Project description:BACKGROUND: Due to their high level of genotypic and phenotypic variability, Mus spretus strains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci. Mus spretus diverged from Mus musculus around 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP) in every 100 base pairs when compared with any of the classical laboratory strains. A genoproteomic approach was used to assess polymorphism of the major milk proteins between SEG/Pas and C57BL/6J, two inbred strains of mice representative of Mus spretus and Mus musculus species, respectively. RESULTS: The milk protein concentration was dramatically reduced in the SEG/Pas strain by comparison with the C57BL/6J strain (34 ± 9 g/L vs. 125 ± 12 g/L, respectively). Nine major proteins were identified in both milks using RP-HPLC, bi-dimensional electrophoresis and MALDI-Tof mass spectrometry. Two caseins (? and ?s1) and the whey acidic protein (WAP), showed distinct chromatographic and electrophoresis behaviours. These differences were partly explained by the occurrence of amino acid substitutions and splicing variants revealed by cDNA sequencing. A total of 34 SNPs were identified in the coding and 3'untranslated regions of the SEG/Pas Csn1s1 (11), Csn2 (7) and Wap (8) genes. In addition, a 3 nucleotide deletion leading to the loss of a serine residue at position 93 was found in the SEG/Pas Wap gene. CONCLUSION: SNP frequencies found in three milk protein-encoding genes between Mus spretus and Mus musculus is twice the values previously reported at the whole genome level. However, the protein structure and post-translational modifications seem not to be affected by SNPs characterized in our study. Splicing mechanisms (cryptic splice site usage, exon skipping, error-prone junction sequence), already identified in casein genes from other species, likely explain the existence of multiple ?s1-casein isoforms both in SEG/Pas and C57BL/6J strains. Finally, we propose a possible mechanism by which the hallmark tandem duplication of a 18-nt exon (14 copies) may have occurred in the mouse genome.
Project description:Copy number variation is an important dimension of genetic diversity and has implications in development and disease. As an important model organism, the mouse is a prime candidate for copy number variant (CNV) characterization, but this has yet to be completed for a large sample size. Here we report CNV analysis of publicly available, high-density microarray data files for 351 mouse tail samples, including 290 mice that had not been characterized for CNVs previously.We found 9634 putative autosomal CNVs across the samples affecting 6.87% of the mouse reference genome. We find significant differences in the degree of CNV uniqueness (single sample occurrence) and the nature of CNV-gene overlap between wild-caught mice and classical laboratory strains. CNV-gene overlap was associated with lipid metabolism, pheromone response and olfaction compared to immunity, carbohydrate metabolism and amino-acid metabolism for wild-caught mice and classical laboratory strains, respectively. Using two subspecies of wild-caught Mus musculus, we identified putative CNVs unique to those subspecies and show this diversity is better captured by wild-derived laboratory strains than by the classical laboratory strains. A total of 9 genic copy number variable regions (CNVRs) were selected for experimental confirmation by droplet digital PCR (ddPCR).The analysis we present is a comprehensive, genome-wide analysis of CNVs in Mus musculus, which increases the number of known variants in the species and will accelerate the identification of novel variants in future studies.
Project description:Rodent betaherpesviruses vary considerably in genomic content, and these variations can result in a distinct pathogenicity. Therefore, the identification of unknown betaherpesviruses in house mice (Mus musculus), the most important rodent host species in basic research, is of importance. During a search for novel herpesviruses in house mice using herpesvirus consensus PCR and attempts to isolate viruses in tissue culture, we identified a previously unknown betaherpesvirus. The primary PCR search in mouse organs revealed the presence of known strains of murine cytomegalovirus (Murid herpesvirus 1) and of Mus musculus rhadinovirus 1 only. However, the novel virus was detected after incubation of organ pieces in fibroblast tissue culture and subsequent PCR analysis of the supernatants. Long-distance PCR amplification including the DNA polymerase and glycoprotein B genes revealed a 3.4 kb sequence that was similar to sequences of rodent cytomegaloviruses. Pairwise sequence comparisons and phylogenetic analyses showed that this newly identified murine virus is most similar to the English isolate of rat cytomegalovirus, thereby raising the possibility that two distinct CMV lineages have evolved in both Mus musculus and Rattus norvegicus.
Project description:This experiment contains the subset of data corresponding to mouse RNA-Seq data from experiment E-GEOD-30352 (http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-30352/), which goal is to understand the dynamics of mammalian transcriptome evolution. To study mammalian transcriptome evolution at high resolution, we generated RNA-Seq data (?3.2 billion Illumina Genome Analyser IIx reads of 76 base pairs) for the polyadenylated RNA fraction of brain (cerebral cortex or whole brain without cerebellum), cerebellum, heart, kidney, liver and testis (usually from one male and one female per somatic tissue and two males for testis) from nine mammalian species: placental mammals (great apes, including humans; rhesus macaque; mouse), marsupials (gray short-tailed opossum) and monotremes (platypus). Corresponding data (?0.3 billion reads) were generated for a bird (red jungle fowl, a non-domesticated chicken) and used as an evolutionary outgroup.
