Mouse Genome Sequence Research Articles

Genome-wide end-sequenced BAC resources for the NOD/MrkTac☆ and NOD/ShiLtJ☆☆ mouse genomes

Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting β-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome, the reduced X chromosome coverage being due to the use of a male donor for this library. Clones from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841 single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29. These BAC libraries represent a powerful resource for functional studies, such as gene targeting in NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments.

Separating derived from ancestral features of mouse and human genomes

To take full advantage of the mouse as a model organism, it is essential to distinguish lineage-specific biology from what is shared between human and mouse. Investigations into shared genetic elements common to both have been well served by the draft human and mouse genome sequences. More recently, the virtually complete euchromatic sequences of the two reference genomes have been finished. These reveal a high ( approximately 5%) level of sequence duplications that had previously been recalcitrant to sequencing and assembly. Within these duplications lie large numbers of rodent- or primate-specific genes. In the present paper, we review the sequence properties of the two genomes, dwelling most on the duplications, deletions and insertions that separate each of them from their most recent common ancestor, approx. 90 million years ago. We consider the differences in gene numbers and repertoires between the two species, and speculate on their contributions to lineage-specific biology. Loss of ancient single-copy genes are rare, as are gains of new functional genes through retrotransposition. Instead, most changes to the gene repertoire have occurred in large multicopy families. It has been proposed that numbers of such 'environmental genes' rise and fall, and their sequences change, as adaptive responses to infection and other environmental pressures, including conspecific competition. Nevertheless, many such genes may be under little or no selection.

Where's the Mouse Info?

The laboratory mouse has long been the species of choice for many animal models of human diseases and for basic research areas (http:// jaxmice.jax.org/research/index.html). In part, this is because of the long-standing genetic resources available for the mouse, but now, also, the development of powerful new techniques for generation of mutations, analysis of complex traits, and high throughput data gathering have made the mouse the preeminent model for the study of human biology and disease. 34 With the completion of the mouse genome sequence, it is now possible to rapidly generate knockouts and knock-in mutations anywhere in the genome, either constitutively or conditionally by using Cre- and Flp-mediated recombination, alterations in gene, or locus copy number. Complex trait analysis now permits the underlying spontaneous genetic predisposition to multigenic diseases to be elucidated. The increased sophistication and miniaturization of physiologic monitoring and diagnostic instrumentation mean that the depth of phenotype data that can be collected is unprecedented. Parallel with this rise is the burgeoning need for ‘‘mouse pathologists,’’ veterinary or medical pathologists who can interpret the histopathologic changes that occur primarily in genetically engineered inbred strains of mice, 8,36 an important aspect of mutant characterization generally referred to as ‘‘phenotyping’’ by molecular biologists and others. Both veterinary pathologists and medical pathologists are capable of performing these functions because of their basic training in mammalian anatomy, normal histology, and pathology. Many pathologists are hesitant to undertake mouse pathology phenotyping because they did not obtain specific training in mouse biology and pathology (although a number of specialized courses are available in the USA and elsewhere) 41 or they are insecure with the fundamental anatomical differences between mice and the domestic mammals and humans with which they are familiar. Anatomic, phenotyping, and both generalized and specific pathology information can be found in many books and review articles* or, by using the modern approach, a great deal of this information is actually online 6 or can be searched for in PubMed. The major topics of interest to

High-throughput insertional mutagenesis screens in mice to identify oncogenic networks

Retroviral insertional mutagenesis screens have been used for many years as a tool for cancer gene discovery. In recent years, completion of the mouse genome sequence as well as improved technologies for cloning and sequencing of retroviral insertions have greatly facilitated the retrieval of more complete data sets from these screens. The concomitant increase of the size of the screens allows researchers to address new questions about the genes and signalling networks involved in tumour development. In addition, the development of new insertional mutagenesis tools such as DNA transposons enables screens for cancer genes in tissues that previously could not be analysed by retroviral insertional mutagenesis.

An Alternatively Spliced Isoform of Non-muscle Myosin II-C Is Not Regulated by Myosin Light Chain Phosphorylation

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC(20)) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC(20) phosphorylation for both actin-activated MgATPase activity and in vitro motility ( Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775 ). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC(20) phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.

The Toca-1-N-WASP Complex Links Filopodial Formation to Endocytosis

The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15Delta95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis.

Analysis of DNA Methylation at the Human Alpha Globin Cluster during Hematopoiesis.

