The annotation of both human and mouse kinomes in UniProtKB/Swiss-Prot: one small step in manual annotation, one giant leap for full comprehension of genomes

Biomolecule phosphorylation by protein kinases is a fundamental cell signaling process in all living cells. Following the comprehensive cataloguing of the protein kinase complement of the human genome (Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science 298, 1912-1934), this review will detail the state-of-the-art human and mouse kinase proteomes as provided in the UniProtKB/Swiss-Prot protein knowledgebase. The sequences of the 480 classical and up to 24 atypical protein kinases now believed to exist in the human genome and 484 classical and up to 24 atypical kinases within the mouse genome have been reviewed and, where necessary, revised. Extensive annotation has been added to each entry. In an era when a wealth of new databases is emerging on the Internet, UniProtKB/Swiss-Prot makes available to the scientific community the most up-to-date and in-depth annotation of these proteins with access to additional external resources linked from within each entry. Incorrect sequence annotations resulting from errors and artifacts have been eliminated. Each entry will be constantly reviewed and updated as new information becomes available with the orthologous enzymes in related species being annotated in a parallel effort and complete kinomes being completed as sequences become available. This ensures that the mammalian kinomes available from UniProtKB/Swiss-Prot are of a consistently high standard with each separate entry acting both as a valuable information resource and a central portal to a wealth of further detail via extensive cross-referencing.     

Ensembl 2002: accommodating comparative genomics

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of human, mouse and other genome sequences, available as either an interactive web site or as flat files. Ensembl also integrates manually annotated gene structures from external sources where available. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. These range from sequence analysis to data storage and visualisation and installations exist around the world in both companies and at academic sites. With both human and mouse genome sequences available and more vertebrate sequences to follow, many of the recent developments in Ensembl have focusing on developing automatic comparative genome analysis and visualisation.     

Strong nucleosomes of mouse genome including recovered centromeric sequences

Recently discovered strong nucleosomes (SNs) characterized by visibly periodical DNA sequences have been found to concentrate in centromeres of Arabidopsis thaliana and in transient meiotic centromeres of Caenorhabditis elegans. To find out whether such affiliation of SNs to centromeres is a more general phenomenon, we studied SNs of the Mus musculus. The publicly available genome sequences of mouse, as well as of practically all other eukaryotes do not include the centromere regions which are difficult to assemble because of a large amount of repeat sequences in the centromeres and pericentromeric regions. We recovered those missing sequences using the data from MNase-seq experiments in mouse embryonic stem cells, where the sequence of DNA inside nucleosomes, including missing regions, was determined by 100-bp paired-end sequencing. Those nucleosome sequences, which are not matching to the published genome sequence, would largely belong to the centromeres. By evaluating SN densities in centromeres and in non-centromeric regions, we conclude that mouse SNs concentrate in the centromeres of telocentric mouse chromosomes, with ~3.9 times excess compared to their density in the rest of the genome. The remaining non-centromeric SNs are harbored mainly by introns and intergenic regions, by retro-transposons, in particular. The centromeric involvement of the SNs opens new horizons for the chromosome and centromere structure studies.     

Predicting the Proportion of Essential Genes in Mouse Duplicates Based on Biased Mouse Knockout Genes

In the yeast or nematode, the proportion of essential genes in duplicates is lower than in singletons (single-copy genes), due to the functional redundancy. One may expect that it should be the same in the mouse genome. However, based on the publicly available mouse knockout data, it was observed that the proportion of essential genes in duplicates is similar to that in singletons. The most straightforward interpretation, as claimed in a recent study, is that duplicate genes may have a negligible role in the mouse genetic robustness. Here we show that in the current mouse knockout dataset, recently duplicated genes have been highly underrepresented, leading to an overestimation of the proportion of essential genes in duplicates. After estimating the duplication time of mouse duplication events, we have developed a simple bias-correcting procedure and shown that the bias-corrected proportion of essential genes in mouse duplicates is significantly lower than that in singletons.     

Now and future of mouse mutagenesis for human disease models

One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse (Mus musculus) is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to de-velop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea (ENU) has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions (epistasis) have been challenged with mutant mice. In addition, chromosomal substitution swains and collaborative cross strains are also available to elucidate the complex Waits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human.     

Next-generation sequencing of experimental mouse strains

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5?years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.     

Analysis of segmental duplications reveals a distinct pattern of continuation-of-synteny between human and mouse genomes

About 5% of the human genome consists of large-scale duplicated segments of almost identical sequences. Segmental duplications (SDs) have been proposed to be involved in non-allelic homologous recombination leading to recurrent genomic variation and disease. It has also been suggested that these SDs are associated with syntenic rearrangements that have shaped the human genome. We have analyzed 14 members of a single family of closely related SDs in the human genome, some of which are associated with common inversion polymorphisms at chromosomes 8p23 and 4p16. Comparative analysis with the mouse genome revealed syntenic inversions for these two human polymorphic loci. In addition, 12 of the 14 SDs, while absent in the mouse genome, occur at the breaks of synteny; suggesting a non-random involvement of these sequences in genome evolution. Furthermore, we observed a syntenic familial relationship between 8 and 12 breakpoint-loci, where broken synteny that ends at one family member resumes at another, even across different chromosomes. Subsequent genome-wide assessment revealed that this relationship, which we named continuation-of-synteny, is not limited to the 8p23 family and occurs 46 times in the human genome with high frequency at specific chromosomes. Our analysis supports a non-random breakage model of genomic evolution with an active involvement of segmental duplications for specific regions of the human genome. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos

Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.     