Project description:BACKGROUND:Identification of protein-protein interactions is an important first step to understand living systems. High-throughput experimental approaches have accumulated large amount of information on protein-protein interactions in human and other model organisms. Such interaction information has been successfully transferred to other species, in which the experimental data are limited. However, the annotation transfer method could yield false positive interologs due to the lack of conservation of interactions when applied to phylogenetically distant organisms. RESULTS:To address this issue, we used phylogenetic profile method to filter false positives in interologs based on the notion that evolutionary conserved interactions show similar patterns of occurrence along the genomes. The approach was applied to Mus musculus, in which the experimentally identified interactions are limited. We first inferred the protein-protein interactions in Mus musculus by using two approaches: i) identifying mouse orthologs of interacting proteins (interologs) based on the experimental protein-protein interaction data from other organisms; and ii) analyzing frequency of mouse ortholog co-occurrence in predicted operons of bacteria. We then filtered possible false-positives in the predicted interactions using the phylogenetic profiles. We found that this filtering method significantly increased the frequency of interacting protein-pairs coexpressed in the same cells/tissues in gene expression omnibus (GEO) database as well as the frequency of interacting protein-pairs shared the similar Gene Ontology (GO) terms for biological processes and cellular localizations. The data supports the notion that phylogenetic profile helps to reduce the number of false positives in interologs. CONCLUSION:We have developed protein-protein interaction database in mouse, which contains 41109 interologs. We have also developed a web interface to facilitate the use of database http://lgsun.grc.nia.nih.gov/mppi/.
Project description:Mus musculus (Rodentia: Muridae) has generally been infected with a rodent hookworm Nippostrongylus brasiliensis. In this report, we present morphological and molecular identification of N. brasiliensis by light and scanning electron microscopy and PCR amplification of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the protein sequences encoded by cox1 gene, respectively. Despite the use of N. brasiliensis in many biochemistry studies from India, their taxonomic identification was not fully understood, especially at the species level, and no molecular data is available in GenBank from India. Sequence analysis of cox1 gene in this study revealed that the present specimen showed close identity with the same species available in GenBank, confirming that the species is N. brasiliensis. This study represents the first record of molecular identification of N. brasiliensis from India and the protein structure to better understand the comparative phylogenetic characteristics.
Project description:BACKGROUND:With the advent of the GeneChip Exon Arrays, it is now possible to extract "exon-level" expression estimates, allowing for detection of alternative splicing events, one of the primary mechanisms of transcript diversity. In the context of (1) a complex trait use case and (2) a human cerebellum vs. heart comparison on previously validated data, we present a transcript-based statistical model and validation framework to allow detection of alternative exon usage (AEU) between different groups. To illustrate the approach, we detect and confirm differences in exon usage in the two of the most widely studied mouse genetic models (the C57BL/6J and DBA/2J inbred strains) and in a human dataset. RESULTS:We developed a computational framework that consists of probe level annotation mapping and statistical modeling to detect putative AEU events, as well as visualization and alignment with known splice events. We show a dramatic improvement (?25 fold) in the ability to detect these events using the appropriate annotation and statistical model which is actually specified at the transcript level, as compared with the transcript cluster/gene-level annotation used on the array. An additional component of this workflow is a probe index that allows ranking AEU candidates for validation and can aid in identification of false positives due to single nucleotide polymorphisms. DISCUSSION:Our work highlights the importance of concordance between the functional unit interrogated (e.g., gene, transcripts) and the entity (e.g., exon, probeset) within the statistical model. The framework we present is broadly applicable to other platforms (including RNAseq).
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Overall design: Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.
Project description:Mouse apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 (mA3), an intracellular antiviral factor, has 2 allelic variations that are linked with different susceptibilities to beta- and gammaretrovirus infections among various mouse strains. In virus-resistant C57BL/6 (B6) mice, mA3 transcripts are more abundant than those in susceptible BALB/c mice both in the spleen and bone marrow. These strains of mice also express mA3 transcripts with different splicing patterns: B6 mice preferentially express exon 5-deficient (?5) mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the ?5 mRNA exerts stronger antiretroviral activities than the full-length protein, how exon 5 affects mA3 antiviral activity, as well as the genetic mechanisms regulating exon 5 inclusion into the mA3 transcripts, remains largely uncharacterized. Here we show that mA3 exon 5 is indeed a functional element that influences protein synthesis at a post-transcriptional level. We further employed in vitro splicing assays using genomic DNA clones to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and the single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary. Distribution of the above polymorphisms among different Mus species indicates that the inclusion of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of this exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function.