The analysis of globin gene regulation has elucidated many of the principles underlying mammalian gene expression. To understand how the epigenetic program that unfolds during hematopoiesis impacts on alpha (α)-globin gene expression, we analyzed DNA methylation at over 200 CpG dinucleotides in 130 kb around the human α-globin gene cluster focussing particularly on a region (12 kb) covering the α2 and α1-globin genes (HBA2 and HBA1), and the upstream regulatory sites (DNAse I hypersensitive sites (HS) -48, HS-40, HS-33, and HS-10); so-called multi-species conserved sequencesregulatory element1-4 (MCS-R1-4). Cultures of primary adult erythroid cells (at three stages: early, intermediate, and late) were analyzed together with non-erythroid human cells (neutrophils, peripheral blood mononuclear cells, and a human embryonic stem (ES) cell line). Using a recently published method involving bisulphite modification and MALDI-TOF Mass Spectrometry we quantified the level of DNA methylation across the alpha globin cluster. Most sequences outside of the CpG islands were methylated. However, the DNA methylation levels between erythroid and non-erythroid cells differed at regulatory elements. MCS-R1 contained only 1 CpG site and the average percentage of DNA methylation for erythroid and non-erythroid was 8% and 40%, respectively. While they were 8% vs. 42% (p=0.428), 11% vs. 99% (p=0.054), and 14% vs. 97% (p=0.001) in MCS-R2 to 4, respectively. To study changes in DNA methylation in a comprehensive, developmental and tissue-specific manner, we used a humanized-mouse model, in which the conserved α-globin syntenic region of mouse genomic sequence is replaced by that of human. This contains all cis-acting sequences required for fully regulated expression of the α-globin genes. Although the alpha globin cluster is expressed efficiently in this model, DNA methylation patterns differed in the 20 kb region encompassing the α-globin genes when comparing primary human cells and primary humanized cells. DNA in humanized cells were relatively hypomethylated both in erythroid and non-erythroid tissues. The methylation levels were higher in testis of the humanized mouse than in other tissues but still not as high as those in normal human non-erythroid cells. The cause of these differences is being investigated. Despite these differences, the humanized-mouse mimicked the patterns of methylation found in regulatory elements in erythroid and nonerythroid cells. This study shows that despite appropriate regulation of gene expression, epigenetic templating may differ between species (human and mouse). DNA methylation at the upstream regulatory elements might be involved in the regulation of α-globin gene expression during erythropoiesis, although it is equally possible that these changes in methylation are generated by passive demethylation secondary to transcription factor binding in erythroid cells. The mechanism(s) underlying demethylation of regulatory elements during differentiation remains to be clarified.

Differences in DBA/1J and DBA/2J reveal lipid QTL genes

Recent advances in mouse genomics have revealed considerable variation in the form of single-nucleotide polymorphisms (SNPs) among common inbred strains. This has made it possible to characterize closely related strains and to identify genes that differ; such genes may be causal for quantitative phenotypes. The mouse strains DBA/1J and DBA/2J differ by just 5.6% at the SNP level. These strains exhibit differences in a number of metabolic and lipid phenotypes, such as plasma levels of triglycerides (TGs) and HDL. A cross between these strains revealed multiple quantitative trait loci (QTLs) in 294 progeny. We identified significant TG QTLs on chromosomes (Chrs) 1, 2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 16, and 19, and significant HDL QTLs on Chrs 3, 9, and 16. Some QTLs mapped to chromosomes with limited variability between the two strains, thus facilitating the identification of candidate genes. We suggest that Tshr is the QTL gene for Chr 12 TG and HDL levels and that Ihh may account for the TG QTL on Chr 1. This cross highlights the advantage of crossing closely related strains for subsequent identification of QTL genes.

Identification of cis-acting promoter sequences required for expression of the glycerol-3-phosphate acyltransferase 1 gene in mice

Glycerol-3-phosphate acyltransferase 1 (GPAT1) is a rate limiting enzyme in de novo glycerophospholipid synthesis. The murine GPAT1 promoter sequence (the “classical” sequence) was reported previously. However, the organization of this DNA sequence does not fully match the mouse genome sequences on NCBI/GenBank. Here we have identified net cis-acting promoter sequences for the mouse GPAT1 gene: promoter 1a which includes part of the classical sequence and the downstream promoter 1b. Promoter 1a facilitates transcription of two alternative GPAT1 transcript variants, GPAT1-V1 and V2, while promoter 1b produces a third transcript variant, GPAT1-V3. Upstream stimulating factor-1 (USF-1) controlled both promoters whereas sterol regulatory element-binding protein-1 (SREBP-1) exclusively regulated promoter 1a activity in vitro. Feeding increased GPAT1-V1 and V2, but not V3 mRNA levels in mouse liver. The obese condition of db/ db mice did not alter the hepatic expression levels of any of the three GPAT1 variants. Feeding enhanced hepatic mRNA levels, intranuclear protein levels and promoter 1a-binding levels of SREBP-1, but not of USF-1. Thus, promoter 1a was exclusively activated by routine feeding in vivo. Our results indicate differential roles of the two promoters in the regulation of hepatic GPAT1 gene expression in mice.

