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Most pseudogenes are generated when an RNA transcript is reverse-transcribed and integrated into the genome at a new location. Pseudogenes are often considered as an imperfect and silent copy of a functional gene because of the accumulation of numerous mutations in their sequence. Here we report the presence of Pfh8-ps, a Phf8 retrotransposed pseudogene in the mouse genome, which has no disruptions in its coding sequence. We show that this pseudogene is mainly transcribed in testis and can produce a PHF8-PS protein in vivo. As the PHF8-PS protein has a well-conserved JmjC domain, we characterized its enzymatic activity and show that PHF8-PS does not have the intrinsic capability to demethylate H3K9me2 in vitro compared to the parental PHF8 protein. Surprisingly, PHF8-PS does not localize in the nucleus like PHF8, but rather is mostly located at the cytoplasm. Finally, our proteomic analysis of PHF8-PS-associated proteins revealed that PHF8-PS interacts not only with mitochondrial proteins, but also with prefoldin subunits (PFDN proteins) that deliver unfolded proteins to the cytosolic chaperonin complex implicated in the folding of cytosolic proteins. Together, our findings highlighted PHF8-PS as a new pseudogene-derived protein with distinct molecular functions from PHF8. [ABSTRACT FROM AUTHOR]

Abstract Background Trimethylation of histone H3 on lysine 9 (H3K9me3) at satellite DNA sequences has been primarily studied at (peri)centromeric regions, where its level shows differences associated with various processes such as development and malignant transformation. However, the dynamics of H3K9me3 at distal satellite DNA repeats has not been thoroughly investigated. Results We exploit the sets of publicly available data derived from chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-Seq), produced by the The Encyclopedia of DNA Elements (ENCODE) project, to analyze H3K9me3 at assembled satellite DNA repeats in genomes of human cell lines and during mouse fetal development. We show that annotated satellite elements are generally enriched for H3K9me3, but its level in cancer cell lines is on average lower than in normal cell lines. We find 407 satellite DNA instances with differential H3K9me3 enrichment between cancer and normal cells including a large 115-kb cluster of GSATII elements on chromosome 12. Differentially enriched regions are not limited to satellite DNA instances, but instead encompass a wider region of flanking sequences. We found no correlation between the levels of H3K9me3 and noncoding RNA at corresponding satellite DNA loci. The analysis of data derived from multiple tissues identified 864 instances of satellite DNA sequences in the mouse reference genome that are differentially enriched between fetal developmental stages. Conclusions Our study reveals significant differences in H3K9me3 level at a subset of satellite repeats between biological states and as such contributes to understanding of the role of satellite DNA repeats in epigenetic regulation during development and carcinogenesis.

• A novel deep learning model for identifying DNA N4-methylcytosine sites in mouse genome. • we introduce the bidirectional gated recurrent units (BGRU) to extract representation from DNA sequence. • Our model's powerful learning ability is demonstrated by visualizing the training process. DNA N4-methylcytosine (4mC) is an important DNA modification and plays a crucial role in a variety of biological processes. Accurate 4mC site identification is fundamental to improving the understanding of 4mC biological functions and mechanisms. However, lots of identification approaches are limited to traditional machine learning, which leads to weak learning ability and a complex feature extraction process. Here, we propose Mouse4mC-BGRU, an advanced deep learning model that utilizes adaptive embedding based on bidirectional gated recurrent units (BGRU). Benchmark results show that our model performs better than the state-of-the-art methods in the prediction of 4mC sites in the mouse genome. By using adaptive features to extract representation, Mouse4mC-BGRU can capture the latent biology information of input sequence, which effectively enhances model representation ability. In addition, we visualize the training process of Mouse4mC-BGRU with dim reduction tools and intuitively show the effectiveness of our model, demonstrating that Mouse4mC-BGRU has great potential to be a powerful and practically useful tool to accurately identify 4mC sites. [ABSTRACT FROM AUTHOR]

N4-methylcytosine (4mC) is one of the most important DNA modifications and involved in regulating cell differentiations and gene expressions. The accurate identification of 4mC sites is necessary to understand various biological functions. In this work, we developed a new computational predictor called i4mC-Mouse to identify 4mC sites in the mouse genome. Herein, six encoding schemes of k-space nucleotide composition (KSNC), k-mer nucleotide composition (Kmer), mono nucleotide binary encoding (MBE), dinucleotide binary encoding, electron–ion interaction pseudo potentials (EIIP) and dinucleotide physicochemical composition were explored that cover different characteristics of DNA sequence information. Subsequently, we built six RF-based encoding models and then linearly combined their probability scores to construct the final predictor. Among the six RF-based models, the Kmer, KSNC, MBE, and EIIP encodings are sufficient, which contributed to 10%, 45%, 25%, and 20% of the prediction performance, respectively. On the independent test the i4mC-Mouse predicted the 4mC sites with accuracy and MCC of 0.816 and 0.633, respectively, which were approximately 2.5% and 5% higher than those of the existing method (4mCpred-EL). For experimental biologists, a freely available web application was implemented at http://kurata14.bio.kyutech.ac.jp/i4mC-Mouse/.

Background: Trimethylation of histone H3 on lysine 9 (H3K9me3) at satellite DNA sequences has been primarily studied at (peri)centromeric regions, where its level shows differences associated with various processes such as development and malignant transformation. However, the dynamics of H3K9me3 at distal satellite DNA repeats has not been thoroughly investigated. Results: We exploit the sets of publicly available data derived from chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-Seq), produced by the The Encyclopedia of DNA Elements (ENCODE) project, to analyze H3K9me3 at assembled satellite DNA repeats in genomes of human cell lines and during mouse fetal development. We show that annotated satellite elements are generally enriched for H3K9me3, but its level in cancer cell lines is on average lower than in normal cell lines. We find 407 satellite DNA instances with differential H3K9me3 enrichment between cancer and normal cells including a large 115-kb cluster of GSATII elements on chromosome 12. Differentially enriched regions are not limited to satellite DNA instances, but instead encompass a wider region of flanking sequences. We found no correlation between the levels of H3K9me3 and noncoding RNA at corresponding satellite DNA loci. The analysis of data derived from multiple tissues identified 864 instances of satellite DNA sequences in the mouse reference genome that are differentially enriched between fetal developmental stages. Conclusions: Our study reveals significant differences in H3K9me3 level at a subset of satellite repeats between biological states and as such contributes to understanding of the role of satellite DNA repeats in epigenetic regulation during development and carcinogenesis. [ABSTRACT FROM AUTHOR]

DNA N4-methylcytosine (4mC) is one of the key epigenetic alterations, playing essential roles in DNA replication, differentiation, cell cycle, and gene expression. To better understand 4mC biological functions, it is crucial to gain knowledge on its genomic distribution. In recent times, few computational studies, in particular machine learning (ML) approaches have been applied in the prediction of 4mC site predictions. Although ML-based methods are promising for 4mC identification in other species, none are available for detecting 4mCs in the mouse genome. Our novel computational approach, called 4mCpred-EL, is the first method for identifying 4mC sites in the mouse genome where four different ML algorithms with a wide range of seven feature encodings are utilized. Subsequently, those feature encodings predicted probabilistic values are used as a feature vector and are once again inputted to ML algorithms, whose corresponding models are integrated into ensemble learning. Our benchmarking results demonstrated that 4mCpred-EL achieved an accuracy and MCC values of 0.795 and 0.591, which significantly outperformed seven other classifiers by more than 1.5−5.9% and 3.2−11.7%, respectively. Additionally, 4mCpred-EL attained an overall accuracy of 79.80%, which is 1.8−5.1% higher than that yielded by seven other classifiers in the independent evaluation. We provided a user-friendly web server, namely 4mCpred-EL which could be implemented as a pre-screening tool for the identification of potential 4mC sites in the mouse genome.

Chromocenters are interphase nuclear landmark structures of constitutive heterochromatin. The tandem repeat (TR)-enriched parts of different chromosomes cluster together in chromocenters. There has been progress in recent years in determining the protein content of chromocenters, although it is not clear which DNA sequences underly constitutive heterochromatin apart from the TRs. The aim of the current work was to find out which DNA sequences besides TRs are involved in chromocenters' formation. Biochemically isolated chromocenters and microdissected centromeric regions were amplified by DOP-PCR, then cloned and sequenced. Alignment to Repbase, the mouse reference genome and WGS databases separated the sequences from both libraries into three groups: (1) sequences with similarity to pericentromere mouse major satellite; (2) sequences without similarity to any repetitive sequences; (3) sequences with similarity to long interspersed nuclear elements (LINEs). LINE-related sequences have a disperse pattern distribution on chromosomes predicted in silico. Selected clones were used for fluorescent in situ hybridization (FISH). The 10 clones tested hybridized to chromocenters and centromeric regions of metaphase chromosomes. These clones were used for double FISH with four known cloned TRs (satDNA, satellite DNA) and a probe specific for the sex chromosomes. The probes bind various chromocenters' regions without overlapping; so, FISH results reveal a complex chromocenter composition. We mapped 18 LINE-derived clones to the RepBase L1 records. Most of them grouped in a ∼2-kb region at the end of the second ORF and 3′ untranslated region (UTR). So, even the limited number of the clones allows us to determine the region of the L1 element that is specific for heterochromatic regions. Although the L1 full-length probe did not hybridize at detectable levels to the heterochromatic region on any chromosome, the 2-kb fragment found is definitely a part of these regions. The precise LINE ∼2-kb fragment is the component of mouse and human constitutive heterochromatin enriched with TRs. The method used for amplification of the probes from two sources of the heterochromatic material uncovered the enrichment of a precise fragment of LINE within chromocenters. [ABSTRACT FROM AUTHOR]

