Fine-scale maps of recombination rates and hotspots in the mouse genome.

PubMed

Brunschwig, Hadassa; Levi, Liat; Ben-David, Eyal; Williams, Robert W; Yakir, Benjamin; Shifman, Sagiv

2012-07-01

Recombination events are not uniformly distributed and often cluster in narrow regions known as recombination hotspots. Several studies using different approaches have dramatically advanced our understanding of recombination hotspot regulation. Population genetic data have been used to map and quantify hotspots in the human genome. Genetic variation in recombination rates and hotspots usage have been explored in human pedigrees, mouse intercrosses, and by sperm typing. These studies pointed to the central role of the PRDM9 gene in hotspot modulation. In this study, we used single nucleotide polymorphisms (SNPs) from whole-genome resequencing and genotyping studies of mouse inbred strains to estimate recombination rates across the mouse genome and identified 47,068 historical hotspots--an average of over 2477 per chromosome. We show by simulation that inbred mouse strains can be used to identify positions of historical hotspots. Recombination hotspots were found to be enriched for the predicted binding sequences for different alleles of the PRDM9 protein. Recombination rates were on average lower near transcription start sites (TSS). Comparing the inferred historical recombination hotspots with the recent genome-wide mapping of double-strand breaks (DSBs) in mouse sperm revealed a significant overlap, especially toward the telomeres. Our results suggest that inbred strains can be used to characterize and study the dynamics of historical recombination hotspots. They also strengthen previous findings on mouse recombination hotspots, and specifically the impact of sequence variants in Prdm9.

Genome-wide comparative analysis reveals human-mouse regulatory landscape and evolution.

PubMed

Denas, Olgert; Sandstrom, Richard; Cheng, Yong; Beal, Kathryn; Herrero, Javier; Hardison, Ross C; Taylor, James

2015-02-14

Because species-specific gene expression is driven by species-specific regulation, understanding the relationship between sequence and function of the regulatory regions in different species will help elucidate how differences among species arise. Despite active experimental and computational research, relationships among sequence, conservation, and function are still poorly understood. We compared transcription factor occupied segments (TFos) for 116 human and 35 mouse TFs in 546 human and 125 mouse cell types and tissues from the Human and the Mouse ENCODE projects. We based the map between human and mouse TFos on a one-to-one nucleotide cross-species mapper, bnMapper, that utilizes whole genome alignments (WGA). Our analysis shows that TFos are under evolutionary constraint, but a substantial portion (25.1% of mouse and 25.85% of human on average) of the TFos does not have a homologous sequence on the other species; this portion varies among cell types and TFs. Furthermore, 47.67% and 57.01% of the homologous TFos sequence shows binding activity on the other species for human and mouse respectively. However, 79.87% and 69.22% is repurposed such that it binds the same TF in different cells or different TFs in the same cells. Remarkably, within the set of repurposed TFos, the corresponding genome regions in the other species are preferred locations of novel TFos. These events suggest exaptation of some functional regulatory sequences into new function. Despite TFos repurposing, we did not find substantial changes in their predicted target genes, suggesting that CRMs buffer evolutionary events allowing little or no change in the TFos - target gene associations. Thus, the small portion of TFos with strictly conserved occupancy underestimates the degree of conservation of regulatory interactions. We mapped regulatory sequences from an extensive number of TFs and cell types between human and mouse using WGA. A comparative analysis of this correspondence unveiled the extent of the shared regulatory sequence across TFs and cell types under study. Importantly, a large part of the shared regulatory sequence is repurposed on the other species. This sequence, fueled by turnover events, provides a strong case for exaptation in regulatory elements.

Augmenting Chinese hamster genome assembly by identifying regions of high confidence.

PubMed

Vishwanathan, Nandita; Bandyopadhyay, Arpan A; Fu, Hsu-Yuan; Sharma, Mohit; Johnson, Kathryn C; Mudge, Joann; Ramaraj, Thiruvarangan; Onsongo, Getiria; Silverstein, Kevin A T; Jacob, Nitya M; Le, Huong; Karypis, George; Hu, Wei-Shou

2016-09-01

Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re-scaffolding and gap-filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as "high confidence regions" which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high-quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Partnering for functional genomics research conference: Abstracts of poster presentations

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1998-06-01

This reports contains abstracts of poster presentations presented at the Functional Genomics Research Conference held April 16--17, 1998 in Oak Ridge, Tennessee. Attention is focused on the following areas: mouse mutagenesis and genomics; phenotype screening; gene expression analysis; DNA analysis technology development; bioinformatics; comparative analyses of mouse, human, and yeast sequences; and pilot projects to evaluate methodologies.

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

Mouse Genome Database: From sequence to phenotypes and disease models

PubMed Central

Richardson, Joel E.; Kadin, James A.; Smith, Cynthia L.; Blake, Judith A.; Bult, Carol J.

2015-01-01

Summary The Mouse Genome Database (MGD, www.informatics.jax.org) is the international scientific database for genetic, genomic, and biological data on the laboratory mouse to support the research requirements of the biomedical community. To accomplish this goal, MGD provides broad data coverage, serves as the authoritative standard for mouse nomenclature for genes, mutants, and strains, and curates and integrates many types of data from literature and electronic sources. Among the key data sets MGD supports are: the complete catalog of mouse genes and genome features, comparative homology data for mouse and vertebrate genes, the authoritative set of Gene Ontology (GO) annotations for mouse gene functions, a comprehensive catalog of mouse mutations and their phenotypes, and a curated compendium of mouse models of human diseases. Here, we describe the data acquisition process, specifics about MGD's key data areas, methods to access and query MGD data, and outreach and user help facilities. genesis 53:458–473, 2015. © 2015 The Authors. Genesis Published by Wiley Periodicals, Inc. PMID:26150326

Principles of regulatory information conservation between mouse and human

DOE PAGES

Cheng, Yong; Ma, Zhihai; Kim, Bong-Hyun; ...

2014-11-19

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human–mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and withmore » genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Lastly, single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.« less

Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

PubMed Central

Schmouth, Jean-François; Bonaguro, Russell J.; Corso-Diaz, Ximena; Simpson, Elizabeth M.

2012-01-01

An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation. PMID:22396661

Identification of structural variation in mouse genomes.

PubMed

Keane, Thomas M; Wong, Kim; Adams, David J; Flint, Jonathan; Reymond, Alexandre; Yalcin, Binnaz

2014-01-01

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Genomic organization and sequence of the Gus-s/sup a/ allele of the murine. beta. -glucuronidase gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Funkenstein, B.; Leary, S.L.; Stein, J.C.

1988-03-01

The Gus-s/sup ..cap alpha../ allele of the mouse ..beta..-glucuronidase gene exhibits a high degree of inducibility by androgens due to its linkage with the Gus-r/sup ..cap alpha../ regulatory locus. The authors isolated Gus-s/sup ..cap alpha../ on a 28-kilobase pair fragment of mouse chromosome 5 and found that it contains 12 exons and 11 intervening sequences spanning 14 kilobase pairs of this genomic segment. The mRNA cap site was identified by ribonuclease protection and primer extension analyses which revealed an unusually short 5' noncoding sequence of 12 nucleotides. Proximal regulatory sequences in the 5'-flanking DNA and the complete sequence of themore » Gus-s/sup ..cap alpha../ mRNA transcript were also determined. Comparison of the amino acid sequence determined from the Gus-s/sup ..cap alpha../ nucleotide sequence with that of human ..beta..-glucuronidase indicated that the two human mRNA species differ due to alternate splicing of an exon homologous to exon 6 of the mouse gene.« less

CIDR

Science.gov Websites

CIDR Skip navigation Home About CIDR General Highlights Newsletter Staff Employment Opportunities Genotyping General Information Genome Wide Association Custom FFPE Sample Options Methylation Linkage Consortium Developed Mouse Whole Genome Sequencing General Information Whole Genome Whole Exome Custom

CIDR

Science.gov Websites

Initiation Application Schedule Service Information and Pricing Services Sample Requirements Pricing SNP Genotyping General Information Genome Wide Association Custom FFPE Sample Options Methylation Linkage Consortium Developed Mouse Whole Genome Sequencing General Information Whole Genome Whole Exome Custom

Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner.

PubMed

Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan; Brent, Michael R

2009-07-01

The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary determinant of alignment accuracy, while heuristics that prevent consideration of certain alignments are a primary determinant of runtime and memory usage. Both accuracy and speed are important considerations in choosing an alignment algorithm, but scoring systems have received much less attention than heuristics. We present Pairagon, a pair hidden Markov model based cDNA-to-genome alignment program, as the most accurate aligner for sequences with high- and low-identity levels. We conducted a series of experiments testing alignment accuracy with varying sequence identity. We first created 'perfect' simulated cDNA sequences by splicing the sequences of exons in the reference genome sequences of fly and human. The complete reference genome sequences were then mutated to various degrees using a realistic mutation simulator and the perfect cDNAs were aligned to them using Pairagon and 12 other aligners. To validate these results with natural sequences, we performed cross-species alignment using orthologous transcripts from human, mouse and rat. We found that aligner accuracy is heavily dependent on sequence identity. For sequences with 100% identity, Pairagon achieved accuracy levels of >99.6%, with one quarter of the errors of any other aligner. Furthermore, for human/mouse alignments, which are only 85% identical, Pairagon achieved 87% accuracy, higher than any other aligner. Pairagon source and executables are freely available at http://mblab.wustl.edu/software/pairagon/

Genomes of the Mouse Collaborative Cross.

PubMed

Srivastava, Anuj; Morgan, Andrew P; Najarian, Maya L; Sarsani, Vishal Kumar; Sigmon, J Sebastian; Shorter, John R; Kashfeen, Anwica; McMullan, Rachel C; Williams, Lucy H; Giusti-Rodríguez, Paola; Ferris, Martin T; Sullivan, Patrick; Hock, Pablo; Miller, Darla R; Bell, Timothy A; McMillan, Leonard; Churchill, Gary A; de Villena, Fernando Pardo-Manuel

2017-06-01

The Collaborative Cross (CC) is a multiparent panel of recombinant inbred (RI) mouse strains derived from eight founder laboratory strains. RI panels are popular because of their long-term genetic stability, which enhances reproducibility and integration of data collected across time and conditions. Characterization of their genomes can be a community effort, reducing the burden on individual users. Here we present the genomes of the CC strains using two complementary approaches as a resource to improve power and interpretation of genetic experiments. Our study also provides a cautionary tale regarding the limitations imposed by such basic biological processes as mutation and selection. A distinct advantage of inbred panels is that genotyping only needs to be performed on the panel, not on each individual mouse. The initial CC genome data were haplotype reconstructions based on dense genotyping of the most recent common ancestors (MRCAs) of each strain followed by imputation from the genome sequence of the corresponding founder inbred strain. The MRCA resource captured segregating regions in strains that were not fully inbred, but it had limited resolution in the transition regions between founder haplotypes, and there was uncertainty about founder assignment in regions of limited diversity. Here we report the whole genome sequence of 69 CC strains generated by paired-end short reads at 30× coverage of a single male per strain. Sequencing leads to a substantial improvement in the fine structure and completeness of the genomes of the CC. Both MRCAs and sequenced samples show a significant reduction in the genome-wide haplotype frequencies from two wild-derived strains, CAST/EiJ and PWK/PhJ. In addition, analysis of the evolution of the patterns of heterozygosity indicates that selection against three wild-derived founder strains played a significant role in shaping the genomes of the CC. The sequencing resource provides the first description of tens of thousands of new genetic variants introduced by mutation and drift in the CC genomes. We estimate that new SNP mutations are accumulating in each CC strain at a rate of 2.4 ± 0.4 per gigabase per generation. The fixation of new mutations by genetic drift has introduced thousands of new variants into the CC strains. The majority of these mutations are novel compared to currently sequenced laboratory stocks and wild mice, and some are predicted to alter gene function. Approximately one-third of the CC inbred strains have acquired large deletions (>10 kb) many of which overlap known coding genes and functional elements. The sequence of these mice is a critical resource to CC users, increases threefold the number of mouse inbred strain genomes available publicly, and provides insight into the effect of mutation and drift on common resources. Copyright © 2017 Srivastava et al.

Genomes of the Mouse Collaborative Cross

PubMed Central

Srivastava, Anuj; Morgan, Andrew P.; Najarian, Maya L.; Sarsani, Vishal Kumar; Sigmon, J. Sebastian; Shorter, John R.; Kashfeen, Anwica; McMullan, Rachel C.; Williams, Lucy H.; Giusti-Rodríguez, Paola; Ferris, Martin T.; Sullivan, Patrick; Hock, Pablo; Miller, Darla R.; Bell, Timothy A.; McMillan, Leonard; Churchill, Gary A.; de Villena, Fernando Pardo-Manuel

2017-01-01

The Collaborative Cross (CC) is a multiparent panel of recombinant inbred (RI) mouse strains derived from eight founder laboratory strains. RI panels are popular because of their long-term genetic stability, which enhances reproducibility and integration of data collected across time and conditions. Characterization of their genomes can be a community effort, reducing the burden on individual users. Here we present the genomes of the CC strains using two complementary approaches as a resource to improve power and interpretation of genetic experiments. Our study also provides a cautionary tale regarding the limitations imposed by such basic biological processes as mutation and selection. A distinct advantage of inbred panels is that genotyping only needs to be performed on the panel, not on each individual mouse. The initial CC genome data were haplotype reconstructions based on dense genotyping of the most recent common ancestors (MRCAs) of each strain followed by imputation from the genome sequence of the corresponding founder inbred strain. The MRCA resource captured segregating regions in strains that were not fully inbred, but it had limited resolution in the transition regions between founder haplotypes, and there was uncertainty about founder assignment in regions of limited diversity. Here we report the whole genome sequence of 69 CC strains generated by paired-end short reads at 30× coverage of a single male per strain. Sequencing leads to a substantial improvement in the fine structure and completeness of the genomes of the CC. Both MRCAs and sequenced samples show a significant reduction in the genome-wide haplotype frequencies from two wild-derived strains, CAST/EiJ and PWK/PhJ. In addition, analysis of the evolution of the patterns of heterozygosity indicates that selection against three wild-derived founder strains played a significant role in shaping the genomes of the CC. The sequencing resource provides the first description of tens of thousands of new genetic variants introduced by mutation and drift in the CC genomes. We estimate that new SNP mutations are accumulating in each CC strain at a rate of 2.4 ± 0.4 per gigabase per generation. The fixation of new mutations by genetic drift has introduced thousands of new variants into the CC strains. The majority of these mutations are novel compared to currently sequenced laboratory stocks and wild mice, and some are predicted to alter gene function. Approximately one-third of the CC inbred strains have acquired large deletions (>10 kb) many of which overlap known coding genes and functional elements. The sequence of these mice is a critical resource to CC users, increases threefold the number of mouse inbred strain genomes available publicly, and provides insight into the effect of mutation and drift on common resources. PMID:28592495

Strategies and tools for whole genome alignments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Couronne, Olivier; Poliakov, Alexander; Bray, Nicolas

2002-11-25

The availability of the assembled mouse genome makespossible, for the first time, an alignment and comparison of two largevertebrate genomes. We have investigated different strategies ofalignment for the subsequent analysis of conservation of genomes that areeffective for different quality assemblies. These strategies were appliedto the comparison of the working draft of the human genome with the MouseGenome Sequencing Consortium assembly, as well as other intermediatemouse assemblies. Our methods are fast and the resulting alignmentsexhibit a high degree of sensitivity, covering more than 90 percent ofknown coding exons in the human genome. We have obtained such coveragewhile preserving specificity. With amore » view towards the end user, we havedeveloped a suite of tools and websites for automatically aligning, andsubsequently browsing and working with whole genome comparisons. Wedescribe the use of these tools to identify conserved non-coding regionsbetween the human and mouse genomes, some of which have not beenidentified by other methods.« less

YAC cloning Mus musculus telomeric DNA: physical, genetic, in situ and STS markers for the distal telomere of chromosome 10.

PubMed

Kipling, D; Wilson, H E; Thomson, E J; Cooke, H J

1995-06-01

Three Mus musculus DBA/2 YAC libraries were constructed using a half-YAC telomere cloning vector. This functional complementation approach yields libraries which include terminal restriction fragments of the mouse genome. Screening all three libraries led to the isolation of 32 independent clones which carry linear YACs containing the mouse terminal repeat sequence, (TTAGGG)n. These YACs provide a resource to isolate regions of the mouse genome close to chromosome termini and excluded from existing conventional YAC libraries. To demonstrate their utility, a hybridization probe was isolated from Mtel-1, the first (TTAGGG)n-containing YAC isolated. This probe detects a approximately 70 kb Kpnl fragment in the mouse genome which is sensitive to pretreatment with BAL31 exonuclease. A PCR-based genetic marker generated from the sequence of this probe maps 4.4 cM from the most distal anchor locus on chromosome 10 in the EUCIB interspecific backcross. STS primers for this locus, D10Hgu1, were used to isolate YAC 110F4 from a commercially available mouse YAC library. Fluorescence in situ hybridization demonstrates that YAC 110F4 hybridizes to the distal telomere of chromosome 10. Clones in this collection of telomere YACs therefore partially overlap clones in conventional YAC libraries, and thus the previously unavailable terminal regions of the mouse genome can now be linked with the developing mouse STS YAC contig. Genetic markers such as D10Hgu1 allow the ends of the mouse genetic map to be defined, thus closing the map.

Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs.

PubMed

Holmes, Roger S; Goldberg, Erwin

2009-10-01

Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals.

Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs

PubMed Central

Holmes, Roger S; Goldberg, Erwin

2009-01-01

Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals. PMID:19679512

Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

A genome survey sequencing of the Java mouse deer (Tragulus javanicus) adds new aspects to the evolution of lineage specific retrotransposons in Ruminantia (Cetartiodactyla).

PubMed

Gallus, S; Kumar, V; Bertelsen, M F; Janke, A; Nilsson, M A

2015-10-25

Ruminantia, the ruminating, hoofed mammals (cow, deer, giraffe and allies) are an unranked artiodactylan clade. Around 50-60 million years ago the BovB retrotransposon entered the ancestral ruminantian genome through horizontal gene transfer. A survey genome screen using 454-pyrosequencing of the Java mouse deer (Tragulus javanicus) and the lesser kudu (Tragelaphus imberbis) was done to investigate and to compare the landscape of transposable elements within Ruminantia. The family Tragulidae (mouse deer) is the only representative of Tragulina and phylogenetically important, because it represents the earliest divergence in Ruminantia. The data analyses show that, relative to other ruminantian species, the lesser kudu genome has seen an expansion of BovB Long INterspersed Elements (LINEs) and BovB related Short INterspersed Elements (SINEs) like BOVA2. In comparison the genome of Java mouse deer has fewer BovB elements than other ruminants, especially Bovinae, and has in addition a novel CHR-3 SINE most likely propagated by LINE-1. By contrast the other ruminants have low amounts of CHR SINEs but high numbers of actively propagating BovB-derived and BovB-propagated SINEs. The survey sequencing data suggest that the transposable element landscape in mouse deer (Tragulina) is unique among Ruminantia, suggesting a lineage specific evolutionary trajectory that does not involve BovB mediated retrotransposition. This shows that the genomic landscape of mobile genetic elements can rapidly change in any lineage. Copyright © 2015 Elsevier B.V. All rights reserved.

Sequence Diversity, Intersubgroup Relationships, and Origins of the Mouse Leukemia Gammaretroviruses of Laboratory and Wild Mice.

PubMed

Bamunusinghe, Devinka; Naghashfar, Zohreh; Buckler-White, Alicia; Plishka, Ronald; Baliji, Surendranath; Liu, Qingping; Kassner, Joshua; Oler, Andrew J; Hartley, Janet; Kozak, Christine A

2016-04-01

Mouse leukemia viruses (MLVs) are found in the common inbred strains of laboratory mice and in the house mouse subspecies ofMus musculus Receptor usage and envelope (env) sequence variation define three MLV host range subgroups in laboratory mice: ecotropic, polytropic, and xenotropic MLVs (E-, P-, and X-MLVs, respectively). These exogenous MLVs derive from endogenous retroviruses (ERVs) that were acquired by the wild mouse progenitors of laboratory mice about 1 million years ago. We analyzed the genomes of seven MLVs isolated from Eurasian and American wild mice and three previously sequenced MLVs to describe their relationships and identify their possible ERV progenitors. The phylogenetic tree based on the receptor-determining regions ofenvproduced expected host range clusters, but these clusters are not maintained in trees generated from other virus regions. Colinear alignments of the viral genomes identified segmental homologies to ERVs of different host range subgroups. Six MLVs show close relationships to a small xenotropic ERV subgroup largely confined to the inbred mouse Y chromosome.envvariations define three E-MLV subtypes, one of which carries duplications of various sizes, sequences, and locations in the proline-rich region ofenv Outside theenvregion, all E-MLVs are related to different nonecotropic MLVs. These results document the diversity in gammaretroviruses isolated from globally distributedMussubspecies, provide insight into their origins and relationships, and indicate that recombination has had an important role in the evolution of these mutagenic and pathogenic agents. Laboratory mice carry mouse leukemia viruses (MLVs) of three host range groups which were acquired from their wild mouse progenitors. We sequenced the complete genomes of seven infectious MLVs isolated from geographically separated Eurasian and American wild mice and compared them with endogenous germ line retroviruses (ERVs) acquired early in house mouse evolution. We did this because the laboratory mouse viruses derive directly from specific ERVs or arise by recombination between different ERVs. The six distinctively different wild mouse viruses appear to be recombinants, often involving different host range subgroups, and most are related to a distinctive, largely Y-chromosome-linked MLV ERV subtype. MLVs with ecotropic host ranges show the greatest variability with extensive inter- and intrasubtype envelope differences and with homologies to other host range subgroups outside the envelope. The sequence diversity among these wild mouse isolates helps define their relationships and origins and emphasizes the importance of recombination in their evolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Extensive Mobilome-Driven Genome Diversification in Mouse Gut-Associated Bacteroides vulgatus mpk

PubMed Central

Lange, Anna; Beier, Sina; Steimle, Alex; Autenrieth, Ingo B.; Huson, Daniel H.; Frick, Julia-Stefanie

2016-01-01

Like many other Bacteroides species, Bacteroides vulgatus strain mpk, a mouse fecal isolate which was shown to promote intestinal homeostasis, utilizes a variety of mobile elements for genome evolution. Based on sequences collected by Pacific Biosciences SMRT sequencing technology, we discuss the challenges of assembling and studying a bacterial genome of high plasticity. Additionally, we conducted comparative genomics comparing this commensal strain with the B. vulgatus type strain ATCC 8482 as well as multiple other Bacteroides and Parabacteroides strains to reveal the most important differences and identify the unique features of B. vulgatus mpk. The genome of B. vulgatus mpk harbors a large and diverse set of mobile element proteins compared with other sequenced Bacteroides strains. We found evidence of a number of different horizontal gene transfer events and a genome landscape that has been extensively altered by different mobilization events. A CRISPR/Cas system could be identified that provides a possible mechanism for preventing the integration of invading external DNA. We propose that the high genome plasticity and the introduced genome instabilities of B. vulgatus mpk arising from the various mobilization events might play an important role not only in its adaptation to the challenging intestinal environment in general, but also in its ability to interact with the gut microbiota. PMID:27071651

A novel bioinformatics method for efficient knowledge discovery by BLSOM from big genomic sequence data.

PubMed

Bai, Yu; Iwasaki, Yuki; Kanaya, Shigehiko; Zhao, Yue; Ikemura, Toshimichi

2014-01-01

With remarkable increase of genomic sequence data of a wide range of species, novel tools are needed for comprehensive analyses of the big sequence data. Self-Organizing Map (SOM) is an effective tool for clustering and visualizing high-dimensional data such as oligonucleotide composition on one map. By modifying the conventional SOM, we have previously developed Batch-Learning SOM (BLSOM), which allows classification of sequence fragments according to species, solely depending on the oligonucleotide composition. In the present study, we introduce the oligonucleotide BLSOM used for characterization of vertebrate genome sequences. We first analyzed pentanucleotide compositions in 100 kb sequences derived from a wide range of vertebrate genomes and then the compositions in the human and mouse genomes in order to investigate an efficient method for detecting differences between the closely related genomes. BLSOM can recognize the species-specific key combination of oligonucleotide frequencies in each genome, which is called a "genome signature," and the specific regions specifically enriched in transcription-factor-binding sequences. Because the classification and visualization power is very high, BLSOM is an efficient powerful tool for extracting a wide range of information from massive amounts of genomic sequences (i.e., big sequence data).

Complete Genome Sequence of Escherichia coli Strain M8, Isolated from ob/ob Mice

PubMed Central

Siddharth, Jay; Membrez, Mathieu; Chakrabarti, Anirikh; Betrisey, Bertrand; Chou, Chieh Jason

2017-01-01

ABSTRACT Escherichia coli is one of the common inhabitants of the mammalian gastrointestinal track. We isolated a strain from an ob/ob mouse and performed whole-genome sequencing, which yielded a chromosome of ~5.1 Mb and three plasmids of ~160 kb, ~6 kb, and ~4 kb. PMID:28572322

Efficient genome editing of differentiated renal epithelial cells.

PubMed

Hofherr, Alexis; Busch, Tilman; Huber, Nora; Nold, Andreas; Bohn, Albert; Viau, Amandine; Bienaimé, Frank; Kuehn, E Wolfgang; Arnold, Sebastian J; Köttgen, Michael

2017-02-01

Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.

Whole Genome Sequence of Two Wild-Derived Mus musculus domesticus Inbred Strains, LEWES/EiJ and ZALENDE/EiJ, with Different Diploid Numbers

PubMed Central

Morgan, Andrew P.; Didion, John P.; Doran, Anthony G.; Holt, James M.; McMillan, Leonard; Keane, Thomas M.; de Villena, Fernando Pardo-Manuel

2016-01-01

Wild-derived mouse inbred strains are becoming increasingly popular for complex traits analysis, evolutionary studies, and systems genetics. Here, we report the whole-genome sequencing of two wild-derived mouse inbred strains, LEWES/EiJ and ZALENDE/EiJ, of Mus musculus domesticus origin. These two inbred strains were selected based on their geographic origin, karyotype, and use in ongoing research. We generated 14× and 18× coverage sequence, respectively, and discovered over 1.1 million novel variants, most of which are private to one of these strains. This report expands the number of wild-derived inbred genomes in the Mus genus from six to eight. The sequence variation can be accessed via an online query tool; variant calls (VCF format) and alignments (BAM format) are available for download from a dedicated ftp site. Finally, the sequencing data have also been stored in a lossless, compressed, and indexed format using the multi-string Burrows-Wheeler transform. All data can be used without restriction. PMID:27765810

Human, Mouse, and Rat Genome Large-Scale Rearrangements: Stability Versus Speciation

PubMed Central

Zhao, Shaying; Shetty, Jyoti; Hou, Lihua; Delcher, Arthur; Zhu, Baoli; Osoegawa, Kazutoyo; de Jong, Pieter; Nierman, William C.; Strausberg, Robert L.; Fraser, Claire M.

2004-01-01

Using paired-end sequences from bacterial artificial chromosomes, we have constructed high-resolution synteny and rearrangement breakpoint maps among human, mouse, and rat genomes. Among the >300 syntenic blocks identified are segments of over 40 Mb without any detected interspecies rearrangements, as well as regions with frequently broken synteny and extensive rearrangements. As closely related species, mouse and rat share the majority of the breakpoints and often have the same types of rearrangements when compared with the human genome. However, the breakpoints not shared between them indicate that mouse rearrangements are more often interchromosomal, whereas intrachromosomal rearrangements are more prominent in rat. Centromeres may have played a significant role in reorganizing a number of chromosomes in all three species. The comparison of the three species indicates that genome rearrangements follow a path that accommodates a delicate balance between maintaining a basic structure underlying all mammalian species and permitting variations that are necessary for speciation. PMID:15364903

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

PubMed Central

2008-01-01

Background The draft mouse (Mus musculus) genome sequence revealed an unexpected proliferation of gene duplicates encoding a family of secretoglobin proteins including the androgen-binding protein (ABP) α, β and γ subunits. Further investigation of 14 α-like (Abpa) and 13 β- or γ-like (Abpbg) undisrupted gene sequences revealed a rich diversity of developmental stage-, sex- and tissue-specific expression. Despite these studies, our understanding of the evolution of this gene family remains incomplete. Questions arise from imperfections in the initial mouse genome assembly and a dearth of information about the gene family structure in other rodents and mammals. Results Here, we interrogate the latest 'finished' mouse (Mus musculus) genome sequence assembly to show that the Abp gene repertoire is, in fact, twice as large as reported previously, with 30 Abpa and 34 Abpbg genes and pseudogenes. All of these have arisen since the last common ancestor with rat (Rattus norvegicus). We then demonstrate, by sequencing homologs from species within the Mus genus, that this burst of gene duplication occurred very recently, within the past seven million years. Finally, we survey Abp orthologs in genomes from across the mammalian clade and show that bursts of Abp gene duplications are not specific to the murid rodents; they also occurred recently in the lagomorph (rabbit, Oryctolagus cuniculus) and ruminant (cattle, Bos taurus) lineages, although not in other mammalian taxa. Conclusion We conclude that Abp genes have undergone repeated bursts of gene duplication and adaptive sequence diversification driven by these genes' participation in chemosensation and/or sexual identification. PMID:18269759

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

PubMed

Laukaitis, Christina M; Heger, Andreas; Blakley, Tyler D; Munclinger, Pavel; Ponting, Chris P; Karn, Robert C

2008-02-12

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

Using optical mapping data for the improvement of vertebrate genome assemblies.

PubMed

Howe, Kerstin; Wood, Jonathan M D

2015-01-01

Optical mapping is a technology that gathers long-range information on genome sequences similar to ordered restriction digest maps. Because it is not subject to cloning, amplification, hybridisation or sequencing bias, it is ideally suited to the improvement of fragmented genome assemblies that can no longer be improved by classical methods. In addition, its low cost and rapid turnaround make it equally useful during the scaffolding process of de novo assembly from high throughput sequencing reads. We describe how optical mapping has been used in practice to produce high quality vertebrate genome assemblies. In particular, we detail the efforts undertaken by the Genome Reference Consortium (GRC), which maintains the reference genomes for human, mouse, zebrafish and chicken, and uses different optical mapping platforms for genome curation.

Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

PubMed

Lee, Kang-Hoon; Lim, Debora; Chiu, Sophia; Greenhalgh, David; Cho, Kiho

2016-04-01

Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficient analysis of mouse genome sequences reveal many nonsense variants

PubMed Central

Steeland, Sophie; Timmermans, Steven; Van Ryckeghem, Sara; Hulpiau, Paco; Saeys, Yvan; Van Montagu, Marc; Vandenbroucke, Roosmarijn E.; Libert, Claude

2016-01-01

Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1. PMID:27147605

Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair

PubMed Central

Gelincik, Ozkan; Blecua, Pedro; Edelmann, Winfried; Kucherlapati, Raju; Zhou, Kathy; Jasin, Maria; Gümüş, Zeynep H.; Lipkin, Steven M.

2017-01-01

Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression. PMID:29069730

FANTOM5 CAGE profiles of human and mouse reprocessed for GRCh38 and GRCm38 genome assemblies.

PubMed

Abugessaisa, Imad; Noguchi, Shuhei; Hasegawa, Akira; Harshbarger, Jayson; Kondo, Atsushi; Lizio, Marina; Severin, Jessica; Carninci, Piero; Kawaji, Hideya; Kasukawa, Takeya

2017-08-29

The FANTOM5 consortium described the promoter-level expression atlas of human and mouse by using CAGE (Cap Analysis of Gene Expression) with single molecule sequencing. In the original publications, GRCh37/hg19 and NCBI37/mm9 assemblies were used as the reference genomes of human and mouse respectively; later, the Genome Reference Consortium released newer genome assemblies GRCh38/hg38 and GRCm38/mm10. To increase the utility of the atlas in forthcoming researches, we reprocessed the data to make them available on the recent genome assemblies. The data include observed frequencies of transcription starting sites (TSSs) based on the realignment of CAGE reads, and TSS peaks that are converted from those based on the previous reference. Annotations of the peak names were also updated based on the latest public databases. The reprocessed results enable us to examine frequencies of transcription initiations on the recent genome assemblies and to refer promoters with updated information across the genome assemblies consistently.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes).

PubMed

Dessimoz, Christophe; Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-09-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes)

PubMed Central

Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-01-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references. PMID:21712341

Extensive Mobilome-Driven Genome Diversification in Mouse Gut-Associated Bacteroides vulgatus mpk.

PubMed

Lange, Anna; Beier, Sina; Steimle, Alex; Autenrieth, Ingo B; Huson, Daniel H; Frick, Julia-Stefanie

2016-04-25

Like many other Bacteroides species, Bacteroides vulgatus strain mpk, a mouse fecal isolate which was shown to promote intestinal homeostasis, utilizes a variety of mobile elements for genome evolution. Based on sequences collected by Pacific Biosciences SMRT sequencing technology, we discuss the challenges of assembling and studying a bacterial genome of high plasticity. Additionally, we conducted comparative genomics comparing this commensal strain with the B. vulgatus type strain ATCC 8482 as well as multiple other Bacteroides and Parabacteroides strains to reveal the most important differences and identify the unique features of B. vulgatus mpk. The genome of B. vulgatus mpk harbors a large and diverse set of mobile element proteins compared with other sequenced Bacteroides strains. We found evidence of a number of different horizontal gene transfer events and a genome landscape that has been extensively altered by different mobilization events. A CRISPR/Cas system could be identified that provides a possible mechanism for preventing the integration of invading external DNA. We propose that the high genome plasticity and the introduced genome instabilities of B. vulgatus mpk arising from the various mobilization events might play an important role not only in its adaptation to the challenging intestinal environment in general, but also in its ability to interact with the gut microbiota. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

PubMed Central

Caignard, Grégory; Eva, Megan M.; van Bruggen, Rebekah; Eveleigh, Robert; Bourque, Guillaume; Malo, Danielle; Gros, Philippe; Vidal, Silvia M.

2014-01-01

Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU) has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses. PMID:25268389

ChIP-seq Accurately Predicts Tissue-Specific Activity of Enhancers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Visel, Axel; Blow, Matthew J.; Li, Zirong

2009-02-01

A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover since they are scattered amongst the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here, we performed chromatin immunoprecipitation with the enhancer-associated protein p300, followed by massively-parallel sequencing, to map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain, and limb tissue. Wemore » tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases revealed reproducible enhancer activity in those tissues predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities and suggest that such datasets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.« less

The genomic landscape shaped by selection on transposable elements across 18 mouse strains.

PubMed

Nellåker, Christoffer; Keane, Thomas M; Yalcin, Binnaz; Wong, Kim; Agam, Avigail; Belgard, T Grant; Flint, Jonathan; Adams, David J; Frankel, Wayne N; Ponting, Chris P

2012-06-15

Transposable element (TE)-derived sequence dominates the landscape of mammalian genomes and can modulate gene function by dysregulating transcription and translation. Our current knowledge of TEs in laboratory mouse strains is limited primarily to those present in the C57BL/6J reference genome, with most mouse TEs being drawn from three distinct classes, namely short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs) and the endogenous retrovirus (ERV) superfamily. Despite their high prevalence, the different genomic and gene properties controlling whether TEs are preferentially purged from, or are retained by, genetic drift or positive selection in mammalian genomes remain poorly defined. Using whole genome sequencing data from 13 classical laboratory and 4 wild-derived mouse inbred strains, we developed a comprehensive catalogue of 103,798 polymorphic TE variants. We employ this extensive data set to characterize TE variants across the Mus lineage, and to infer neutral and selective processes that have acted over 2 million years. Our results indicate that the majority of TE variants are introduced though the male germline and that only a minority of TE variants exert detectable changes in gene expression. However, among genes with differential expression across the strains there are twice as many TE variants identified as being putative causal variants as expected. Most TE variants that cause gene expression changes appear to be purged rapidly by purifying selection. Our findings demonstrate that past TE insertions have often been highly deleterious, and help to prioritize TE variants according to their likely contribution to gene expression or phenotype variation.

HuMiChip: Development of a Functional Gene Array for the Study of Human Microbiomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tu, Q.; Deng, Ye; Lin, Lu

Microbiomes play very important roles in terms of nutrition, health and disease by interacting with their hosts. Based on sequence data currently available in public domains, we have developed a functional gene array to monitor both organismal and functional gene profiles of normal microbiota in human and mouse hosts, and such an array is called human and mouse microbiota array, HMM-Chip. First, seed sequences were identified from KEGG databases, and used to construct a seed database (seedDB) containing 136 gene families in 19 metabolic pathways closely related to human and mouse microbiomes. Second, a mother database (motherDB) was constructed withmore » 81 genomes of bacterial strains with 54 from gut and 27 from oral environments, and 16 metagenomes, and used for selection of genes and probe design. Gene prediction was performed by Glimmer3 for bacterial genomes, and by the Metagene program for metagenomes. In total, 228,240 and 801,599 genes were identified for bacterial genomes and metagenomes, respectively. Then the motherDB was searched against the seedDB using the HMMer program, and gene sequences in the motherDB that were highly homologous with seed sequences in the seedDB were used for probe design by the CommOligo software. Different degrees of specific probes, including gene-specific, inclusive and exclusive group-specific probes were selected. All candidate probes were checked against the motherDB and NCBI databases for specificity. Finally, 7,763 probes covering 91.2percent (12,601 out of 13,814) HMMer confirmed sequences from 75 bacterial genomes and 16 metagenomes were selected. This developed HMM-Chip is able to detect the diversity and abundance of functional genes, the gene expression of microbial communities, and potentially, the interactions of microorganisms and their hosts.« less

The Importance of Biological Databases in Biological Discovery.

PubMed

Baxevanis, Andreas D; Bateman, Alex

2015-06-19

Biological databases play a central role in bioinformatics. They offer scientists the opportunity to access a wide variety of biologically relevant data, including the genomic sequences of an increasingly broad range of organisms. This unit provides a brief overview of major sequence databases and portals, such as GenBank, the UCSC Genome Browser, and Ensembl. Model organism databases, including WormBase, The Arabidopsis Information Resource (TAIR), and those made available through the Mouse Genome Informatics (MGI) resource, are also covered. Non-sequence-centric databases, such as Online Mendelian Inheritance in Man (OMIM), the Protein Data Bank (PDB), MetaCyc, and the Kyoto Encyclopedia of Genes and Genomes (KEGG), are also discussed. Copyright © 2015 John Wiley & Sons, Inc.

Single-Stranded γPNAs for In Vivo Site-Specific Genome Editing via Watson-Crick Recognition

PubMed Central

Bahal, Raman; Quijano, Elias; McNeer, Nicole Ali; Liu, Yanfeng; Bhunia, Dinesh C.; López-Giráldez, Francesco; Fields, Rachel J.; Saltzman, W. Mark; Ly, Danith H.; Glazer, Peter M.

2014-01-01

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction. PMID:25174576

Single-stranded γPNAs for in vivo site-specific genome editing via Watson-Crick recognition.

PubMed

Bahal, Raman; Quijano, Elias; McNeer, Nicole A; Liu, Yanfeng; Bhunia, Dinesh C; Lopez-Giraldez, Francesco; Fields, Rachel J; Saltzman, William M; Ly, Danith H; Glazer, Peter M

2014-01-01

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.

Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases

PubMed Central

Fanslow, Danielle A.; Wirt, Stacey E.; Barker, Jenny C.; Connelly, Jon P.; Porteus, Matthew H.; Dann, Christina Tenenhaus

2014-01-01

Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse “GS” (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro. PMID:25409432

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research.

PubMed

Miano, Joseph M; Zhu, Qiuyu Martin; Lowenstein, Charles J

2016-06-01

Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of laboratories could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any laboratory can quickly assemble reagents for developing new mouse models for cardiovascular research. Here, we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for 2-component and 3-component CRISPR editing are summarized with many applications in mice including frameshift mutations, deletion of enhancers and noncoding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions. © 2016 American Heart Association, Inc.

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

PubMed Central

Miano, Joseph M.; Zhu, Qiuyu Martin; Lowenstein, Charles J.

2016-01-01

Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of labs could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any lab can quickly assemble reagents for developing new mouse models for cardiovascular research. Here we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for two-component and three-component CRISPR editing are summarized with a number of applications in mice including frameshift mutations, deletion of enhancers and non-coding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions. PMID:27102963

CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

PubMed Central

Naito, Yuki; Hino, Kimihiro; Bono, Hidemasa; Ui-Tei, Kumiko

2015-01-01

Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25414360

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

PubMed

Maggi, Elaine C; Gravina, Silvia; Cheng, Haiying; Piperdi, Bilal; Yuan, Ziqiang; Dong, Xiao; Libutti, Steven K; Vijg, Jan; Montagna, Cristina

2018-01-01

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

PubMed Central

Grey, Corinne; Clément, Julie A.J.; Buard, Jérôme; Leblanc, Benjamin; Gut, Ivo; Gut, Marta; Duret, Laurent

2017-01-01

In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis. PMID:28336543

Structure and polymorphism of the mouse myelin/oligodendrocyte glycoprotein gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daubas, P.; Pham-Dinh, D.; Dautigny, A.

1994-09-01

The authors have isolated and characterized genomic clones containing the mouse myelin/oligodendrocyte glycoprotein (MOG) gene. It spans a region of 12.5 kb and consists of eight exons. Its exon-intron structure differs from that of classical MHC-class I genes, with which it is linked in the mouse genome. Nucleotide sequencing of the 5{prime} flanking region revelas that it contains several putative protein-binding sites, some of them in common with other myelin gene promoters. One intragenic polymorphism has been identified: it consists of a GA repeat, defining at least three alleles in mouse inbred strains, and is easily detectable using the polymerasemore » chain reaction method.« less

The Fecal Viral Flora of Wild Rodents

PubMed Central

Phan, Tung G.; Kapusinszky, Beatrix; Wang, Chunlin; Rose, Robert K.; Lipton, Howard L.; Delwart, Eric L.

2011-01-01

The frequent interactions of rodents with humans make them a common source of zoonotic infections. To obtain an initial unbiased measure of the viral diversity in the enteric tract of wild rodents we sequenced partially purified, randomly amplified viral RNA and DNA in the feces of 105 wild rodents (mouse, vole, and rat) collected in California and Virginia. We identified in decreasing frequency sequences related to the mammalian viruses families Circoviridae, Picobirnaviridae, Picornaviridae, Astroviridae, Parvoviridae, Papillomaviridae, Adenoviridae, and Coronaviridae. Seventeen small circular DNA genomes containing one or two replicase genes distantly related to the Circoviridae representing several potentially new viral families were characterized. In the Picornaviridae family two new candidate genera as well as a close genetic relative of the human pathogen Aichi virus were characterized. Fragments of the first mouse sapelovirus and picobirnaviruses were identified and the first murine astrovirus genome was characterized. A mouse papillomavirus genome and fragments of a novel adenovirus and adenovirus-associated virus were also sequenced. The next largest fraction of the rodent fecal virome was related to insect viruses of the Densoviridae, Iridoviridae, Polydnaviridae, Dicistroviriade, Bromoviridae, and Virgaviridae families followed by plant virus-related sequences in the Nanoviridae, Geminiviridae, Phycodnaviridae, Secoviridae, Partitiviridae, Tymoviridae, Alphaflexiviridae, and Tombusviridae families reflecting the largely insect and plant rodent diet. Phylogenetic analyses of full and partial viral genomes therefore revealed many previously unreported viral species, genera, and families. The close genetic similarities noted between some rodent and human viruses might reflect past zoonoses. This study increases our understanding of the viral diversity in wild rodents and highlights the large number of still uncharacterized viruses in mammals. PMID:21909269

The detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamic organization of the human and mouse genomes.

PubMed

Knoch, Tobias A; Wachsmuth, Malte; Kepper, Nick; Lesnussa, Michael; Abuseiris, Anis; Ali Imam, A M; Kolovos, Petros; Zuin, Jessica; Kockx, Christel E M; Brouwer, Rutger W W; van de Werken, Harmen J G; van IJcken, Wilfred F J; Wendt, Kerstin S; Grosveld, Frank G

2016-01-01

The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture ( T2C ), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.

Genomic interval engineering of mice identified a novel modulator of triglyceride production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Y.; Jong, M.C.; Frazer, K.A.

1999-10-01

To accelerate the biological annotation of novel genes discovered in sequenced of mammalian genomes, we are creating large deletions in the mouse genome targeted to include clusters of such genes. Here we describe the targeted deletion of a 450 kb region on mouse chromosome 11 which, based on computational analysis of the deleted murine sequences and human 5q orthologous sequences, codes for nine putative genes. Mice homozygous for the deletion had a variety of abnormalities including severe hypertriglyceridemia, hepatic and cardiac enlargement, growth retardation and premature mortality. Analysis of triglyceride metabolism in these animals demonstrated a several-fold increase in hepaticmore » very-low density lipoprotein (VLDL) triglyceride secretion, the most prevalent mechanism responsible for hypertriglyceridemia in humans. A series of mouse BAC and human YAC transgenes covering different intervals of the 450 kb deleted region were assessed for their ability to complement the deletion induced abnormalities. These studies revealed that OCTN2, a gene recently shown to play a role in carnitine transport, was able to correct the triglyceride abnormalities. The discovery of this previously unappreciated relationship between OCTN2, carnitine and hepatic triglyceride production is of particular importance due to the clinical consequence of hypertriglyceridemia and the paucity of genes known to modulate triglyceride secretion.« less

Creating reference gene annotation for the mouse C57BL6/J genome assembly.

PubMed

Mudge, Jonathan M; Harrow, Jennifer

2015-10-01

Annotation on the reference genome of the C57BL6/J mouse has been an ongoing project ever since the draft genome was first published. Initially, the principle focus was on the identification of all protein-coding genes, although today the importance of describing long non-coding RNAs, small RNAs, and pseudogenes is recognized. Here, we describe the progress of the GENCODE mouse annotation project, which combines manual annotation from the HAVANA group with Ensembl computational annotation, alongside experimental and in silico validation pipelines from other members of the consortium. We discuss the more recent incorporation of next-generation sequencing datasets into this workflow, including the usage of mass-spectrometry data to potentially identify novel protein-coding genes. Finally, we will outline how the C57BL6/J genebuild can be used to gain insights into the variant sites that distinguish different mouse strains and species.

Sequencing and comparative genomic analysis of 1227 Felis catus cDNA sequences enriched for developmental, clinical and nutritional phenotypes

PubMed Central

2012-01-01

Background The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated. Results We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes. Conclusions The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information. PMID:22257742

Genome-wide compendium and functional assessment of in vivo heart enhancers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dickel, Diane E.; Barozzi, Iros; Zhu, Yiwen

Whole-genome sequencing is identifying growing numbers of non-coding variants in human disease studies, but the lack of accurate functional annotations prevents their interpretation. We describe the genome-wide landscape of distant-acting enhancers active in the developing and adult human heart, an organ whose impairment is a predominant cause of mortality and morbidity. Using integrative analysis of > 35 epigenomic data sets from mouse and human pre-and postnatal hearts we created a comprehensive reference of > 80,000 putative human heart enhancers. To illustrate the importance of enhancers in the regulation of genes involved in heart disease, we deleted the mouse orthologs ofmore » two human enhancers near cardiac myosin genes. In both cases, we observe in vivo expression changes and cardiac phenotypes consistent with human heart disease. Our study provides a comprehensive catalogue of human heart enhancers for use in clinical whole-genome sequencing studies and highlights the importance of enhancers for cardiac function.« less

Genome-wide compendium and functional assessment of in vivo heart enhancers

DOE PAGES

Dickel, Diane E.; Barozzi, Iros; Zhu, Yiwen; ...

2016-10-05

Whole-genome sequencing is identifying growing numbers of non-coding variants in human disease studies, but the lack of accurate functional annotations prevents their interpretation. We describe the genome-wide landscape of distant-acting enhancers active in the developing and adult human heart, an organ whose impairment is a predominant cause of mortality and morbidity. Using integrative analysis of > 35 epigenomic data sets from mouse and human pre-and postnatal hearts we created a comprehensive reference of > 80,000 putative human heart enhancers. To illustrate the importance of enhancers in the regulation of genes involved in heart disease, we deleted the mouse orthologs ofmore » two human enhancers near cardiac myosin genes. In both cases, we observe in vivo expression changes and cardiac phenotypes consistent with human heart disease. Our study provides a comprehensive catalogue of human heart enhancers for use in clinical whole-genome sequencing studies and highlights the importance of enhancers for cardiac function.« less

Genome-wide compendium and functional assessment of in vivo heart enhancers

PubMed Central

Dickel, Diane E.; Barozzi, Iros; Zhu, Yiwen; Fukuda-Yuzawa, Yoko; Osterwalder, Marco; Mannion, Brandon J.; May, Dalit; Spurrell, Cailyn H.; Plajzer-Frick, Ingrid; Pickle, Catherine S.; Lee, Elizabeth; Garvin, Tyler H.; Kato, Momoe; Akiyama, Jennifer A.; Afzal, Veena; Lee, Ah Young; Gorkin, David U.; Ren, Bing; Rubin, Edward M.; Visel, Axel; Pennacchio, Len A.

2016-01-01

Whole-genome sequencing is identifying growing numbers of non-coding variants in human disease studies, but the lack of accurate functional annotations prevents their interpretation. We describe the genome-wide landscape of distant-acting enhancers active in the developing and adult human heart, an organ whose impairment is a predominant cause of mortality and morbidity. Using integrative analysis of >35 epigenomic data sets from mouse and human pre- and postnatal hearts we created a comprehensive reference of >80,000 putative human heart enhancers. To illustrate the importance of enhancers in the regulation of genes involved in heart disease, we deleted the mouse orthologs of two human enhancers near cardiac myosin genes. In both cases, we observe in vivo expression changes and cardiac phenotypes consistent with human heart disease. Our study provides a comprehensive catalogue of human heart enhancers for use in clinical whole-genome sequencing studies and highlights the importance of enhancers for cardiac function. PMID:27703156

A multiplicity of factors contributes to selective RNA polymerase III occupancy of a subset of RNA polymerase III genes in mouse liver

PubMed Central

Canella, Donatella; Bernasconi, David; Gilardi, Federica; LeMartelot, Gwendal; Migliavacca, Eugenia; Praz, Viviane; Cousin, Pascal; Delorenzi, Mauro; Hernandez, Nouria; Hernandez, Nouria; Delorenzi, Mauro; Deplancke, Bart; Desvergne, Béatrice; Guex, Nicolas; Herr, Winship; Naef, Felix; Rougemont, Jacques; Schibler, Ueli; Deplancke, Bart; Guex, Nicolas; Herr, Winship; Guex, Nicolas; Andersin, Teemu; Cousin, Pascal; Gilardi, Federica; Gos, Pascal; Le Martelot, Gwendal; Lammers, Fabienne; Canella, Donatella; Gilardi, Federica; Raghav, Sunil; Fabbretti, Roberto; Fortier, Arnaud; Long, Li; Vlegel, Volker; Xenarios, Ioannis; Migliavacca, Eugenia; Praz, Viviane; Guex, Nicolas; Naef, Felix; Rougemont, Jacques; David, Fabrice; Jarosz, Yohan; Kuznetsov, Dmitry; Liechti, Robin; Martin, Olivier; Ross, Frederick; Sinclair, Lucas; Cajan, Julia; Krier, Irina; Leleu, Marion; Migliavacca, Eugenia; Molina, Nacho; Naldi, Aurélien; Rey, Guillaume; Symul, Laura; Guex, Nicolas; Naef, Felix; Rougemont, Jacques; Bernasconi, David; Delorenzi, Mauro; Andersin, Teemu; Canella, Donatella; Gilardi, Federica; Le Martelot, Gwendal; Lammers, Fabienne; Raghav, Sunil

2012-01-01

The genomic loci occupied by RNA polymerase (RNAP) III have been characterized in human culture cells by genome-wide chromatin immunoprecipitations, followed by deep sequencing (ChIP-seq). These studies have shown that only ∼40% of the annotated 622 human tRNA genes and pseudogenes are occupied by RNAP-III, and that these genes are often in open chromatin regions rich in active RNAP-II transcription units. We have used ChIP-seq to characterize RNAP-III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver RNAP-III-occupied loci including a conserved mammalian interspersed repeat (MIR) as a potential regulator of an RNAP-III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse RNAP-III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence, can strongly affect RNAP-III occupancy of tRNA genes. They reveal correlations with various genomic features that explain the observed variation of 81% of tRNA scores. In mouse liver, loci represented in the NCBI37/mm9 genome assembly that are clearly occupied by RNAP-III comprise 50 Rn5s (5S RNA) genes, 14 known non-tRNA RNAP-III genes, nine Rn4.5s (4.5S RNA) genes, and 29 SINEs. Moreover, out of the 433 annotated tRNA genes, half are occupied by RNAP-III. Transfer RNA gene expression levels reflect both an underlying genomic organization conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes. PMID:22287103

Genome sequences of a mouse-avirulent and a mouse-virulent strain of Ross River virus.

PubMed

Faragher, S G; Meek, A D; Rice, C M; Dalgarno, L

1988-04-01

The nucleotide sequence of the genomic RNA of a mouse-avirulent strain of Ross River virus, RRV NB5092 (isolated in 1969), has been determined and the corresponding sequence for the prototype mouse-virulent strain, RRV T48 (isolated in 1959), has been completed. The RRV NB5092 genome is approximately 11,674 nucleotides in length, compared with 11,853 nucleotides for RRV T48. RRV NB5092 and RRV T48 have the same genome organization. For both viruses an untranslated region of 80 nucleotides at the 5' end of the genome is followed by a 7440-nucleotide open reading frame which is interrupted after 5586 nucleotides by a single opal termination codon. By homology with other alphaviruses, the 5586-nucleotide open reading frame encodes the nonstructural proteins nsP1, nsP2, and nsP3; a fourth nonstructural protein, nsP4, is produced by read-through of the opal codon. The RRV nonstructural proteins show strong homology with the corresponding proteins of Sindbis virus and Semliki Forest virus in terms of size, net charge, and hydropathy characteristics. However, homology is not uniform between or within the proteins; nsP1, nsP2, and nsP4 contain extended domains which are highly conserved between alphaviruses, while the C-terminal region of nsP3 shows little conservation in sequence or length between alphaviruses. An untranslated "junction" region of 44 nucleotides (for RRV NB5092) or 47 nucleotides (for RRV T48) separates the nonstructural and structural protein coding regions. The structural proteins (capsid-E3-E2-6K-E1) are translated from an open reading frame of 3762 nucleotides which is followed by a 3'-untranslated region of approximately 348 nucleotides (for RRV NB5092) or 524 nucleotides (for RRV T48). Excluding deletions and insertions, the genomes of RRV NB5092 and RRV T48 differ at 284 nucleotides, representing a sequence divergence of 2.38%. Sequence deletions or insertions were found only in the noncoding regions and include a 173-nucleotide deletion in the 3'-untranslated region of RRV NB5092, compared with RRV T48. In the coding regions, most of the nucleotide differences are silent; there are 36 amino acid differences in the nonstructural proteins and 12 in the structural proteins. The distribution of amino acid differences between the two RRV strains correlates with the location of domains which are poorly conserved in sequence between alphaviruses. The possible role of amino acid differences in envelope glycoproteins E1 and E2 in determining the different antigenic and biological properties of RRV NB5092 and RRV T48 is discussed.

Nanopore DNA Sequencing and Genome Assembly on the International Space Station.

PubMed

Castro-Wallace, Sarah L; Chiu, Charles Y; John, Kristen K; Stahl, Sarah E; Rubins, Kathleen H; McIntyre, Alexa B R; Dworkin, Jason P; Lupisella, Mark L; Smith, David J; Botkin, Douglas J; Stephenson, Timothy A; Juul, Sissel; Turner, Daniel J; Izquierdo, Fernando; Federman, Scot; Stryke, Doug; Somasekar, Sneha; Alexander, Noah; Yu, Guixia; Mason, Christopher E; Burton, Aaron S

2017-12-21

We evaluated the performance of the MinION DNA sequencer in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. Samples contained equimolar mixtures of genomic DNA from lambda bacteriophage, Escherichia coli (strain K12, MG1655) and Mus musculus (female BALB/c mouse). Nine sequencing runs were performed aboard the ISS over a 6-month period, yielding a total of 276,882 reads with no apparent decrease in performance over time. From sequence data collected aboard the ISS, we constructed directed assemblies of the ~4.6 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% consensus pairwise identity, respectively; de novo assembly of the E. coli genome from raw reads yielded a single contig comprising 99.9% of the genome at 98.6% consensus pairwise identity. Simulated real-time analyses of in-flight sequence data using an automated bioinformatic pipeline and laptop-based genomic assembly demonstrated the feasibility of sequencing analysis and microbial identification aboard the ISS. These findings illustrate the potential for sequencing applications including disease diagnosis, environmental monitoring, and elucidating the molecular basis for how organisms respond to spaceflight.

Comparative analysis of the 5{prime} genomic and promoter regions between the mouse (Hdh) and human Huntington disease (HD) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalchman, M.; Lin, B.; Nasir, J.

1994-09-01

The mouse homologue of the Huntington disease gene (Hdh) has recently been cloned and mapped to a region of synteny with the human, on mouse chromosome 5. The two genes share a high degree of both coding (90% amino acid) and nucleotide (86.2%) identity. We have subsequently performed a detailed comparison of the genomic organization of the 5{prime} region of the two genes encompassing the promoter region and first five exons of both the human and mouse genes. The comparative sequence analysis of the promoter region between HD and Hdh reveals two highly conserved regions. One region (-56 to -118)more » (+1 is the ATG start codon), shared 84% nucleotide identity and another region (-130 to -206) had 81% nucleotide identity. Nine putative Sp1 sites appear in the human promoter region contrasted with only 3 in a similar region in the mouse. Furthermore, 17 and 20 base pair direct repeats present in the HD 5{prime} region are absent in the similar Hdh region. Although both the mouse and human intron/exon boundaries conform to the GT/AG rule, the intron sizes between HD and Hdh are markedly different. The first four introns in Hdh are 15, 7, 5 and 0.5 kb compared to sizes of 10, 15, 7 and 0.5 kb, respectively. Comparison between the mouse and human intronic sequences immediately adjacent to the first five exons (excluding exon 1) reveals only about 46 to 50% identity within the first 60 bp of intronic sequence. Furthermore, we have identified novel polymorphic di-, tri- and tetra-nucleotide repeats in Hdh introns of various mouse strains that are not present in the human. For example, polymorphic CT repeats are present in introns 2 and 4 of Hdh and a novel mouse 56 AAG trinucleotide repeat (interrupted by an AAGG) is also located within intron 2. This information concerning the promoter and genomic organization of both HD and Hdh is critical for designing appropriate gene targetting vectors for studying the normal function of the HD and Hdh genes in model systems.« less

Genomic organization, expression, and chromosome localization of a third aurora-related kinase gene, Aie1.

PubMed

Hu, H M; Chuang, C K; Lee, M J; Tseng, T C; Tang, T K

2000-11-01

We previously reported two novel testis-specific serine/threonine kinases, Aie1 (mouse) and AIE2 (human), that share high amino acid identities with the kinase domains of fly aurora and yeast Ipl1. Here, we report the entire intron-exon organization of the Aie1 gene and analyze the expression patterns of Aie1 mRNA during testis development. The mouse Aie1 gene spans approximately 14 kb and contains seven exons. The sequences of the exon-intron boundaries of the Aie1 gene conform to the consensus sequences (GT/AG) of the splicing donor and acceptor sites of most eukaryotic genes. Comparative genomic sequencing revealed that the gene structure is highly conserved between mouse Aie1 and human AIE2. However, much less homology was found in the sequence outside the kinase-coding domains. The Aie1 locus was mapped to mouse chromosome 7A2-A3 by fluorescent in situ hybridization. Northern blot analysis indicates that Aie1 mRNA likely is expressed at a low level on day 14 and reaches its plateau on day 21 in the developing postnatal testis. RNA in situ hybridization indicated that the expression of the Aie1 transcript was restricted to meiotically active germ cells, with the highest levels detected in spermatocytes at the late pachytene stage. These findings suggest that Aie1 plays a role in spermatogenesis.

Deletion of ultraconserved elements yields viable mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahituv, Nadav; Zhu, Yiwen; Visel, Axel

2007-07-15

Ultraconserved elements have been suggested to retainextended perfect sequence identity between the human, mouse, and ratgenomes due to essential functional properties. To investigate thenecessities of these elements in vivo, we removed four non-codingultraconserved elements (ranging in length from 222 to 731 base pairs)from the mouse genome. To maximize the likelihood of observing aphenotype, we chose to delete elements that function as enhancers in amouse transgenic assay and that are near genes that exhibit markedphenotypes both when completely inactivated in the mouse as well as whentheir expression is altered due to other genomic modifications.Remarkably, all four resulting lines of mice lackingmore » these ultraconservedelements were viable and fertile, and failed to reveal any criticalabnormalities when assayed for a variety of phenotypes including growth,longevity, pathology and metabolism. In addition more targeted screens,informed by the abnormalities observed in mice where genes in proximityto the investigated elements had been altered, also failed to revealnotable abnormalities. These results, while not inclusive of all thepossible phenotypic impact of the deleted sequences, indicate thatextreme sequence constraint does not necessarily reflect crucialfunctions required for viability.« less

A critical assessment of Mus musculus gene function prediction using integrated genomic evidence

PubMed Central

Peña-Castillo, Lourdes; Tasan, Murat; Myers, Chad L; Lee, Hyunju; Joshi, Trupti; Zhang, Chao; Guan, Yuanfang; Leone, Michele; Pagnani, Andrea; Kim, Wan Kyu; Krumpelman, Chase; Tian, Weidong; Obozinski, Guillaume; Qi, Yanjun; Mostafavi, Sara; Lin, Guan Ning; Berriz, Gabriel F; Gibbons, Francis D; Lanckriet, Gert; Qiu, Jian; Grant, Charles; Barutcuoglu, Zafer; Hill, David P; Warde-Farley, David; Grouios, Chris; Ray, Debajyoti; Blake, Judith A; Deng, Minghua; Jordan, Michael I; Noble, William S; Morris, Quaid; Klein-Seetharaman, Judith; Bar-Joseph, Ziv; Chen, Ting; Sun, Fengzhu; Troyanskaya, Olga G; Marcotte, Edward M; Xu, Dong; Hughes, Timothy R; Roth, Frederick P

2008-01-01

Background: Several years after sequencing the human genome and the mouse genome, much remains to be discovered about the functions of most human and mouse genes. Computational prediction of gene function promises to help focus limited experimental resources on the most likely hypotheses. Several algorithms using diverse genomic data have been applied to this task in model organisms; however, the performance of such approaches in mammals has not yet been evaluated. Results: In this study, a standardized collection of mouse functional genomic data was assembled; nine bioinformatics teams used this data set to independently train classifiers and generate predictions of function, as defined by Gene Ontology (GO) terms, for 21,603 mouse genes; and the best performing submissions were combined in a single set of predictions. We identified strengths and weaknesses of current functional genomic data sets and compared the performance of function prediction algorithms. This analysis inferred functions for 76% of mouse genes, including 5,000 currently uncharacterized genes. At a recall rate of 20%, a unified set of predictions averaged 41% precision, with 26% of GO terms achieving a precision better than 90%. Conclusion: We performed a systematic evaluation of diverse, independently developed computational approaches for predicting gene function from heterogeneous data sources in mammals. The results show that currently available data for mammals allows predictions with both breadth and accuracy. Importantly, many highly novel predictions emerge for the 38% of mouse genes that remain uncharacterized. PMID:18613946

Initial sequence and comparative analysis of the cat genome

PubMed Central

Pontius, Joan U.; Mullikin, James C.; Smith, Douglas R.; Lindblad-Toh, Kerstin; Gnerre, Sante; Clamp, Michele; Chang, Jean; Stephens, Robert; Neelam, Beena; Volfovsky, Natalia; Schäffer, Alejandro A.; Agarwala, Richa; Narfström, Kristina; Murphy, William J.; Giger, Urs; Roca, Alfred L.; Antunes, Agostinho; Menotti-Raymond, Marilyn; Yuhki, Naoya; Pecon-Slattery, Jill; Johnson, Warren E.; Bourque, Guillaume; Tesler, Glenn; O’Brien, Stephen J.

2007-01-01

The genome sequence (1.9-fold coverage) of an inbred Abyssinian domestic cat was assembled, mapped, and annotated with a comparative approach that involved cross-reference to annotated genome assemblies of six mammals (human, chimpanzee, mouse, rat, dog, and cow). The results resolved chromosomal positions for 663,480 contigs, 20,285 putative feline gene orthologs, and 133,499 conserved sequence blocks (CSBs). Additional annotated features include repetitive elements, endogenous retroviral sequences, nuclear mitochondrial (numt) sequences, micro-RNAs, and evolutionary breakpoints that suggest historic balancing of translocation and inversion incidences in distinct mammalian lineages. Large numbers of single nucleotide polymorphisms (SNPs), deletion insertion polymorphisms (DIPs), and short tandem repeats (STRs), suitable for linkage or association studies were characterized in the context of long stretches of chromosome homozygosity. In spite of the light coverage capturing ∼65% of euchromatin sequence from the cat genome, these comparative insights shed new light on the tempo and mode of gene/genome evolution in mammals, promise several research applications for the cat, and also illustrate that a comparative approach using more deeply covered mammals provides an informative, preliminary annotation of a light (1.9-fold) coverage mammal genome sequence. PMID:17975172

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

SNPs in putative regulatory regions identified by human mouse comparative sequencing and transcription factor binding site data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Banerjee, Poulabi; Bahlo, Melanie; Schwartz, Jody R.

2002-01-01

Genome wide disease association analysis using SNPs is being explored as a method for dissecting complex genetic traits and a vast number of SNPs have been generated for this purpose. As there are cost and throughput limitations of genotyping large numbers of SNPs and statistical issues regarding the large number of dependent tests on the same data set, to make association analysis practical it has been proposed that SNPs should be prioritized based on likely functional importance. The most easily identifiable functional SNPs are coding SNPs (cSNPs) and accordingly cSNPs have been screened in a number of studies. SNPs inmore » gene regulatory sequences embedded in noncoding DNA are another class of SNPs suggested for prioritization due to their predicted quantitative impact on gene expression. The main challenge in evaluating these SNPs, in contrast to cSNPs is a lack of robust algorithms and databases for recognizing regulatory sequences in noncoding DNA. Approaches that have been previously used to delineate noncoding sequences with gene regulatory activity include cross-species sequence comparisons and the search for sequences recognized by transcription factors. We combined these two methods to sift through mouse human genomic sequences to identify putative gene regulatory elements and subsequently localized SNPs within these sequences in a 1 Megabase (Mb) region of human chromosome 5q31, orthologous to mouse chromosome 11 containing the Interleukin cluster.« less

Consensus coding sequence (CCDS) database: a standardized set of human and mouse protein-coding regions supported by expert curation

PubMed Central

Pujar, Shashikant; O’Leary, Nuala A; Farrell, Catherine M; Mudge, Jonathan M; Wallin, Craig; Diekhans, Mark; Barnes, If; Bennett, Ruth; Berry, Andrew E; Cox, Eric; Davidson, Claire; Goldfarb, Tamara; Gonzalez, Jose M; Hunt, Toby; Jackson, John; Joardar, Vinita; Kay, Mike P; Kodali, Vamsi K; McAndrews, Monica; McGarvey, Kelly M; Murphy, Michael; Rajput, Bhanu; Rangwala, Sanjida H; Riddick, Lillian D; Seal, Ruth L; Webb, David; Zhu, Sophia; Aken, Bronwen L; Bult, Carol J; Frankish, Adam; Pruitt, Kim D

2018-01-01

Abstract The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community. PMID:29126148

Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB)

PubMed Central

Pamp, Sünje J.; Harrington, Eoghan D.; Quake, Stephen R.; Relman, David A.; Blainey, Paul C.

2012-01-01

Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which “holdfasts” and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract. PMID:22434425

Number and location of mouse mammary tumor virus proviral DNA in mouse DNA of normal tissue and of mammary tumors.

PubMed Central

Groner, B; Hynes, N E

1980-01-01

The Southern DNA filter transfer technique was used to characterize the genomic location of the mouse mammary tumor proviral DNA in different inbred strains of mice. Two of the strains (C3H and CBA) arose from a cross of a Bagg albino (BALB/c) mouse and a DBA mouse. The mouse mammary tumor virus-containing restriction enzyme DNA fragments of these strains had similar patterns, suggesting that the proviruses of these mice are in similar genomic locations. Conversely, the pattern arising from the DNA of the GR mouse, a strain genetically unrelated to the others, appeared different, suggesting that its mouse mammary tumor proviruses are located in different genomic sites. The structure of another gene, that coding for beta-globin, was also compared. The mice strains which we studied can be categorized into two classes, expressing either one or two beta-globin proteins. The macroenvironment of the beta-globin gene appeared similar among the mice strains belonging to one genetic class. Female mice of the C3H strain exogenously transmit mouse mammary tumor virus via the milk, and their offspring have a high incidence of mammary tumor occurrence. DNA isolated from individual mammary tumors taken from C3H mice or from BALB/c mice foster nursed on C3H mothers was analyzed by the DNA filter transfer technique. Additional mouse mammary tumor virus-containing fragments were found in the DNA isolated from each mammary tumor. These proviral sequences were integrated into different genomic sites in each tumor. Images PMID:6245257

Genomic sequences of murine gamma B- and gamma C-crystallin-encoding genes: promoter analysis and complete evolutionary pattern of mouse, rat and human gamma-crystallins.

PubMed

Graw, J; Liebstein, A; Pietrowski, D; Schmitt-John, T; Werner, T

1993-12-22

The murine genes, gamma B-cry and gamma C-cry, encoding the gamma B- and gamma C-crystallins, were isolated from a genomic DNA library. The complete nucleotide (nt) sequences of both genes were determined from 661 and 711 bp, respectively, upstream from the first exon to the corresponding polyadenylation sites, comprising more than 2650 and 2890 bp, respectively. The new sequences were compared to the partial cDNA sequences available for the murine gamma B-cry and gamma C-cry, as well as to the corresponding genomic sequences from rat and man, at both the nt and predicted amino acid (aa) sequence levels. In the gamma B-cry promoter region, a canonical CCAAT-box, a TATA-box, putative NF-I and C/EBP sites were detected. An R-repeat is inserted 366 bp upstream from the transcription start point. In contrast, the gamma C-cry promoter does not contain a CCAAT-box, but some other putative binding sites for transcription factors (AP-2, UBP-1, LBP-1) were located by computer analysis. The promoter regions of all six gamma-cry from mouse, rat and human, except human psi gamma F-cry, were analyzed for common sequence elements. A complex sequence element of about 70-80 bp was found in the proximal promoter, which contains a gamma-cry-specific and almost invariant sequence (crygpel) of 14 nt, and ends with the also invariant TATA-box. Within the complex sequence element, a minimum of three further features specific for the gamma A-, gamma B- and gamma D/E/F-cry genes can be defined, at least two of which were recently shown to be functional. In addition to these four sequence elements, a subtype-specific structure of inverted repeats with different-sized spacers can be deduced from the multiple sequence alignment. A phylogenetic analysis based on the promoter region, as well as the complete exon 3 of all gamma-cry from mouse, rat and man, suggests separation of only five gamma-cry subtypes (gamma A-, gamma B-, gamma C-, gamma D- and gamma E/F-cry) prior to species separation.

Cross-referencing yeast genetics and mammalian genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hieter, P.; Basset, D.; Boguski, M.

1994-09-01

We have initiated a project that will systematically transfer information about yeast genes onto the genetic maps of mice and human beings. Rapidly expanding human EST data will serve as a source of candidate human homologs that will be repeatedly searched using yeast protein sequence queries. Search results will be automatically reported to participating labs. Human cDNA sequences from which the ESTs are derived will be mapped at high resolution in the human and mouse genomes. The comparative mapping information cross-references the genomic position of novel human cDNAs with functional information known about the cognate yeast genes. This should facilitatemore » the initial identification of genes responsible for mammalian mutant phenotypes, including human disease. In addition, the identification of mammalian homologs of yeast genes provides reagents for determining evolutionary conservation and for performing direct experiments in multicellular eukaryotes to enhance study of the yeast protein`s function. For example, ESTs homologous to CDC27 and CDC16 were identified, and the corresponding cDNA clones were obtained from ATTC, completely sequenced, and mapped on human and mouse chromosomes. In addition, the CDC17hs cDNA has been used to raise antisera to the CDC27Hs protein and used in subcellular localization experiments and junctional studies in mammalian cells. We have received funding from the National Center for Human Genome Research to provide a community resource which will establish comprehensive cross-referencing among yeast, human, and mouse loci. The project is set up as a service and information on how to communicate with this effort will be provided.« less

Characterization of Aeromonas hydrophila wound pathotypes by comparative genomic and functional analyses of virulence genes.

PubMed

Grim, Christopher J; Kozlova, Elena V; Sha, Jian; Fitts, Eric C; van Lier, Christina J; Kirtley, Michelle L; Joseph, Sandeep J; Read, Timothy D; Burd, Eileen M; Tall, Ben D; Joseph, Sam W; Horneman, Amy J; Chopra, Ashok K; Shak, Joshua R

2013-04-23

Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A. hydrophila (E2). To better understand the differences between pathogenic and environmental strains of A. hydrophila, we conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966(T), and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966(T). The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966(T) and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, we characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity. Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms.

FANTOM5 CAGE profiles of human and mouse samples.

PubMed

Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-Ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A; Babina, Magda; Baillie, J Kenneth; Barnett, Timothy C; Beckhouse, Anthony G; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J; Clevers, Hans C; Davis, Carrie A; Detmar, Michael; Dohi, Taeko; Edge, Albert S B; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C; Faulkner, Geoffrey J; Ferrai, Carmelo; Fisher, Malcolm E; Forrester, Lesley M; Fujita, Rie; Furusawa, Jun-Ichi; Geijtenbeek, Teunis B; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J; Hume, David A; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I; Kempfle, Judith S; Kenna, Tony J; Kere, Juha; Khachigian, Levon M; Kitamura, Toshio; Klein, Sarah; Klinken, S Peter; Knox, Alan J; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-Sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J; Motohashi, Hozumi; Mummery, Christine L; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A; Winteringham, Louise N; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide

2017-08-29

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

FANTOM5 CAGE profiles of human and mouse samples

PubMed Central

Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A.; Babina, Magda; Baillie, J. Kenneth; Barnett, Timothy C.; Beckhouse, Anthony G.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J.; Clevers, Hans C.; Davis, Carrie A.; Detmar, Michael; Dohi, Taeko; Edge, Albert S.B.; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C.; Faulkner, Geoffrey J.; Ferrai, Carmelo; Fisher, Malcolm E.; Forrester, Lesley M.; Fujita, Rie; Furusawa, Jun-ichi; Geijtenbeek, Teunis B.; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J.; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J.; Hume, David A.; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klein, Sarah; Klinken, S. Peter; Knox, Alan J.; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J.; Motohashi, Hozumi; Mummery, Christine L.; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W.; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M.; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A.; Winteringham, Louise N.; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G.; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O.; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R.R.; Hayashizaki, Yoshihide

2017-01-01

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities. PMID:28850106

Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa.

PubMed

Erkek, Serap; Hisano, Mizue; Liang, Ching-Yeu; Gill, Mark; Murr, Rabih; Dieker, Jürgen; Schübeler, Dirk; van der Vlag, Johan; Stadler, Michael B; Peters, Antoine H F M

2013-07-01

In mammalian spermatozoa, most but not all of the genome is densely packaged by protamines. Here we reveal the molecular logic underlying the retention of nucleosomes in mouse spermatozoa, which contain only 1% residual histones. We observe high enrichment throughout the genome of nucleosomes at CpG-rich sequences that lack DNA methylation. Residual nucleosomes are largely composed of the histone H3.3 variant and are trimethylated at Lys4 of histone H3 (H3K4me3). Canonical H3.1 and H3.2 histones are also enriched at CpG-rich promoters marked by Polycomb-mediated H3K27me3, a modification predictive of gene repression in preimplantation embryos. Histone variant-specific nucleosome retention in sperm is strongly associated with nucleosome turnover in round spermatids. Our data show evolutionary conservation of the basic principles of nucleosome retention in mouse and human sperm, supporting a model of epigenetic inheritance by nucleosomes between generations.

Genome-wide ENU mutagenesis for the discovery of novel male fertility regulators.

PubMed

Jamsai, Duangporn; O'Bryan, Moira K

2010-06-01

The completion of genome sequencing projects has provided an extensive knowledge of the contents of the genomes of human, mouse, and many other organisms. Despite this, the function of most of the estimated 25,000 human genes remains largely unknown. Attention has now turned to elucidating gene function and identifying biological pathways that contribute to human diseases, including male infertility. Our understanding of the genetic regulation of male fertility has been accelerated through the use of genetically modified mouse models including knockout, knock-in, gene-trapped, and transgenic mice. Such reverse genetic approaches however, require some fore-knowledge of a gene's function and, as such, bias against the discovery of completely novel genes and biological pathways. To facilitate high throughput gene discovery, genome-wide mouse mutagenesis via the use of a potent chemical mutagen, N-ethyl-N-nitrosourea (ENU), has been developed over the past decade. This forward genetic, or phenotype-driven, approach relies upon observing a phenotype first, then subsequently defining the underlining genetic defect. Mutations are randomly introduced into the mouse genome via ENU exposure. Through a controlled breeding scheme, mutations causing a phenotype of interest (e.g., male infertility) are then identified by linkage analysis and candidate gene sequencing. This approach allows for the possibility of revealing comprehensive phenotype-genotype relationships for a range of genes and pathways i.e. in addition to null alleles, mice containing partial loss of function or gain-of-function mutations, can be recovered. Such point mutations are likely to be more reflective of those that occur within the human population. Many research groups have successfully used this approach to generate infertile mouse lines and some novel male fertility genes have been revealed. In this review, we focus on the utility of ENU mutagenesis for the discovery of novel male fertility regulators.

Lessons learned from the initial sequencing of the pig genome: comparative analysis of an 8 Mb region of pig chromosome 17

PubMed Central

Hart, Elizabeth A; Caccamo, Mario; Harrow, Jennifer L; Humphray, Sean J; Gilbert, James GR; Trevanion, Steve; Hubbard, Tim; Rogers, Jane; Rothschild, Max F

2007-01-01

Background We describe here the sequencing, annotation and comparative analysis of an 8 Mb region of pig chromosome 17, which provides a useful test region to assess coverage and quality for the pig genome sequencing project. We report our findings comparing the annotation of draft sequence assembled at different depths of coverage. Results Within this region we annotated 71 loci, of which 53 are orthologous to human known coding genes. When compared to the syntenic regions in human (20q13.13-q13.33) and mouse (chromosome 2, 167.5 Mb-178.3 Mb), this region was found to be highly conserved with respect to gene order. The most notable difference between the three species is the presence of a large expansion of zinc finger coding genes and pseudogenes on mouse chromosome 2 between Edn3 and Phactr3 that is absent from pig and human. All of our annotation has been made publicly available in the Vertebrate Genome Annotation browser, VEGA. We assessed the impact of coverage on sequence assembly across this region and found, as expected, that increased sequence depth resulted in fewer, longer contigs. One-third of our annotated loci could not be fully re-aligned back to the low coverage version of the sequence, principally because the transcripts are fragmented over several contigs. Conclusion We have demonstrated the considerable advantages of sequencing at increased read depths and discuss the implications that lower coverage sequence may have on subsequent comparative and functional studies, particularly those involving complex loci such as GNAS. PMID:17705864

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

PubMed

Sangiuolo, Federica; Scaldaferri, Maria Lucia; Filareto, Antonio; Spitalieri, Paola; Guerra, Lorenzo; Favia, Maria; Caroppo, Rosa; Mango, Ruggiero; Bruscia, Emanuela; Gruenert, Dieter C; Casavola, Valeria; De Felici, Massimo; Novelli, Giuseppe

2008-01-01

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.

Mouse scrapie responsive gene 1 (Scrg1): genomic organization, physical linkage to sap30, genetic mapping on chromosome 8, and expression in neuronal primary cell cultures.

PubMed

Dron, M; Tartare, X; Guillo, F; Haik, S; Barbin, G; Maury, C; Tovey, M; Dandoy-Dron, F

2000-11-15

We have previously reported a transcript of a novel mouse gene (Scrg1) with increased expression in transmissible spongiform encephalopathies and the cloning of the human mRNA analogue. In this paper, we present the genomic organization of the mouse and human SCRG1 loci, which exhibit a high degree of conservation. The genes are composed of three exons; the two downstream exons contain the protein coding region. The mouse gene is expressed in brain tissue essentially as a 0.7-kb message but also as a minor 2.6-kb mRNA. We have sequenced 20 kb of DNA at the mouse Scrg1 locus and found that the longer transcript is the prolongation of the 0.7-kb mRNA to a polyadenylation site located about 2 kb further downstream. Sequencing revealed that the mouse Scrg1 gene is physically linked to Sap30, a gene that encodes a protein of the histone deacetylase complex, and genetic linkage mapping assigned the localization of Scrg1 to chromosome 8 between Ant1 and Hmg2. Northern blot analysis showed that Scrg1 is under strict developmental control in mouse embryo and is expressed by cells of neuronal origin in vitro. Comparison of the rat, mouse, and human SCRG1 proteins identified a box of 35 identical contiguous amino acids and a characteristic cysteine distribution pattern defining a new protein signature. Copyright 2000 Academic Press.

Sequence, molecular properties, and chromosomal mapping of mouse lumican

NASA Technical Reports Server (NTRS)

Funderburgh, J. L.; Funderburgh, M. L.; Hevelone, N. D.; Stech, M. E.; Justice, M. J.; Liu, C. Y.; Kao, W. W.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1995-01-01

PURPOSE. Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS. Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS. Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS. This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

Characterization of genetic variability of Venezuelan equine encephalitis viruses

DOE PAGES

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.; ...

2016-04-07

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

Consensus coding sequence (CCDS) database: a standardized set of human and mouse protein-coding regions supported by expert curation.

PubMed

Pujar, Shashikant; O'Leary, Nuala A; Farrell, Catherine M; Loveland, Jane E; Mudge, Jonathan M; Wallin, Craig; Girón, Carlos G; Diekhans, Mark; Barnes, If; Bennett, Ruth; Berry, Andrew E; Cox, Eric; Davidson, Claire; Goldfarb, Tamara; Gonzalez, Jose M; Hunt, Toby; Jackson, John; Joardar, Vinita; Kay, Mike P; Kodali, Vamsi K; Martin, Fergal J; McAndrews, Monica; McGarvey, Kelly M; Murphy, Michael; Rajput, Bhanu; Rangwala, Sanjida H; Riddick, Lillian D; Seal, Ruth L; Suner, Marie-Marthe; Webb, David; Zhu, Sophia; Aken, Bronwen L; Bruford, Elspeth A; Bult, Carol J; Frankish, Adam; Murphy, Terence; Pruitt, Kim D

2018-01-04

The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

The genomic sequence of ectromelia virus, the causative agent of mousepox.

PubMed

Chen, Nanhai; Danila, Maria I; Feng, Zehua; Buller, R Mark L; Wang, Chunlin; Han, Xiaosi; Lefkowitz, Elliot J; Upton, Chris

2003-12-05

Ectromelia virus is the causative agent of mousepox, an acute exanthematous disease of mouse colonies in Europe, Japan, China, and the U.S. The Moscow, Hampstead, and NIH79 strains are the most thoroughly studied with the Moscow strain being the most infectious and virulent for the mouse. In the late 1940s mousepox was proposed as a model for the study of the pathogenesis of smallpox and generalized vaccinia in humans. Studies in the last five decades from a succession of investigators have resulted in a detailed description of the virologic and pathologic disease course in genetically susceptible and resistant inbred and out-bred mice. We report the DNA sequence of the left-hand end, the predicted right-hand terminal repeat, and central regions of the genome of the Moscow strain of ectromelia virus (approximately 177,500 bp), which together with the previously sequenced right-hand end, yields a genome of 209,771 bp. We identified 175 potential genes specifying proteins of between 53 and 1924 amino acids, and 29 regions containing sequences related to genes predicted in other poxviruses, but unlikely to encode for functional proteins in ectromelia virus. The translated protein sequences were compared with the protein database for structure/function relationships, and these analyses were used to investigate poxvirus evolution and to attempt to explain at the cellular and molecular level the well-characterized features of the ectromelia virus natural life cycle.

Sequence analysis of chromosome 1 revealed different selection patterns between Chinese wild mice and laboratory strains.

PubMed

Xu, Fuyi; Hu, Shixian; Chao, Tianzhu; Wang, Maochun; Li, Kai; Zhou, Yuxun; Xu, Hongyan; Xiao, Junhua

2017-10-01

Both natural and artificial selection play a critical role in animals' adaptation to the environment. Detection of the signature of selection in genomic regions can provide insights for understanding the function of specific phenotypes. It is generally assumed that laboratory mice may experience intense artificial selection while wild mice more natural selection. However, the differences of selection signature in the mouse genome and underlying genes between wild and laboratory mice remain unclear. In this study, we used two mouse populations: chromosome 1 (Chr 1) substitution lines (C1SLs) derived from Chinese wild mice and mouse genome project (MGP) sequenced inbred strains and two selection detection statistics: Fst and Tajima's D to identify the signature of selection footprint on Chr 1. For the differentiation between the C1SLs and MGP, 110 candidate selection regions containing 47 protein coding genes were detected. A total of 149 selection regions which encompass 7.215 Mb were identified in the C1SLs by Tajima's D approach. While for the MGP, we identified nearly twice selection regions (243) compared with the C1SLs which accounted for 13.27 Mb Chr 1 sequence. Through functional annotation, we identified several biological processes with significant enrichment including seven genes in the olfactory transduction pathway. In addition, we searched the phenotypes associated with the 47 candidate selection genes identified by Fst. These genes were involved in behavior, growth or body weight, mortality or aging, and immune systems which align well with the phenotypic differences between wild and laboratory mice. Therefore, the findings would be helpful for our understanding of the phenotypic differences between wild and laboratory mice and applications for using this new mouse resource (C1SLs) for further genetics studies.

Applications of Gene Targeting Technology to Mental Retardation and Developmental Disability Research

ERIC Educational Resources Information Center

Pimenta, Aurea F.; Levitt, Pat

2005-01-01

The human and mouse genome projects elucidated the sequence and position map of innumerous genes expressed in the central nervous system (CNS), advancing our ability to manipulate these sequences and create models to investigate regulation of gene expression and function. In this article, we reviewed gene targeting methodologies with emphasis on…

Livestock in biomedical research: history, current status and future prospective.

PubMed

Polejaeva, Irina A; Rutigliano, Heloisa M; Wells, Kevin D

2016-01-01

Livestock models have contributed significantly to biomedical and surgical advances. Their contribution is particularly prominent in the areas of physiology and assisted reproductive technologies, including understanding developmental processes and disorders, from ancient to modern times. Over the past 25 years, biomedical research that traditionally embraced a diverse species approach shifted to a small number of model species (e.g. mice and rats). The initial reasons for focusing the main efforts on the mouse were the availability of murine embryonic stem cells (ESCs) and genome sequence data. This powerful combination allowed for precise manipulation of the mouse genome (knockouts, knockins, transcriptional switches etc.) leading to ground-breaking discoveries on gene functions and regulation, and their role in health and disease. Despite the enormous contribution to biomedical research, mouse models have some major limitations. Their substantial differences compared with humans in body and organ size, lifespan and inbreeding result in pronounced metabolic, physiological and behavioural differences. Comparative studies of strategically chosen domestic species can complement mouse research and yield more rigorous findings. Because genome sequence and gene manipulation tools are now available for farm animals (cattle, pigs, sheep and goats), a larger number of livestock genetically engineered (GE) models will be accessible for biomedical research. This paper discusses the use of cattle, goats, sheep and pigs in biomedical research, provides an overview of transgenic technology in farm animals and highlights some of the beneficial characteristics of large animal models of human disease compared with the mouse. In addition, status and origin of current regulation of GE biomedical models is also reviewed.

mySyntenyPortal: an application package to construct websites for synteny block analysis.

PubMed

Lee, Jongin; Lee, Daehwan; Sim, Mikang; Kwon, Daehong; Kim, Juyeon; Ko, Younhee; Kim, Jaebum

2018-06-05

Advances in sequencing technologies have facilitated large-scale comparative genomics based on whole genome sequencing. Constructing and investigating conserved genomic regions among multiple species (called synteny blocks) are essential in the comparative genomics. However, they require significant amounts of computational resources and time in addition to bioinformatics skills. Many web interfaces have been developed to make such tasks easier. However, these web interfaces cannot be customized for users who want to use their own set of genome sequences or definition of synteny blocks. To resolve this limitation, we present mySyntenyPortal, a stand-alone application package to construct websites for synteny block analyses by using users' own genome data. mySyntenyPortal provides both command line and web-based interfaces to build and manage websites for large-scale comparative genomic analyses. The websites can be also easily published and accessed by other users. To demonstrate the usability of mySyntenyPortal, we present an example study for building websites to compare genomes of three mammalian species (human, mouse, and cow) and show how they can be easily utilized to identify potential genes affected by genome rearrangements. mySyntenyPortal will contribute for extended comparative genomic analyses based on large-scale whole genome sequences by providing unique functionality to support the easy creation of interactive websites for synteny block analyses from user's own genome data.

Comparative sequence analysis of a region on human chromosome 13q14, frequently deleted in B-cell chronic lymphocytic leukemia, and its homologous region on mouse chromosome 14.

PubMed

Kapanadze, B; Makeeva, N; Corcoran, M; Jareborg, N; Hammarsund, M; Baranova, A; Zabarovsky, E; Vorontsova, O; Merup, M; Gahrton, G; Jansson, M; Yankovsky, N; Einhorn, S; Oscier, D; Grandér, D; Sangfelt, O

2000-12-15

Previous studies have indicated the presence of a putative tumor suppressor gene on human chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have recently identified a minimally deleted region encompassing parts of two adjacent genes, termed LEU1 and LEU2 (leukemia-associated genes 1 and 2), and several additional transcripts. In addition, 50 kb centromeric to this region we have identified another gene, LEU5/RFP2. To elucidate further the complex genomic organization of this region, we have identified, mapped, and sequenced the homologous region in the mouse. Fluorescence in situ hybridization analysis demonstrated that the region maps to mouse chromosome 14. The overall organization and gene order in this region were found to be highly conserved in the mouse. Sequence comparison between the human deletion hotspot region and its homologous mouse region revealed a high degree of sequence conservation with an overall score of 74%. However, our data also show that in terms of transcribed sequences, only two of those, human LEU2 and LEU5/RFP2, are clearly conserved, strengthening the case for these genes as putative candidate B-CLL tumor suppressor genes.

Phylogenomic Insights into Mouse Evolution Using a Pseudoreference Approach

PubMed Central

Sarver, Brice A.J.; Keeble, Sara; Cosart, Ted; Tucker, Priscilla K.; Dean, Matthew D.

2017-01-01

Comparative genomic studies are now possible across a broad range of evolutionary timescales, but the generation and analysis of genomic data across many different species still present a number of challenges. The most sophisticated genotyping and down-stream analytical frameworks are still predominantly based on comparisons to high-quality reference genomes. However, established genomic resources are often limited within a given group of species, necessitating comparisons to divergent reference genomes that could restrict or bias comparisons across a phylogenetic sample. Here, we develop a scalable pseudoreference approach to iteratively incorporate sample-specific variation into a genome reference and reduce the effects of systematic mapping bias in downstream analyses. To characterize this framework, we used targeted capture to sequence whole exomes (∼54 Mbp) in 12 lineages (ten species) of mice spanning the Mus radiation. We generated whole exome pseudoreferences for all species and show that this iterative reference-based approach improved basic genomic analyses that depend on mapping accuracy while preserving the associated annotations of the mouse reference genome. We then use these pseudoreferences to resolve evolutionary relationships among these lineages while accounting for phylogenetic discordance across the genome, contributing an important resource for comparative studies in the mouse system. We also describe patterns of genomic introgression among lineages and compare our results to previous studies. Our general approach can be applied to whole or partitioned genomic data and is easily portable to any system with sufficient genomic resources, providing a useful framework for phylogenomic studies in mice and other taxa. PMID:28338821

UCbase 2.0: ultraconserved sequences database (2014 update)

PubMed Central

Lomonaco, Vincenzo; Martoglia, Riccardo; Mandreoli, Federica; Anderlucci, Laura; Emmett, Warren; Bicciato, Silvio; Taccioli, Cristian

2014-01-01

UCbase 2.0 (http://ucbase.unimore.it) is an update, extension and evolution of UCbase, a Web tool dedicated to the analysis of ultraconserved sequences (UCRs). UCRs are 481 sequences >200 bases sharing 100% identity among human, mouse and rat genomes. They are frequently located in genomic regions known to be involved in cancer or differentially expressed in human leukemias and carcinomas. UCbase 2.0 is a platform-independent Web resource that includes the updated version of the human genome annotation (hg19), information linking disorders to chromosomal coordinates based on the Systematized Nomenclature of Medicine classification, a query tool to search for Single Nucleotide Polymorphisms (SNPs) and a new text box to directly interrogate the database using a MySQL interface. To facilitate the interactive visual interpretation of UCR chromosomal positioning, UCbase 2.0 now includes a graph visualization interface directly linked to UCSC genome browser. Database URL: http://ucbase.unimore.it PMID:24951797

Genes Altered by Intracisternal A Particles in Mouse Mammary Tumorigenesis

DTIC Science & Technology

1997-07-01

mouse Mus musculus as well as most other rodents (1). They are defective retroviruses which contain 3’ and 5’ long terminal repeat (LTR) sequences and... musculus (C57BL/6J) X Mus spretus backcross was obtained for The Jackson Laboratory (Bar Harbor, Maine) and used for localization of the pl7b(kokopelli...understand the nature of the potential mutation found in the tumors I decided to localize pl7b within the mouse genome. I screened a Mus musculus musculus X

Lineage-specific genomics: Frequent birth and death in the human genome: The human genome contains many lineage-specific elements created by both sequence and functional turnover.

PubMed

Young, Robert S

2016-07-01

Frequent evolutionary birth and death events have created a large quantity of biologically important, lineage-specific DNA within mammalian genomes. The birth and death of DNA sequences is so frequent that the total number of these insertions and deletions in the human population remains unknown, although there are differences between these groups, e.g. transposable elements contribute predominantly to sequence insertion. Functional turnover - where the activity of a locus is specific to one lineage, but the underlying DNA remains conserved - can also drive birth and death. However, this does not appear to be a major driver of divergent transcriptional regulation. Both sequence and functional turnover have contributed to the birth and death of thousands of functional promoters in the human and mouse genomes. These findings reveal the pervasive nature of evolutionary birth and death and suggest that lineage-specific regions may play an important but previously underappreciated role in human biology and disease. © 2016 The Authors BioEssays Published by WILEY Periodicals, Inc.

The mutational landscape of MYCN, Lin28b and ALK F1174L driven murine neuroblastoma mimics human disease.

PubMed

De Wilde, Bram; Beckers, Anneleen; Lindner, Sven; Kristina, Althoff; De Preter, Katleen; Depuydt, Pauline; Mestdagh, Pieter; Sante, Tom; Lefever, Steve; Hertwig, Falk; Peng, Zhiyu; Shi, Le-Ming; Lee, Sangkyun; Vandermarliere, Elien; Martens, Lennart; Menten, Björn; Schramm, Alexander; Fischer, Matthias; Schulte, Johannes; Vandesompele, Jo; Speleman, Frank

2018-02-02

Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems ( ALK, Th- MYCN, Dbh- MYCN and Lin28b ). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.

Comparative transgenic analysis of enhancers from the human SHOX and mouse Shox2 genomic regions.

PubMed

Rosin, Jessica M; Abassah-Oppong, Samuel; Cobb, John

2013-08-01

Disruption of presumptive enhancers downstream of the human SHOX gene (hSHOX) is a frequent cause of the zeugopodal limb defects characteristic of Léri-Weill dyschondrosteosis (LWD). The closely related mouse Shox2 gene (mShox2) is also required for limb development, but in the more proximal stylopodium. In this study, we used transgenic mice in a comparative approach to characterize enhancer sequences in the hSHOX and mShox2 genomic regions. Among conserved noncoding elements (CNEs) that function as enhancers in vertebrate genomes, those that are maintained near paralogous genes are of particular interest given their ancient origins. Therefore, we first analyzed the regulatory potential of a genomic region containing one such duplicated CNE (dCNE) downstream of mShox2 and hSHOX. We identified a strong limb enhancer directly adjacent to the mShox2 dCNE that recapitulates the expression pattern of the endogenous gene. Interestingly, this enhancer requires sequences only conserved in the mammalian lineage in order to drive strong limb expression, whereas the more deeply conserved sequences of the dCNE function as a neural enhancer. Similarly, we found that a conserved element downstream of hSHOX (CNE9) also functions as a neural enhancer in transgenic mice. However, when the CNE9 transgenic construct was enlarged to include adjacent, non-conserved sequences frequently deleted in LWD patients, the transgene drove expression in the zeugopodium of the limbs. Therefore, both hSHOX and mShox2 limb enhancers are coupled to distinct neural enhancers. This is the first report demonstrating the activity of cis-regulatory elements from the hSHOX and mShox2 genomic regions in mammalian embryos.

Sequencing, Annotation and Analysis of the Syrian Hamster (Mesocricetus auratus) Transcriptome

PubMed Central

Tchitchek, Nicolas; Safronetz, David; Rasmussen, Angela L.; Martens, Craig; Virtaneva, Kimmo; Porcella, Stephen F.; Feldmann, Heinz

2014-01-01

Background The Syrian hamster (golden hamster, Mesocricetus auratus) is gaining importance as a new experimental animal model for multiple pathogens, including emerging zoonotic diseases such as Ebola. Nevertheless there are currently no publicly available transcriptome reference sequences or genome for this species. Results A cDNA library derived from mRNA and snRNA isolated and pooled from the brains, lungs, spleens, kidneys, livers, and hearts of three adult female Syrian hamsters was sequenced. Sequence reads were assembled into 62,482 contigs and 111,796 reads remained unassembled (singletons). This combined contig/singleton dataset, designated as the Syrian hamster transcriptome, represents a total of 60,117,204 nucleotides. Our Mesocricetus auratus Syrian hamster transcriptome mapped to 11,648 mouse transcripts representing 9,562 distinct genes, and mapped to a similar number of transcripts and genes in the rat. We identified 214 quasi-complete transcripts based on mouse annotations. Canonical pathways involved in a broad spectrum of fundamental biological processes were significantly represented in the library. The Syrian hamster transcriptome was aligned to the current release of the Chinese hamster ovary (CHO) cell transcriptome and genome to improve the genomic annotation of this species. Finally, our Syrian hamster transcriptome was aligned against 14 other rodents, primate and laurasiatheria species to gain insights about the genetic relatedness and placement of this species. Conclusions This Syrian hamster transcriptome dataset significantly improves our knowledge of the Syrian hamster's transcriptome, especially towards its future use in infectious disease research. Moreover, this library is an important resource for the wider scientific community to help improve genome annotation of the Syrian hamster and other closely related species. Furthermore, these data provide the basis for development of expression microarrays that can be used in functional genomics studies. PMID:25398096

Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression

PubMed Central

Parks, Matthew M.; Kurylo, Chad M.; Dass, Randall A.; Bojmar, Linda; Lyden, David; Vincent, C. Theresa; Blanchard, Scott C.

2018-01-01

The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome’s molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease. PMID:29503865

Human Hrs, a tyrosine kinase substrate in growth factor-stimulated cells: cDNA cloning and mapping of the gene to chromosome 17.

PubMed

Lu, L; Komada, M; Kitamura, N

1998-06-15

Hrs is a 115kDa zinc finger protein which is rapidly tyrosine phosphorylated in cells stimulated with various growth factors. We previously purified the protein from a mouse cell line and cloned its cDNA. In the present study, we cloned a human Hrs cDNA from a human placenta cDNA library by cross-hybridization, using the mouse cDNA as a probe, and determined its nucleotide sequence. The human Hrs cDNA encoded a 777-amino-acid protein whose sequence was 93% identical to that of mouse Hrs. Northern blot analysis showed that the Hrs mRNA was about 3.0kb long and was expressed in all the human adult and fetal tissues tested. In addition, we showed by genomic Southern blot analysis that the human Hrs gene was a single-copy gene with a size of about 20kb. Furthermore, the human Hrs gene was mapped to chromosome 17 by Southern blotting of genomic DNAs from human/rodent somatic cell hybrids. Copyright 1998 Elsevier Science B.V. All rights reserved.

Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

PubMed Central

Sundaram, Vasavi; Choudhary, Mayank N. K.; Pehrsson, Erica; Xing, Xiaoyun; Fiore, Christopher; Pandey, Manishi; Maricque, Brett; Udawatta, Methma; Ngo, Duc; Chen, Yujie; Paguntalan, Asia; Ray, Tammy; Hughes, Ava; Cohen, Barak A.; Wang, Ting

2017-01-01

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. PMID:28348391

Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria.

PubMed

Salzman, Nita H; de Jong, Hendrik; Paterson, Yvonne; Harmsen, Hermie J M; Welling, Gjalt W; Bos, Nicolaas A

2002-11-01

Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of these sequences, 25% (10/40) corresponded to described intestinal organisms of the mouse, including Lactobacillus spp., Helicobacter spp., segmented filamentous bacteria and members of the altered Schaedler flora (ASF360, ASF361, ASF502 and ASF519); 75% (30/40) represented novel sequences. A large number (11/40) of the novel sequences revealed a new operational taxonomic unit (OTU) belonging to the Cytophaga-Flavobacter-Bacteroides phylum, which the authors named 'mouse intestinal bacteria'. 16S rRNA probes were developed for this new OTU. Upon analysis of the novel sequences, eight were found to cluster within the Eubacterium rectale-Clostridium coccoides group and three clustered within the Bacteroides group. One of the novel sequences was distantly related to Verrucomicrobium spinosum and one was distantly related to Bacillus mycoides. Oligonucleotide probes specific for the 16S rRNA of these novel clones were generated. Using a combination of four previously described and four newly designed probes, approximately 80% of bacteria recovered from the murine large intestine and 71% of bacteria recovered from the murine caecum could be identified by fluorescence in situ hybridization (FISH).

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

PubMed

Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

1986-02-01

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations.

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

PubMed Central

Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

1986-01-01

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations. Images PMID:3003377

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

PubMed Central

Chang, D D; Clayton, D A

1986-01-01

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species. Images PMID:3785226

Translating human genetics into mouse: the impact of ultra-rapid in vivo genome editing.

PubMed

Aida, Tomomi; Imahashi, Risa; Tanaka, Kohichi

2014-01-01

Gene-targeted mutant animals, such as knockout or knockin mice, have dramatically improved our understanding of the functions of genes in vivo and the genetic diversity that characterizes health and disease. However, the generation of targeted mice relies on gene targeting in embryonic stem (ES) cells, which is a time-consuming, laborious, and expensive process. The recent groundbreaking development of several genome editing technologies has enabled the targeted alteration of almost any sequence in any cell or organism. These technologies have now been applied to mouse zygotes (in vivo genome editing), thereby providing new avenues for simple, convenient, and ultra-rapid production of knockout or knockin mice without the need for ES cells. Here, we review recent achievements in the production of gene-targeted mice by in vivo genome editing. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Genomic anatomy of the Tyrp1 (brown) deletion complex

PubMed Central

Smyth, Ian M.; Wilming, Laurens; Lee, Angela W.; Taylor, Martin S.; Gautier, Phillipe; Barlow, Karen; Wallis, Justine; Martin, Sancha; Glithero, Rebecca; Phillimore, Ben; Pelan, Sarah; Andrew, Rob; Holt, Karen; Taylor, Ruth; McLaren, Stuart; Burton, John; Bailey, Jonathon; Sims, Sarah; Squares, Jan; Plumb, Bob; Joy, Ann; Gibson, Richard; Gilbert, James; Hart, Elizabeth; Laird, Gavin; Loveland, Jane; Mudge, Jonathan; Steward, Charlie; Swarbreck, David; Harrow, Jennifer; North, Philip; Leaves, Nicholas; Greystrong, John; Coppola, Maria; Manjunath, Shilpa; Campbell, Mark; Smith, Mark; Strachan, Gregory; Tofts, Calli; Boal, Esther; Cobley, Victoria; Hunter, Giselle; Kimberley, Christopher; Thomas, Daniel; Cave-Berry, Lee; Weston, Paul; Botcherby, Marc R. M.; White, Sharon; Edgar, Ruth; Cross, Sally H.; Irvani, Marjan; Hummerich, Holger; Simpson, Eleanor H.; Johnson, Dabney; Hunsicker, Patricia R.; Little, