Mapping of theKHSRPGene to a Region of Conserved Synteny on Human Chromosome 19p13.3 and Mouse Chromosome 17

The K homology-type splicing regulatory protein, KSRP, activates splicing through intronic splicing enhancer sequences. It is highly expressed in neural cells and is required for the neural-specific splicing of the c-src N1 exon. In this study, we mapped the gene (gene symbolsKHSRPandKhsrp) to human chromosome 19 by using radiation hybrid panels and to mouse chromosome 17 by studying an interspecific backcross panel. HumanKHSRPis a positional candidate gene for familial febrile convulsion and Cayman type cerebellar ataxia. Comparative analysis of the human and mouse genomes indicates that theKHSRPgene is located in regions of conserved synteny between the two species.     

A Postimplantation Lethal Mutation Induced by Transgene Insertion on Mouse Chromosome 8

We have produced three lines of transgenic mice that contain additional copies of the mouse phosphoglycerate kinase 1 (Pgk1) gene. Two of these lines, 94-A and 94-K, which are descendants of a common founder, did not produce liveborn progeny carrying two copies of these transgenes (i.e., A/A, K/K, or A/K). Genotyping of midgestation embryos showed that A/K embryos are dead by Embryonic Day 10. Comparison of the level of transgene expression in the three transgenic lines ruled out PGK1 toxicity as the cause of death of A/A, A/K, and K/K embryos. The death of A/A, K/K, and A/K transgenic mice was therefore attributed to an insertional mutation disrupting a gene or genes essential for normal embryogenesis. Analysis of the structure of the 94-A and 94-K transgenes indicated that they differ in the number of tandem repeats and in the positions of the transgene-cellular DNA junctions. To determine if the two transgenes represent a single integration followed by a rearrangement or two independent integration events, we cloned the endogenous sequences surrounding the 94-A and 94-K transgene insertion sites. Restriction analysis of the isolated genomic clones indicated that the endogenous sequences abutting the 3′ ends of the 94-A and 94-K transgenes are separated by less than 20 kb, providing strong support for the single integration model. Further analysis indicated that the 94-A transgene is associated with a deletion of at least 18 kb and is located in the vicinity of a widely transcribed endogenous gene. Chromosomal mapping of the endogenous sequences flanking the 94-A and 94-K transgene insertions using mouse-hamster somatic cell hybrids and a (C57BL/6J × SPRET/Ei)F1 × SPRET/Ei backcross panel allowed us to assign the 94-A(K) transgene insertion to the subcentral region of mouse chromosome 8.     

Tissue Distribution, Genomic Structure, and Chromosome Mapping of Mouse and Human Eukaryotic Initiation Factor 4E-Binding Proteins 1 and 2

Two related eukaryotic initiation factor-4E binding proteins (4E-BP1 and 4E-BP2) were recently characterized for their capacity to bind specifically to eIF4E and inhibit its function. Here, we determined the cDNA sequence, tissue distribution, genomic structure, and chromosome localization of murine and human 4E-BP1 and 4E-BP2. Mouse 4E-BP1 and 4E-BP2 consist of 117 and 120 amino acids and exhibit 91.5 and 95.0% identity, respectively, to their human homologues. 4E-BP1 mRNA is expressed in most tissues, but is most abundant in adipose tissue, pancreas, and skeletal muscle, while 4E-BP2 mRNA is ubiquitously expressed. The structures of the mouse 4E-BP1 and 4E-BP2 were determined. The 4E-BP1 gene consists of three exons and spans ∼16 kb. In addition, two 4E-BP1 pseudogenes exist in the mouse genome. The 4E-BP2 gene spans approximately 20 kb and exhibits an identical genomic organization to that of 4E-BP1, with the protein coding portion of the gene divided into three exons. There are no pseudogenes for 4E-BP2. The chromosomal locations of 4E-BP1 and 4E-BP2 were determined in both mice and humans by fluorescencein situhybridization analysis. Mouse 4E-BP1 and 4E-BP2 map to chromosomes 8 (A4-B1) and 10 (B4-B5), respectively, and human 4E-BP1 and 4E-BP2 localize to chromosomes 8p12 and 10q21–q22, respectively.     

Genomic Structure and Chromosomal Mapping of the MousenovGene

Thenovgene encodes a cysteine-rich protein that is overexpressed in avian nephroblastomas. It is a member of the CCN family of proteins, all of which are involved in cell growth. Genomic and cDNA clones encompassing the mousenovgene have been isolated and characterized. The mousenovgene is highly conserved with the human and chicknovgenes at the level of nucleotide sequence and genomic organization. The exon structure reflects the modular organization of the NOV protein in a number of structural domains. These are highly conserved with other members of the CCN family, as is the distribution of 38 of its 40 cysteine residues. Thenovgene maps to chromosome 15, between D15 Mit 153 and D15 Mit 183, in a region of conserved synteny with human chromosome 8.     

The Structure of a Conserved Region of Porcine Genome,Represented in Human Genome by Chromosome 17

Radiation mapping of nine genes (H3F3B, HLR1, MYL4,STAT5B, THRA1, TOP2A, MCP1, NF1, and MPO) to porcine chromosome 12 was carried out. Also, subchromosomal location of the NF1 gene along with the two loci containing the DNA sequences homologous to the DNA of the two human BAC clones was determined. The NF1 position was ascertained via microdissection of chromosome 12 with subsequent PCR amplification of the gene fragment with specific primers. BAC clones were mapped using FISH. Comparative analysis of the gene order in porcine chromosome 12 and in the homologous human chromosome 17 was performed. It was demonstrated that the gene orders in these chromosomes differed relative to the position of the MPO gene.     

Deletion Mapping of Four Loci Defined by N-Ethyl-N-Nitrosourea-Induced Postimplantation-Lethal Mutations within the Pid-Hbb Region of Mouse Chromosome 7

As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7, four loci [l(7)1Rn, l(7)2Rn, l(7)3Rn, l(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the mouse chromosome-7 albino (c) locus. Each locus was assigned to a defined subregion of the deletion map at the distal end of the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pid and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethals was found to arrest development after uterine implantation; the specific time of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outside of and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals defined by deletion breakpoints suggests a number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segment of the human genome (perhaps human 11q13.5).     

Searching for Genomic Region of High-Fat Diet-Induced Type 2 Diabetes in Mouse Chromosome 2 by Analysis of Congenic Strains

SMXA-5 mice are a high-fat diet-induced type 2 diabetes animal model established from non-diabetic SM/J and A/J mice. By using F2 intercross mice between SMXA-5 and SM/J mice under feeding with a high-fat diet, we previously mapped a major diabetogenic QTL (T2dm2sa) on chromosome 2. We then produced the congenic strain (SM.A-T2dm2sa (R0), 20.8–163.0 Mb) and demonstrated that the A/J allele of T2dm2sa impaired glucose tolerance and increased body weight and body mass index in the congenic strain compared to SM/J mice. We also showed that the combination of T2dm2sa and other diabetogenic loci was needed to develop the high-fat diet-induced type 2 diabetes. In this study, to narrow the potential genomic region containing the gene(s) responsible for T2dm2sa, we constructed R1 and R2 congenic strains. Both R1 (69.6–163.0 Mb) and R2 (20.8–128.2 Mb) congenic mice exhibited increases in body weight and abdominal fat weight and impaired glucose tolerance compared to SM/J mice. The R1 and R2 congenic analyses strongly suggested that the responsible genes existed in the overlapping genomic interval (69.6–128.2 Mb) between R1 and R2. In addition, studies using the newly established R1A congenic strain showed that the narrowed genomic region (69.6–75.4 Mb) affected not only obesity but also glucose tolerance. To search for candidate genes within the R1A genomic region, we performed exome sequencing analysis between SM/J and A/J mice and extracted 4 genes (Itga6, Zak, Gpr155, and Mtx2) with non-synonymous coding SNPs. These four genes might be candidate genes for type 2 diabetes caused by gene-gene interactions. This study indicated that one of the genes responsible for high-fat diet-induced diabetes exists in the 5.8 Mb genomic interval on mouse chromosome 2.     