Background: Retrotransposons are mobile elements that have a high impact on shaping the mammalian genomes. Since the availability of whole genomes, genomic analyses have provided novel insights into retrotransposon biology. However, many retrotransposon families and their possible genomic impact have not yet been analysed. Results: Here, we analysed the structural features, the genomic distribution and the evolutionary history of mouse VL30 LTR-retrotransposons. In total, we identified 372 VL30 sequences categorized as 86 full-length and 49 truncated copies as well as 237 solo LTRs, with non-random chromosomal distribution. Full-length VL30s were highly conserved elements with intact retroviral replication signals, but with no protein-coding capacity. Analysis of LTRs revealed a high number of common transcription factor binding sites, possibly explaining the known inducible and tissue-specific expression of individual elements. The overwhelming majority of full-length and truncated elements (82/86 and 40/49, respectively) contained one or two specific motifs required for binding of the VL30 RNA to the poly-pyrimidine tract-binding protein-associated splicing factor (PSF). Phylogenetic analysis revealed three VL30 groups with the oldest emerging ~17.5 Myrs ago, while the other two were characterized mostly by new genomic integrations. Most VL30 sequences were found integrated either near, adjacent or inside transcription start sites, or into introns or at the 3' end of genes. In addition, a significant number of VL30s were found near Krueppel-associated box (KRAB) genes functioning as potent transcriptional repressors. Conclusion: Collectively, our study provides data on VL30s related to their: (a) number and structural features involved in their transcription that play a role in steroidogenesis and oncogenesis; (b) evolutionary history and potential for retrotransposition; and (c) unique genomic distribution and impact on gene expression. [ABSTRACT FROM AUTHOR]

Abstract The current proliferation of mammalian genomes is creating a nomenclature issue caused by naming genes based on their best BLAST hit to a gene in another annotated genome. The rat genome is relying heavily on the mouse genome for nomenclature, but not all rat genes have direct orthologues in the mouse; often, there are paralogous groups of genes -- due to expansions of that gene subfamily in one or the other genome. Many of these genes have already been assigned names in the rat, so that renaming them based on BLAST scores leads to duplicate sets of names. The supposed orthology created by name sharing across genomes is not always found. These inaccurate names are appearing in frequently used sites, such as the University of California Santa Cruz Genome Browser. The example of rat cytochrome P450 (Cyp) genes is presented here, but other gene families are also likely to be affected.

Abstract Why is agreement on one particular name for each gene important? As one genome after another becomes sequenced, it is imperative to consider the complexity of genes, genetic architecture, gene expression, gene-gene and gene-product interactions and evolutionary relatedness across species. To agree on a particular gene name not only makes one's own research easier, it aids automated text-mining algorithms and search engines, which are increasingly employed to find relationships in the millions of abstracts in the medical research literature and sequence databases. A common nomenclature system will also be helpful to the present generation, as well as future generations, of graduate students and postdoctoral fellows who are about to enter genomics research. In this paper, the authors present some problems that arose when two separate research communities decided to choose the same root, CYP, for naming their gene families. They then offer a logical solution, by renaming the cyclophilin genes with a common root, such a cyn- in Caenorhabditis and CYN- in mammals (Cyn in mouse), and using evolutionary divergence to cluster genes of the highest level of relatedness.

Abstract Genome comparisons are behind the powerful new annotation methods being developed to find all human genes, as well as genes from other genomes. Genomes are now frequently being studied in pairs to provide cross-comparison datasets. This 'Noah's Ark' approach often reveals unsuspected genes and may support the deletion of false-positive predictions. Joining mouse and human as the cross-comparison dataset for the first two mammals are: two Drosophila species, D. melanogaster and D. pseudoobscura; two sea squirts, Ciona intestinalis and Ciona savignyi; four yeast (Saccharomyces) species; two nematodes, Caenorhabditis elegans and Caenorhabditis briggsae; and two pufferfish (Takefugu rubripes and Tetraodon nigroviridis). Even genomes like yeast and C. elegans, which have been known for more than five years, are now being significantly improved. Methods developed for yeast or nematodes will now be applied to mouse and human, and soon to additional mammals such as rat and dog, to identify all the mammalian protein-coding genes. Current large disparities between human Unigene predictions (127,835 genes) and gene-scanning methods (45,000 genes) still need to be resolved. This will be the challenge during the next few years.