A catalog of nonsynonymous polymorphism on mouse Chromosome 16

Numerous phenotypic traits differ among inbred mice, and the genetic diversity of inbred strains has been exploited in studies of quantitative trait loci (QTL). Sequencing the mouse genome has resulted in improved tools for the study of QTL, but a comprehensive catalog of sequence variants between strains would be of great value in identifying and testing potentially causative alleles. A/J DNA was included in the Celera shotgun sequence of the mouse genome and C57BL/6 DNA was sequenced by an international consortium. We have resequenced A/J and B6 DNA to cover nearly all of the protein-coding portions of mouse Chromosome 16, revealing that there are 106 nonsynonymous substitutions in 74 of the 779 genes on the chromosome. The pattern of substitution is more similar to the spectrum of benign polymorphism in the human population than it is to human disease-causing mutations. In mouse, polymorphic variants tend to be associated with one another on large haplotypes; this pattern also holds true for nonsynonymous polymorphism. However, sufficient fragmentation of haplotypes is present to suggest that only a very-high-resolution haplotype map will enable effective inference of alleles in additional strains. SNP data have been submitted to dbSNP with ssid No. 46531525-46532013.     

A proposal to sequence the genome of a garter snake (Thamnophis sirtalis)

Here we develop an argument in support of sequencing a garter snake (Thamnophis sirtalis) genome, and outline a plan to accomplish this. This snake is a common, widespread, nonvenomous North American species that has served as a model for diverse studies in evolutionary biology, physiology, genomics, behavior and coevolution. The anole lizard is currently the only genome sequence available for a non-avian reptile. Thus, the garter snake at this time would be the first available snake genome sequence and as such would provide much needed comparative representation of non-avian reptilian genomes, and would also allow critical new insights for vertebrate comparative genomic studies. We outline the major areas of discovery that the availability of the garter snake genome would enable, and describe a plan for whole-genome sequencing.     

Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.

We established a mouse Ltk- cell line that contains within its genome a herpes simplex virus thymidine kinase gene (tk) that had been disrupted by the insertion of the recognition sequence for yeast endonuclease I-SceI. The artificially introduced 18 bp I-SceI recognition sequence was likely a unique sequence in the genome of the mouse cell line. To assess whether an induced double-strand break (DSB) in the genomic tk gene would be repaired preferentially by gene targeting or non-homologous recombination, we electroporated the mouse cell line with endonuclease I-SceI alone, one of two different gene targeting constructs alone, or with I-SceI in conjunction with each of the two targeting constructs. Each targeting construct was, in principle, capable of correcting the defective genomic tk sequence via homologous recombination. tk+ colonies were recovered following electroporation of cells with I-SceI in the presence or absence of a targeting construct. Through the detection of small deletions at the I-SceI recognition sequence in the mouse genome, we present evidence that a specific DSB can be introduced into the genome of a living mammalian cell by yeast endonuclease I-SceI. We further report that a DSB in the genome of a mouse Ltk- cell is repaired preferentially by non-homologous end-joining rather than by targeted homologous recombination with an exogenous donor sequence. The potential utility of this system is discussed.     

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project. Most of clones entirely sequenced in this study were selected by computer-assisted analysis of terminal sequences of the cDNAs. The average size of the 404 cDNA sequences reached 5.3 kb and that of the deduced amino acid sequences from these cDNAs was 868 amino acid residues. The results of sequence analyses of these clones showed that single mouse KIAA cDNAs bridged two different human KIAA cDNAs in some cases, which indicated that these two human KIAA cDNAs were derived from single genes although they had been supposed to originate from different genes. Furthermore, we successfully mapped all the mouse KIAA cDNAs along the genome using a recently published mouse genome draft sequence.     

Deconstructing Mus gemischus: advances in understanding ancestry, structure, and variation in the genome of the laboratory mouse

The laboratory mouse is an artificial construct with a complex relationship to its natural ancestors. In 2002, the mouse became the first mammalian model organism with a reference genome. Importantly, the mouse genome sequence was assembled from data on a single inbred laboratory strain, C57BL/6. Several large-scale genetic variant discovery efforts have been conducted, resulting in a catalog of tens of millions of SNPs and structural variants. High-density genotyping arrays covering a subset of those variants have been used to produce hundreds of millions of genotypes in laboratory stocks and a small number of wild mice. These landmark resources now enable us to determine relationships among laboratory mice, assign local ancestry at fine scale, resolve important controversies, and identify a new set of challenges—most importantly, the troubling scarcity of genetic data on the very natural populations from which the laboratory mouse was derived. Our aim with this review is to provide the reader with an historical context for the mouse as a model organism and to explain how practical decisions made in the past have influenced both the architecture of the laboratory mouse genome and the design and execution of current large-scale resources. We also provide examples on how the accomplishments of the past decade can be used by researchers to streamline the use of mice in their experiments and correctly interpret results. Finally, we propose future steps that will enable the mouse community to extend its successes in the decade to come.     

The use of nuclear transfer to produce transgenic pigs

Manipulation of the pig genome has the potential to improve pig production and offers powerful biomedical applications. Genetic manipulation of mammals has been possible for over two decades, but the technology available has proven both difficult and inefficient. The development of new techniques to enhance efficiency and overcome the complications of random insertion is of importance. Nuclear transfer combined with homologous recombination provides a possible solution: precise genetic modifications in the pig genome may be induced via homologous recombination, and viable offspring can be produced by nuclear transfer using cultured transfected cell lines. The technique is still ineffective, but it is believed to have immense potential. One area that would benefit from the technology is that of xenotransplantation: transgenic pigs are expected to be available as organ donors in the foreseeable future.