organization, expression and chromosomal localization of the mouse Slug ( Slugh) gene

The Slug gene encodes a zinc finger protein implicated in the generation and migration of neural crest cells in several vertebrate species. Here we describe the genomic organization and chromosomal localization of the mouse Slug ( Slugh) gene. The mouse Slug gene consists of three exons spanning approx. 4 kb. Northern blot analysis of RNA isolated from several tissues of adult mice revealed the presence of a single 2.1 kb transcript. The chromosomal location of mouse Slug was determined by interspecific backcross analysis. The mapping results indicated that Slugh is located in the proximal region of mouse chromosome 16.

Deletion mapping of the head tilt (het) gene in mice: a vestibular mutation causing specific absence of otoliths.

Head tilt (het) is a recessive mutation in mice causing vestibular dysfunction. Homozygotes display abnormal responses to position change and linear acceleration and cannot swim. However, they are not deaf. het was mapped to the proximal region of mouse chromosome 17, near the T locus. Here we report anatomical characterization of het mutants and high resolution mapping using a set of chromosome deletions. The defect in het mutants is limited to the utricle and saccule of the inner ear, which completely lack otoliths. The unique specificity of the het mutation provides an opportunity to better understand the development of the vestibular system. Complementation analyses with a collection of embryonic stem (ES)- and germ cell-induced deletions localized het to an interval near the centromere of chromosome 17 that was indivisible by recombination mapping. This approach demonstrates the utility of chromosome deletions as reagents for mapping and characterizing mutations, particularly in situations where recombinational mapping is inadequate.