New insights into SRY regulation through identification of 5' conserved sequences

BackgroundSRY is the pivotal gene initiating male sex determination in most mammals, but how its expression is regulated is still not understood. In this study we derived novel SRY 5' flanking genomic sequence data from bovine and caprine genomic BAC clones.ResultsWe identified four intervals of high homology upstream of SRY by comparison of human, bovine, pig, goat and mouse genomic sequences. These conserved regions contain putative binding sites for a large number of known transcription factor families, including several that have been implicated previously in sex determination and early gonadal development.ConclusionOur results reveal potentially important SRY regulatory elements, mutations in which might underlie cases of idiopathic human XY sex reversal.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Genetic and Molecular Basis of QTL of Diabetes in Mouse: Genes and Polymorphisms

A systematic study has been conducted of all available reports in PubMed and OMIM (Online Mendelian Inheritance in Man) to examine the genetic and molecular basis of quantitative genetic loci (QTL) of diabetes with the main focus on genes and polymorphisms. The major question is, What can the QTL tell us? Specifically, we want to know whether those genome regions differ from other regions in terms of genes relevant to diabetes. Which genes are within those QTL regions, and, among them, which genes have already been linked to diabetes? whether more polymorphisms have been associated with diabetes in the QTL regions than in the non-QTL regions.Our search revealed a total of 9038 genes from 26 type 1 diabetes QTL, which cover 667,096,006 bp of the mouse genomic sequence. On one hand, a large number of candidate genes are in each of these QTL; on the other hand, we found that some obvious candidate genes of QTL have not yet been investigated. Thus, the comprehensive search of candidate genes for known QTL may provide unexpected benefit for identifying QTL genes for diabetes.

A race through the maze of genomic evidence

A race through the maze of genomic evidence

Estrogen Receptor Regulation of Carbonic Anhydrase XII through a Distal Enhancer in Breast Cancer

The expression of carbonic anhydrase XII (CA12), a gene that encodes a zinc metalloenzyme responsible for acidification of the microenvironment of cancer cells, is highly correlated with estrogen receptor alpha (ER alpha) in human breast tumors. Here, we show that CA12 is robustly regulated by estrogen via ER alpha in breast cancer cells, and that this regulation involves a distal estrogen-responsive enhancer region. Upon the addition of estradiol, ER alpha binds directly to this distal enhancer in vivo, resulting in the recruitment of RNA polymerase II and steroid receptor coactivators SRC-2 and SRC-3, and changes in histone acetylation. Mutagenesis of an imperfect estrogen-responsive element within this enhancer region abolishes estrogen-dependent activity, and chromosome conformation capture and chromatin immunoprecipitation assays show that this distal enhancer communicates with the transcriptional start site of the CA12 gene via intrachromosomal looping upon hormone treatment. This distal enhancer element is observed in the homologous mouse genomic sequence, and the expression of the mouse homologue, Car12, is rapidly and robustly stimulated by estradiol in the mouse uterus in vivo, suggesting that the ER regulation of CA12 is mechanistically and evolutionarily conserved. Our findings highlight the crucial role of ER in the regulation of the CA12 gene, and provide insight into the transcriptional regulatory mechanism that accounts for the strong association of CA12 and ER in human breast cancers.

Unusually rapid evolution of Neuroligin-4 in mice

Neuroligins (NLs) are postsynaptic cell-adhesion molecules that are implicated in humans in autism spectrum disorders because the genes encoding NL3 and NL4 are mutated in rare cases of familial autism. NLs are highly conserved evolutionarily, except that no NL4 was detected in the currently available mouse genome sequence assemblies. We now demonstrate that mice express a distant NL4 variant that rapidly evolved from other mammalian NL4 genes and that exhibits sequence variations even between different mouse strains. Despite its divergence, mouse NL4 binds neurexins and is transported into dendritic spines, suggesting that the core properties of NLs are retained in this divergent NL isoform. The selectively rapid evolution of NL4 in mice suggests that its function in the brain is under less stringent control than that of other NLs, shedding light on why its mutation in autism spectrum disorder patients is not lethal, but instead leads to a discrete developmental brain disorder.

Molecular cloning and structural organization of the gene encoding the mouse lysosomal di- N-acetylchitobiase ( ctbs)

The lysosomal enzyme di- N-acetylchitobiase hydrolyzes N-acetylglucosamine from the reducing-end of the N, N′ diacetylchitobiose core of N-linked-oligosaccharides. The presence of chitobiase in the tissues of different species is probably responsible for differences in the structure of oligosaccharides accumulated in the lysosomal storage disease beta-mannosidosis. The disease has so far been described in humans, cats, cattle and goats. Low chitobiase activity has been observed in the tissues of ruminants and it has been hypothesized that in cattle this low level of expression is due to evolutionary changes in the promoter region. A cDNA encoding the mouse chitobiase has been isolated, sequenced and its identity confirmed by expression in COS-7 cells. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of seven exons within the chitobiase gene. The gene spans about 15 kb and a single transcription initiation site was determined by 5′RACE. Chitobiase is differentially and ubiquitously expressed in mouse tissues as demonstrated by qRT-PCR analysis. Chitobiase is differentially expressed at lower levels in bovine tissues. In two bovine tissues (heart and muscle) mRNA was not detectable. Mouse and bovine promoters have been isolated and sequenced and their activities compared. The activity of the bovine promoter is very low and might explain the low activity of chitobiase observed in cattle.

Rapid bursts of androgen-binding protein (Abp) gene duplication occurred independently in diverse mammals.

BackgroundThe draft mouse (Mus musculus) genome sequence revealed an unexpected proliferation of gene duplicates encoding a family of secretoglobin proteins including the androgen-binding protein (ABP) α, β and γ subunits. Further investigation of 14 α-like (Abpa) and 13 β- or γ-like (Abpbg) undisrupted gene sequences revealed a rich diversity of developmental stage-, sex- and tissue-specific expression. Despite these studies, our understanding of the evolution of this gene family remains incomplete. Questions arise from imperfections in the initial mouse genome assembly and a dearth of information about the gene family structure in other rodents and mammals.ResultsHere, we interrogate the latest 'finished' mouse (Mus musculus) genome sequence assembly to show that the Abp gene repertoire is, in fact, twice as large as reported previously, with 30 Abpa and 34 Abpbg genes and pseudogenes. All of these have arisen since the last common ancestor with rat (Rattus norvegicus). We then demonstrate, by sequencing homologs from species within the Mus genus, that this burst of gene duplication occurred very recently, within the past seven million years. Finally, we survey Abp orthologs in genomes from across the mammalian clade and show that bursts of Abp gene duplications are not specific to the murid rodents; they also occurred recently in the lagomorph (rabbit, Oryctolagus cuniculus) and ruminant (cattle, Bos taurus) lineages, although not in other mammalian taxa.ConclusionWe conclude that Abp genes have undergone repeated bursts of gene duplication and adaptive sequence diversification driven by these genes' participation in chemosensation and/or sexual identification.

Cloning and characterization of the promoter of Hugl-2, the human homologue of Drosophila lethal giant larvae ( lgl) polarity gene

The human lgl gene, Hugl-2 ( llgl2, Lgl2), codes for a cytoskeletal protein involved in regulating cell polarity. Here, we report the identification and functional characterization of the promoter region (∼1.2 kb) of the Hugl-2 gene. Luciferase expression assays show a high basal Hugl-2 promoter activity in different cell lines and primary human hepatocytes. Truncations of the promoter identified a GC-rich region important for this activity. Alignment of human and mouse genomic sequences demonstrate that this is an evolutionary conserved region fcontaining putative binding sites for several transcription factors including Elk-1 and Sp-1. Mithramycin A reduces Hugl-2 expression indicating Sp-1 transcription factors activate Hugl-2. Treatment of primary hepatocytes with epidermal growth factor (EGF) suppresses Hugl-2, suggesting regulation by the EGF-signaling pathway. Downregulation of Hugl-2 by EGF may contribute to loss of cell polarity and tumour progression, therefore supporting a tumour suppressor role for Hugl-2.

The vertebrate genome annotation (Vega) database

The Vertebrate Genome Annotation (Vega) database (http://vega.sanger.ac.uk) was first made public in 2004 and has been designed to view manual annotation of human, mouse and zebrafish genomic sequences produced at the Wellcome Trust Sanger Institute. Since its initial release, the number of human annotated loci has more than doubled to close to 33 000 and now contains comprehensive annotation on 20 of the 24 human chromosomes, four whole mouse chromosomes and around 40% of the zebrafish Danio rerio genome. In addition, we offer manual annotation of a number of haplotype regions in mouse and human and regions of comparative interest in pig and dog that are unique to Vega.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Mitochondrial Lipid Metabolism and Function Resulting in a Deficient Mitochondrial Bioenergetic Phenotype

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.