High-Resolution Mapping of a Genetic Locus Regulating Preferential Carbohydrate Intake,Total Kilocalories,and Food Volume on Mouse Chromosome 17

The specific genes regulating the quantitative variation in macronutrient preference and food intake are virtually unknown. We fine mapped a previously identified mouse chromosome 17 region harboring quantitative trait loci (QTL) with large effects on preferential macronutrient intake-carbohydrate (Mnic1), total kilcalories (Kcal2), and total food volume (Tfv1) using interval-specific strains. These loci were isolated in the [C57BL/6J.CAST/EiJ-17.1-(D17Mit19-D17Mit50); B6.CAST-17.1] strain, possessing a ∼40.1 Mb region of CAST DNA on the B6 genome. In a macronutrient selection paradigm, the B6.CAST-17.1 subcongenic mice eat 30% more calories from the carbohydrate-rich diet, ∼10% more total calories, and ∼9% more total food volume per body weight. In the current study, a cross between carbohydrate-preferring B6.CAST-17.1 and fat-preferring, inbred B6 mice was used to generate a subcongenic-derived F₂ mapping population; genotypes were determined using a high-density, custom SNP panel. Genetic linkage analysis substantially reduced the 95% confidence interval for Mnic1 (encompassing Kcal2 and Tfv1) from 40.1 to 29.5 Mb and more precisely established its boundaries. Notably, no genetic linkage for self-selected fat intake was detected, underscoring the carbohydrate-specific effect of this locus. A second key finding was the separation of two energy balance QTLs: Mnic1/Kcal2/Tfv1 for food intake and a newly discovered locus regulating short term body weight gain. The Mnic1/Kcal2/Tfv1 QTL was further de-limited to 19.0 Mb, based on the absence of nutrient intake phenotypes in subcongenic HQ17IIa mice. Analyses of available sequence data and gene ontologies, along with comprehensive expression profiling in the hypothalamus of non-recombinant, cast/cast and b6/b6 F₂ controls, focused our attention on candidates within the QTL interval. Zfp811, Zfp870, and Btnl6 showed differential expression and also contain stop codons, but have no known biology related to food intake regulation. The genes Decr2, Ppard and Agapt1 are more appealing candidates because of their involvement in lipid metabolism and down-regulation in carbohydrate-preferring animals.     

Aryl Hydrocarbon Hydroxylase Induction by Benzo[a]anthracene: Regulatory Gene Localized to the Distal Portion of Mouse Chromosome 17

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase inducibility by benzo[a]anthracene was studied in 29 somatic cell hybrid clones, developed by fusing mouse spleen or peritoneal cells from four different inbred strains with hypoxanthine phosphoribosyltransferase-deficient Chinese hamster E36 cells. Karyotype analysis plus 25 markers assigned to 16 autosomes and the X chromosome were examined. In 28 of the 29 clones, the presence or absence of inducibility is associated with the presence or absence, respectively, of mouse chromosome 17.—Liver microsomal aryl hydrocarbon hydroxylase induction by 3-methylcholanthrene or benzo[a]anthracene was assessed in appropriate backcrosses with the Mus musculus molossinus, M. m. castaneus, MOR/Cv, PL/J, SM/J and DBA/2J inbred strains and in 13 NX8 recombinant inbred lines. Twenty-seven biochemical genetic markers representing all but four autosomes were tested for possible linkage with the hydroxylase inducibility, and no linkage was found. The hepatic Ah receptor was quantitated in 26 BXD recombinant inbred lines; the Ah phenotype did not match exactly any of the more than 70 genes with established strain distribution patterns representing 12 autosomes and at least five unlinked markers.—It is concluded that a major gene controlling aryl hydrocarbon hydroxylase inducibility by benzo[a]anthracene is located on chromosome 17. Because there is no significant linkage with any of three biochemical markers in the upper third of the chromosome, we conclude that the inducibility gene is located in the distal 40% of mouse chromosome 17. Whether this trait represents the Ah locus, i.e., the gene encoding the cytosolic Ah receptor, will require further study.     

Association Mapping and Haplotype Analysis of a 3.1-Mb Genomic Region Involved in Fusarium Head Blight Resistance on Wheat Chromosome 3BS

A previous study provided an in-depth understanding of molecular population genetics of European and Asian wheat gene pools using a sequenced 3.1-Mb contig (ctg954) on chromosome 3BS. This region is believed to carry the Fhb1 gene for response to Fusarium head blight. In this study, 266 wheat accessions were evaluated in three environments for Type II FHB response based on the single floret inoculation method. Hierarchical clustering (UPGMA) based on a Manhattan dissimilarity matrix divided the accessions into eight groups according to five FHB-related traits which have a high correlation between them; Group VIII comprised six accessions with FHB response levels similar to variety Sumai 3. Based on the compressed mixed linear model (MLM), association analysis between five FHB-related traits and 42 molecular markers along the 3.1-Mb region revealed 12 significant association signals at a threshold of P<0.05. The highest proportion of phenotypic variation (6.2%) in number of diseased spikelets (NDS) occurred at locus cfb6059, and the physical distance was about 2.9 Kb between umn10 and this marker. Haplotype block (HapB) analysis using a sliding window LD of 5 markers, detected six HapBs in the 3.1-Mb region at r²>0.1 and P<0.001 between random closely linked markers. F-tests among Haps with frequencies >0.05 within each HapB at r²>0.1 and P<0.001 showed significant differences between the Hap carried by FHB resistant resources, such as Sumai 3 and Wangshuibai, and susceptible genotypes in HapB3 and HapB6. These results suggest that Fhb1 is located within HapB6, with the possibility that another gene is located at or near HapB3. SSR markers and Haps detected in this study will be helpful in further understanding the genetic basis of FHB resistance, and provide useful information for marker-assisted selection of Fhb1 in wheat breeding.     

Conserved Linkage within a 4-Cm Region of Mouse Chromosome 9 and Human Chromosome 11

A six-point cross was carried out to determine the gene order and distances among loci on mouse chromosome 9. Our results are consistent with the following arrangement: centromere – Lap-1 – (1.2 ± 0.8) – Es-17 – (3.0 ± 1.0) – Ups – (1.3 ± 0.7) – Alp-1 – (23.1 ± 3.4) – Mod-1 – (10.9 ± 2.6) – Acy-1 . This study provides the first estimate of the distances between Es-17, Ups and Alp-1. Exceptions to the preferred association of alleles of Es-17 and Ups have been found in three feral populations and one inbred strain. Evidence is presented for the homology of this chromosome region with the ESA4 – UPS – APO-AI region on the long arm of human chromosome 11.     

Genetic and Physical Mapping of the Dreher Locus on Mouse Chromosome 1

Mutations in the mouse dreher (dr) gene cause skeletal defects, hyperactivity, abnormal gait, deafness, white belly spotting, and hypoplasia of Müllerian duct derivatives. To map dr to high resolution, we utilized two crosses. Initially, we analyzed an intersubspecific intercross to construct a detailed genetic map of simple sequence length polymorphism markers within a 6.3-cM region surrounding the dr locus. Subsequently, we analyzed a second intersubspecific intercross segregating for the dr(6J) allele, which positioned dr within a 0.13-cM region between Rxrg and D1Mit370. A physical contig of BAC clones spanning the dr critical region was constructed, and eight potential dr candidate genes were excluded by genetic or physical mapping. Together these results lay the foundation for positional cloning of the dr gene.     

Genetic and Molecular Mapping of Chromosome Region 85a in Drosophila Melanogaster

Chromosome region 85A contains at least 12 genetic complementation groups, including the genes dhod, pink and hunchback. In order to better understand the organization of this chromosomal segment and to permit molecular studies of these genes, we have carried out a genetic analysis coupled with a chromosome walk to isolate the DNA containing these genes. Complementation tests with chromosomal deficiencies permitted unambiguous ordering of most of the complementation groups identified within the 85A region. Recombinant bacteriophage clones were isolated that collectively span over 120 kb of 85A DNA and these were used to produce a molecular map of the region. The breakpoint sites of a number of 85A chromosome rearrangements were localized on the molecular map, thereby delimiting regions of the DNA that contain the various genetic complementation groups.     

Normal Testis Determination in the Mouse Depends on Genetic Interaction of a Locus on Chromosome 17 and the Y Chromosome

We previously described a locus on chromosome (Chr) 17 of the mouse that is critical for normal testis development. This locus was designated "T-associated sex reversal" (Tas) because it segregated with the dominant brachyury allele hairpin tail (Thp) and caused gonads of C57BL/6J XY, Thp/+ individuals to develop as ovaries or ovotestes rather than as testes. To clarify the inheritance of Tas, we investigated the effects of T-Orleans (TOrl), another brachyury mutation, on gonad development. We found that gonads of C57BL/6J XY, Thp/+ and TOrl/+ mice develop ovarian tissue if the Y chromosome is derived from the AKR/J inbred strain, whereas normal testicular development occurs in the presence of a Y chromosome derived from the C57BL/6J inbred strain. From these observations we conclude that: (1) Tas is located in a region on Chr 17 common to the deletions associated with Thp, and TOrl, and (2) the Y-linked testis determining gene, Tdy, carried by the AKR/J inbred strain differs from that of the C57BL/6J inbred strain. We suggest that in mammals Tdy is not the sole testis determinant because autosomal loci must be genetically compatible with Tdy for normal testicular development.     

Fine Mapping of a GWAS-Derived Obesity Candidate Region on Chromosome 16p11.2

IntroductionLarge-scale genome-wide association studies (GWASs) have identified 97 chromosomal loci associated with increased body mass index in population-based studies on adults. One of these SNPs, rs7359397, tags a large region (approx. 1MB) with high linkage disequilibrium (r²>0.7), which comprises five genes (SH2B1, APOBR, sulfotransferases: SULT1A1 and SULT1A2, TUFM). We had previously described a rare mutation in SH2B1 solely identified in extremely obese individuals but not in lean controls.MethodsThe coding regions of the genes APOBR, SULT1A1, SULT1A2, and TUFM were screened for mutations (dHPLC, SSCP, Sanger re-sequencing) in 95 extremely obese children and adolescents. Detected non-synonymous variants were genotyped (TaqMan SNP Genotyping, MALDI TOF, PCR-RFLP) in independent large study groups (up to 3,210 extremely obese/overweight cases, 485 lean controls and 615 obesity trios). In silico tools were used for the prediction of potential functional effects of detected variants.ResultsExcept for TUFM we detected non-synonymous variants in all screened genes. Two polymorphisms rs180743 (APOBR p.Pro428Ala) and rs3833080 (APOBR p.Gly369_Asp370del9) showed nominal association to (extreme) obesity (uncorrected p = 0.003 and p = 0.002, respectively). In silico analyses predicted a functional implication for rs180743 (APOBR p.Pro428Ala). Both APOBR variants are located in the repetitive region with unknown function.ConclusionVariants in APOBR contributed as strongly as variants in SH2B1 to the association with extreme obesity in the chromosomal region chr16p11.2. In silico analyses implied no functional effect of several of the detected variants. Further in vitro or in vivo analyses on the functional implications of the obesity associated variants are warranted.     

Phenobarbital Induction of Aldehyde Dehydrogenase Type 2 mRNA in Mouse Liver: A Candidate Region on Chromosome 7 for a Putative Regulatory Gene

Phenobarbital (PB) strongly induces in the liver the expression of many genes encoding detoxication enzymes, such as the aldehyde dehydrogenase type 2 in the mouse (Aldh2). With the aim of identifying genes involved in this response, we have undertaken an approach based on a genetic analysis in mice. In a previous report, the genetic analysis of both the C57BL/6J (B6) x DBA/2J (D2) F1 and the (F1 x F1) F2 led us to the hypothesis that Aldh2 responsiveness to PB was under the control of one major locus independent of the structural gene. In the present study, the genetic analysis of the inducibility by PB of Aldh2 in the backcross population B6D2F1 x D2 has allowed us to confirm the involvement of a major regulatory gene in this mechanism. By searching for genetic linkage between this locus and a series of microsatellites DNA markers, we obtained indicative evidence for a region on chromosome 7, which may carry this gene.     

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

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