Genomic features such as rate of recombination and differentiation have been suggested to play a role in species divergence. However, the relationship of these phenomena to functional organization of the genome in the context of reproductive isolation remains unexplored. Here, we examine genomic characteristics of the species boundaries between two house mouse subspecies (Mus musculus musculus/M. m. domesticus). These taxa form a narrow semipermeable zone of secondary contact across Central Europe. Due to the incomplete nature of reproductive isolation, gene flow in the zone varies across the genome. We present an analysis of genomic differentiation, rate of recombination, and functional composition of genes relative to varying amounts of introgression. We assessed introgression using 1,316 autosomal single nucleotide polymorphism markers, previously genotyped in hybrid populations from three transects. We found a significant relationship between amounts of introgression and both genomic differentiation and rate of recombination with genomic regions of reduced introgression associated with higher genomic differentiation and lower rates of recombination, and the opposite for genomic regions of extensive introgression. We also found a striking functional polarization of genes based on where they are expressed in the cell. Regions of elevated introgression exhibit a disproportionate number of genes involved in signal transduction functioning at the cell periphery, among which olfactory receptor genes were found to be the most prominent group. Conversely, genes expressed intracellularly and involved in DNA binding were the most prevalent in regions of reduced introgression. We hypothesize that functional organization of the genome is an important driver of species divergence. [ABSTRACT FROM AUTHOR]

SUMMARY With the emergence of popular next-generation sequencing (NGS)-based genome-wide protocols such as chromatin immunoprecipitation followed by sequencing (ChIP-Seq) and RNA-Seq, there is a growing need for research and infrastructure to support the requirement of effectively analyzing NGS data. Such research and infrastructure do not replace but complement algorithmic advances developments in analyzing NGS data. We present a runtime environment, Distributed Application Runtime Environment, that supports the scalable, flexible, and extensible composition of capabilities that cover the primary requirements of NGS-based analytics. In this work, we use BFAST as a representative stand-alone tool used for NGS data analysis and a ChIP-Seq pipeline as a representative pipeline-based approach to analyze the computational requirements. We analyze the performance characteristics of BFAST and understand its dependency on different input parameters. The computational complexity of genome-wide mapping using BFAST, amongst other factors, depends upon the size of a reference genome and the data size of short reads. Characterizing the performance suggests that the mapping benefits from both scaling-up (increased fine-grained parallelism) and scaling-out (task-level parallelism - local and distributed). For certain problem instances, scaling-out can be a more efficient approach than scaling-up. On the basis of investigations using the pipeline for ChIP-Seq, we also discuss the importance of dynamical execution of tasks. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Copy number variation (CNV), an important source of diversity in genomic structure, is frequently found in clusters called CNV regions (CNVRs). CNVRs are strongly associated with segmental duplications (SDs), but the composition of these complex repetitive structures remains unclear. Results: We conducted self-comparative-plot analysis of all mouse chromosomes using the high-speed and largescale- homology search algorithm SHEAP. For eight chromosomes, we identified various types of large SD as tartanchecked patterns within the self-comparative plots. A complex arrangement of diagonal split lines in the selfcomparative- plots indicated the presence of large homologous repetitive sequences. We focused on one SD on chromosome 13 (SD13M), and developed SHEPHERD, a stepwise ab initio method, to extract longer repetitive elements and to characterize repetitive structures in this region. Analysis using SHEPHERD showed the existence of 60 core elements, which were expected to be the basic units that form SDs within the repetitive structure of SD13M. The demonstration that sequences homologous to the core elements (>70% homology) covered approximately 90% of the SD13M region indicated that our method can characterize the repetitive structure of SD13M effectively. Core elements were composed largely of fragmented repeats of a previously identified type, such as long interspersed nuclear elements (LINEs), together with partial genic regions. Comparative genome hybridization array analysis showed that whereas 42 core elements were components of CNVR that varied among mouse strains, 8 did not vary among strains (constant type), and the status of the others could not be determined. The CNV-type core elements contained significantly larger proportions of long terminal repeat (LTR) types of retrotransposon than the constant-type core elements, which had no CNV. The higher divergence rates observed in the CNV-type core elements than in the constant type indicate that the CNV-type core elements have a longer evolutionary history than constant-type core elements in SD13M. Conclusions: Our methodology for the identification of repetitive core sequences simplifies characterization of the structures of large SDs and detailed analysis of CNV. The results of detailed structural and quantitative analyses in this study might help to elucidate the biological role of one of the SDs on chromosome 13. [ABSTRACT FROM AUTHOR]

Abstract: Endogenous retroviral elements (EREs), a family of transposable elements, constitute a substantial fraction of mammalian genomes. It is expected that profiles of the ERE sequences and their genomic locations are unique for each individual. Comprehensive characterization of the EREs’ genomic locations and their biological properties is essential for understanding their roles in the pathophysiology of the host. In this study, we identified and mapped putative EREs (a total of 111 endogenous retroviruses [ERVs] and 488 solo long terminal repeats [sLTRs]) within the C57BL/6J mouse genome. The biological properties of individual ERE isolates (both ERVs and sLTRs) were then characterized in the following aspects: transcription potential, tropism trait, coding potential, recombination event, integration age, and primer binding site for replication. In addition, a suite of database management system programs was developed to organize and update the data acquired from current and future studies and to make the data accessible via internet. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

While many studies of cis-elements CArG bound by serum response factor (SRF) are in progress, little is known about the positional distribution of the functional CArG elements around the transcription start site (TSS) of genes that they influence. We use a validated CArG data set to calculate the distance distribution of functional CArG elements around the TSS. Distances between adjacent CArGs were also analyzed. We compare these distributions with those derived using a control set of randomly selected CArGs (that were not experimentally validated for function). Our results show that most functional CArG elements (108 of 152, 71%) exist upstream of the annotated TSS, with copy number increasing as one moves closer to the TSS. Moreover, the average number of the CArG elements in the CArG-containing genes is significantly more than that in the control genes. Our study extends earlier bioinformatic analyses of functional CArG elements and provides an application of comparative sequence data to the identification of transcription factor binding sites. Bien que plusieurs études soient en cours sur les boîtes cis CArG sur lesquelles se lie le facteur de réponse au sérum (SRF), la distribution des boîtes CArG fonctionnelles autour des sites d&x2019;initiation de la transcription (TSS) des gènes qu&x2019;ils régulent est peu connue. Les auteurs ont employé un jeu validé de boîtes CArG pour calculer la distribution des distances entre les boîtes CArG fonctionnelles et le TSS. Les distances entre boîtes CArG voisines ont également été analysées. Les auteurs comparent ces distributions à celles obtenues en employant un jeu témoin constitué de boîtes CArG choisies aléatoirement (n&x2019;ayant pas été validées expérimentalement pour leur fonction). Les résultats montrent que la plupart des boîtes CArG fonctionnelles (108 de 152, 71 %) sont situées en amont du TSS annoté et que le nombre de copies augmente plus on se rapproche du TSS. De plus, le nombre moyen de boîtes CArG au sein de gènes qui comptent de tels éléments est significativement plus élevé que chez les témoins. Cette étude s&x2019;inscrit dans la suite d&x2019;analyses bioinformatiques antérieures sur les boîtes CArG fonctionnelles et fournit un exemple de l&x2019;utilisation d&x2019;une analyse comparée de séquences pour l&x2019;identification de sites de liaison de facteurs de transcription. [ABSTRACT FROM AUTHOR]

Abstract: The mouse genome consists of five known families of SINEs: B1, B2, B4/RSINE, ID, and MIR. Using RT-PCR we identified a germ-line transcript that demonstrates 92.7% sequence identity to ID (excluding primer sequence), yet a BLAST search identified numerous matches of 100% sequence identity. We analyzed four of these elements for their presence in orthologous genes in strains and subspecies of Mus musculus as well as other species of Mus using a PCR-based assay. All four analyzed elements were identified either only in M. musculus or exclusively in both M. musculus and M. domesticus, indicative of recent integrations. In conjunction with the identification of transcripts, we present an active ID-like group of elements that is not derived from the proposed BC1 master gene of ID elements. A BLAST of the rat genome indicated that these elements were not in the rat. Therefore, this family of SINEs has recently evolved, and since it has thus far been observed mainly in M. musculus, we refer to this family as MMIDL. [Copyright &y& Elsevier]

Abstract: The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to the embryonic genetic program. Utilizing DNA microarray data, we looked for genes that are expressed only during ZGA and found Zscan4, whose expression is restricted to late 2-cell stage embryos. Sequence analysis of genomic DNA and cDNA clones revealed nine paralogous genes tightly clustered in 0.85 Mb on mouse chromosome 7. Three genes are not transcribed and are thus considered pseudogenes. Among the six expressed genes named Zscan4a-Zscan4f, three – Zscan4c, Zscan4d, and Zscan4f – encode full-length ORFs with 506 amino acids. Zscan4d is a predominant transcript at the late 2-cell stage, whereas Zscan4c is a predominant transcript in embryonic stem (ES) cells. No transcripts of any Zscan4 genes are detected in any other cell types. Reduction of Zscan4 transcript levels by siRNAs delays the progression from the 2-cell to the 4-cell stage and produces blastocysts that fail to implant or proliferate in blastocyst outgrowth culture. Zscan4 thus seems to be essential for preimplantation development. [Copyright &y& Elsevier]

Abstract: A new species of non-coding RNA, microRNAs (miRNAs) has been identified that may regulate the expression of as many as one third to one half of all protein encoding genes. MicroRNAs are found throughout mammalian genomes, but an association between the location of these miRNAs and regions of genomic instability (or fragile sites) in humans has been suggested . In this review we discuss the possible role of altered miRNA expression on human cancer and conduct an analysis correlating the physical location of murine miRNAs with sites of genetic alteration in mouse models of cancer. [Copyright &y& Elsevier]

Based on the alignment of 12 sequences of protein motifs that interact with the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress response 1), we performed genome-wide searches of the sequence [S/G/V]RFx[V/I]xx[V/I/T/S]xx, where x represents any amino acid. The "Mus musculus" search resulted in the identification of 131 mouse proteins containing 137 SPAK/OSR1 putative binding motifs. Similar numbers were found for human, zebrafish, fruit fly, and worm. A little more than half of the mouse proteins containing SPAK/OSR1 binding domains (53%) were also identified in the human search, whereas ~17-18% of these common hits were identified in the zebrafish search. The mouse proteins could be divided into two broad categories: 2/3 had an identified function, whereas 1/3 were either predicted or of unknown function. The known proteins were grouped as transport proteins, other membrane proteins, kinases, phosphatases, cytoskeletal, ribosomal, nuclear, enzymes, and others. Analysis of the location of the SPAK/OSR1 binding motif within the protein sequence revealed distribution throughout the entire length, but with preference to the extreme amino- or carboxyl termini for a large number of proteins. Analysis of the amino acid composition of the motifs revealed a preponderance of serine residues at positions 5, 6, 7, and 8. In summary, our new search found and thus confirms the 12 proteins previously shown to interact with the kinases and identifies 119 potential new targets for SPAK and OSR1 in the mouse proteome. [ABSTRACT FROM AUTHOR]

It is increasingly evident that there is a widespread antisense transcription in the human and other eukaryotic genomes. However, the concept of general antisense expression is rarely investigated. We retrieved and correlated the expression of sense and antisense sequences of 1182 mouse transcripts to assess the prevalence of antisense transcription. We contrasted 20 Affymetrix MGU74A version 1 mouse genome chips to 12 MGU74A version 2 chips. For these 1182 transcripts, the version 1 chips contained the antisense sequences of the transcripts presented on the version 2 chips. The original data was taken from the GEO database. As the Affymetrix data is semi quantitative, the relative levels of antisense partners were analysed. We detected antisense transcription with an overall magnitude of 43% relative to sense transcription in the investigated transcripts. The average MGU74A version 1 expression is shifted towards smaller expression values (MGU74A version 1: 525.1; version 2: 1219.1; t-test: p < 0.001). A direct correlation between sense and antisense expression values could not be observed. Genes with high inverse expression values may be correlated to the investigated condition: genes where sense/control and control/antisense ratios were above two may be included in the pathogenetic pathways associated with dystrophin deficiency. The ratio of sense to antisense transcription varied between different chromosomes. We conclude that antisense transcription is a common phenomenon in the mouse genome and may have indirect regulatory functions. [ABSTRACT FROM AUTHOR]

Solitary LTR loci are the predominant form of LTR retrotransposons in most eukaryotic genomes. They originate from recombination between the two LTRs of an ancestral retrovirus and are therefore incapable of transposition. Despite this inactivity, they appear to have a substantial impact on the host genome. Here we use the murine RMER10 LTR family as an example to describe how such elements can reshape regions of the genome through multiple mutations on an evolutionary time scale. Specifically, we use phylogenetic analysis of multiple copies of RMER10 in rodent species, as well as comparisons of orthologous pairs in mouse and rat, to argue that insertions of members of this family have locally induced the emergence of tandem repeat loci as well as many indels. Analysis of structural aspects of these sequences (secondary structures and transcription factors signals) may explain why RMER10 can become endogenous “mutagenic” factors through induction of replication fork blockages and/or error-prone repair of aberrant DNA structures. This hypothesis is also consistent with features of other interspersed repeated elements. [ABSTRACT FROM AUTHOR]

Genomic amplifications and deletions, the consequence of somatic variation, are a hallmark of human cancer. Such variation has also been observed between ‘normal’ individuals, as well as in individuals with congenital disorders. Thus, copy number measurement is likely to be an important tool for the analysis of genetic variation, genetic disease, and cancer. We developed representational oligonucleotide microarray analysis, a high-resolution comparative genomic hybridization methodology, with this aim in mind, and reported its use in the study of humans. Here we report the development of a representational oligonucleotide microarray analysis microarray for the genomic analysis of the mouse, an important model system for many genetic diseases and cancer. This microarray was designed based on the sequence assembly MM3, and contains ≈84,000 probes randomly distributed throughout the mouse genome. We demonstrate the use of this array to identify copy number changes in mouse cancers, as well to determine copy number variation between inbred strains of mice. Because restriction endonuclease digestion of genomic DNA is an integral component of our method, differences due to polymorphisms at the restriction enzyme cleavage sites are also observed between strains, and these can be useful to follow the inheritance of loci between crosses of different strains. [ABSTRACT FROM AUTHOR]

Processed pseudogenes are generated by reverse transcription of a functional gene. They are generally nonfunctional after their insertion and, as a consequence, are no longer subjected to the selective constraints associated with functional genes. Because of this property they can be used as neutral markers in molecular evolution. In this work, we investigated the relationship between the evolution of GC content in recently inserted processed pseudogenes and the local recombination pattern in two mammalian genomes (human and mouse). We confirmed, using original markers, that recombination drives GC content in the human genome and we demonstrated that this is also true for the mouse genome despite lower recombination rates. Finally, we discussed the consequences on isochores evolution and the contrast between the human and the mouse pattern. [ABSTRACT FROM AUTHOR]

Abstract: Mouse and rat genomic sequences permit us to obtain a global view of evolutionary rearrangements that have occurred between the two species and to define hallmarks that might underlie these events. We present a comparative study of the sequence assemblies of mouse and rat genomes and report an enrichment of rodent-specific segmental duplications in regions where synteny is not preserved. We show that segmental duplications present higher rates of molecular evolution and that genes in rearranged regions have evolved faster than those located elsewhere. Previous studies have shown that synteny breakpoints between the mouse and the human genomes are enriched in human segmental duplications, suggesting a causative connection between such structures and evolutionary rearrangements. Our work provides further evidence to support the role of segmental duplications in chromosomal rearrangements in the evolution of the architecture of mammalian chromosomes and in the speciation processes that separate the mouse and the rat. [Copyright &y& Elsevier]

Abstract: The majority of gene-targeting experiments in mice are performed in 129Sv-derived embryonic stem (ES) cell lines, which are generally considered to be more reliable at colonizing the germ line than ES cells derived from other strains. Gene targeting is reliant on homologous recombination of a targeting vector with the host ES cell genome. The efficiency of recombination is affected by many factors, including the isogenicity (H. te Riele et al., 1992, Proc. Natl. Acad. Sci. USA 89, 5128–5132) and the length of homologous sequence of the targeting vector and the location of the target locus. Here we describe the double-end sequencing and mapping of 84,507 bacterial artificial chromosomes (BACs) generated from AB2.2 ES cell DNA (129S7/SvEvBrd-Hprt b-m2). We have aligned these BACs against the mouse genome and displayed them on the Ensembl genome browser, DAS: 129S7/AB2.2. This library has an average insert size of 110.68 kb and average depth of genome coverage of 3.63- and 1.24-fold across the autosomes and sex chromosomes, respectively. Over 97% of the mouse genome and 99.1% of Ensembl genes are covered by clones from this library. This publicly available BAC resource can be used for the rapid construction of targeting vectors via recombineering. Furthermore, we show that targeting vectors containing DNA recombineered from this BAC library can be used to target genes efficiently in several 129-derived ES cell lines. [Copyright &y& Elsevier]

Abstract: The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a “mutant mouse library” that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical. [Copyright &y& Elsevier]

Abstract: CpG island is a GC-rich motif occurred in gene promoter region, which can play important roles in gene silencing and imprinting. Here, we present a set of discriminant functions that can recognize the structural and compositional features of CpG islands in the putative promoter regions (PPRs) of human and mouse immunoglobulin (Ig) genes. We showed that the PPRs of both human and mouse Ig genes irrespective of gene chromosomal localization are apparently CpG island poor, with a low percentage of the CpG islands overlapped with the transcription start site (TSS). The human Ig genes that have CpG islands in the PPRs show a very narrow range of CpG densities. 47% of the Ig genes fall in the range of 3.5–4 CpGs/100 bp. In contrast, the non-Ig genes examined have a wide range of the density of CpG island, with 10.5% having the density of 8.1–15 CpGs/100 bp. Meantime, five patterns of the CpG distributions within the CpG islands have been classified: Pat A, B, C, D, and E. 21.6% and 10.8% of the Ig genes fall into the Pat B and Pat D groups, respectively, which were significantly higher than the non-Ig genes examined (8.2% and 3.8%). Moreover, the length of CpG islands is shorter in human Ig genes than in non-Ig genes but is much longer than in mouse orthologues. These findings provide a clear picture of non-neutral and nonrandom occurrence of the CpG islands in the PPRs of human and mouse Ig genes, which facilitate rational recommendations regarding their nomenclature. [Copyright &y& Elsevier]

Conservation of microRNAs (miRNAs) among species suggests that they bear conserved biological functions. However, sequencing of new miRNAs has not always been accompanied by a search for orthologues in other species. I report herein the results of a systematic search for interspecies orthologues of miRNA precursors, leading to the identification of 35 human and 45 mouse new putative miRNA genes. MicroRNA tracks were written to visualize miRNAs in human and mouse genomes on the UCSC Genome Browser. Based on their localization, miRNA precursors can be excised either from introns or exons of mRNAs. When intronic miRNAs are antisense to the apparent host gene, they appear to originate from ill-characterized antisense transcription units. Exonic miRNAs are, in general, nonprotein-coding, poorly conserved genes in sense orientation. In three cases, the excision of an miRNA from a protein-coding mRNA might lead to the degradation of the rest of the transcript. Moreover, three new examples of miRNAs fully complementary to an mRNA are reported. Among these, miR135a might control the stability and/or translation of an alternative form of theglycerate kinasemRNA by RNA interference. I also discuss the presence of human miRNAs in introns of paralogous genes and in miRNA clusters. [ABSTRACT FROM AUTHOR]

Exon shuffling, a major mechanism of gene evolution, scrambles existing sequences to create new genes. However, is it possible for an exon to be created from scratch? Here we conduct a survey of rat and mouse genomes and identify 2,302 putative rodent-specific exons absent from the human genome. Analysis of rodent transcripts supporting these exons indicates that over half appear to be alternatively spliced in genes orthologous between rodents and human. This study demonstrates the importance of sequencing genomes from multiple species to accurately document the evolution of gene structure. [ABSTRACT FROM AUTHOR]

We study global fluctuations of the guanine and cytosine base content (GC%) in mouse genomic DNA using spectral analyses. Power spectra S(f) of GC% fluctuations in all nineteen autosomal and two sex chromosomes are observed to have the universal functional form S(f)~1/fα (α≈1) over several orders of magnitude in the frequency range 10-710-5 cycle/base) shows a flattened power-law function with α<1 across all twenty-one chromosomes. The substitution of about 38% interspersed repeats does not affect the functional form of S(f), indicating that these are not predominantly responsible for the long-ranged multi-scale GC% fluctuations in mammalian genomes. Several biological implications of the large-scale GC% fluctuation are discussed, including neutral evolutionary history by DNA duplication, chromosomal bands, spatial distribution of transcription units (genes), replication timing, and recombination hot spots. [ABSTRACT FROM AUTHOR]

To date, 12 phospholipase C (PLC) isozymes have been identified in mammals, and they are divided into five classes, β-, γ-, δ-, #x03B5;-, and ζ-type. PLCδ-type is reported to be composed of four isozymes, PLCδ1–δ4. Here we report that a screening for mouse PLCδ2 from a BAC library with primers that amplify a specific region of bovine PLCδ2 resulted in isolation of one clone containing the mouse PLCδ4 gene. Furthermore, a database search revealed that there is only one gene corresponding to PLCδ2 and PLCδ4 in the mouse and human genomes, indicating that bovine PLCδ2 is a homologue of human and mouse PLCδ4. However, PLCδ2 Western blot analysis with a widely used commercial anti-PLCδ2 antibody showed an expression pattern distinct from that of PLCδ4 in wild-type mice. In addition, an 80-kDa band, which was recognized by antibody against PLCδ2, was smaller than an 85-kDa band detected by anti-PLCδ4 antibody, and the 80-kDa band was detectable in lysates of brain, testis, and spleen from PLCδ4-deficient mice. We also found that immunoprecipitates from brain lysates with this PLCδ2 antibody contained no PLC activity. From these data, we conclude that bovine PLCδ2 is a homologue of human and mouse PLCδ4, and that three isozymes (δ1, δ3, and δ4) exist in the PLCδ family. [Copyright &y& Elsevier]