Dynamic instability of the major urinary protein gene family revealed by genomic and phenotypic comparisons between C57 and 129 strain mice

Targeted sequencing, manual genome annotation, phylogenetic analysis and mass spectrometry were used to characterise major urinary proteins (MUPs) and the Mup clusters of two strains of inbred mice

Pseudo–Messenger RNA: Phantoms of the Transcriptome

The mammalian transcriptome harbours shadowy entities that resist classification and analysis. In analogy with pseudogenes, we define pseudo–messenger RNA to be RNA molecules that resemble protein-coding mRNA, but cannot encode full-length proteins owing to disruptions of the reading frame. Using a rigorous computational pipeline, which rules out sequencing errors, we identify 10,679 pseudo–messenger RNAs (approximately half of which are transposon-associated) among the 102,801 FANTOM3 mouse cDNAs: just over 10% of the FANTOM3 transcriptome. These comprise not only transcribed pseudogenes, but also disrupted splice variants of otherwise protein-coding genes. Some may encode truncated proteins, only a minority of which appear subject to nonsense-mediated decay. The presence of an excess of transcripts whose only disruptions are opal stop codons suggests that there are more selenoproteins than currently estimated. We also describe compensatory frameshifts, where a segment of the gene has changed frame but remains translatable. In summary, we survey a large class of non-standard but potentially functional transcripts that are likely to encode genetic information and effect biological processes in novel ways. Many of these transcripts do not correspond cleanly to any identifiable object in the genome, implying fundamental limits to the goal of annotating all functional elements at the genome sequence level

Harmonizing model organism data in the Alliance of Genome Resources.

The Alliance of Genome Resources (the Alliance) is a combined effort of 7 knowledgebase projects: Saccharomyces Genome Database, WormBase, FlyBase, Mouse Genome Database, the Zebrafish Information Network, Rat Genome Database, and the Gene Ontology Resource. The Alliance seeks to provide several benefits: better service to the various communities served by these projects; a harmonized view of data for all biomedical researchers, bioinformaticians, clinicians, and students; and a more sustainable infrastructure. The Alliance has harmonized cross-organism data to provide useful comparative views of gene function, gene expression, and human disease relevance. The basis of the comparative views is shared calls of orthology relationships and the use of common ontologies. The key types of data are alleles and variants, gene function based on gene ontology annotations, phenotypes, association to human disease, gene expression, protein-protein and genetic interactions, and participation in pathways. The information is presented on uniform gene pages that allow facile summarization of information about each gene in each of the 7 organisms covered (budding yeast, roundworm Caenorhabditis elegans, fruit fly, house mouse, zebrafish, brown rat, and human). The harmonized knowledge is freely available on the alliancegenome.org portal, as downloadable files, and by APIs. We expect other existing and emerging knowledge bases to join in the effort to provide the union of useful data and features that each knowledge base currently provides

Genetic Analysis of Completely Sequenced Disease-Associated MHC Haplotypes Identifies Shuffling of Segments in Recent Human History

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II–related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR–DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations

Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing

Structural and functional annotation of the porcine immunome

Background: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.[br/] Results: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.[br/] Conclusions: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue-like organization in primary osteoblast cultures

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue-like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue-like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H-thymidine labelling and autoradiography, indicate that greater than 98% of the pre-confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down-regulated cell growth associated genes. Post-proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial-derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue-like organization

Sequencing and characterization of the FVB/NJ mouse genome.

ABSTRACT: BACKGROUND: The FVB/NJ mouse strain has its origins in a colony of outbred Swiss mice established in 1935 at the National Institutes of Health. Mice derived from this source were selectively bred for sensitivity to histamine diphosphate and the B strain of Friend leukemia virus. This led to the establishment of the FVB/N inbred strain, which was subsequently imported to the Jackson Laboratory and designated FVB/NJ. The FVB/NJ mouse has several distinct characteristics, such as large pronuclear morphology, vigorous reproductive performance, and consistently large litters that make it highly desirable for transgenic strain production and general purpose use. RESULTS: Using next-generation sequencing technology, we have sequenced the genome of FVB/NJ to approximately 50-fold coverage, and have generated a comprehensive catalog of single nucleotide polymorphisms, small insertion/deletion polymorphisms, and structural variants, relative to the reference C57BL/6J genome. We have examined a previously identified quantitative trait locus for atherosclerosis susceptibility on chromosome 10 and identify several previously unknown candidate causal variants. CONCLUSION: The sequencing of the FVB/NJ genome and generation of this catalog has increased the number of known variant sites in FVB/NJ by a factor of four, and will help accelerate the identification of the precise molecular variants that are responsible for phenotypes observed in this widely used strain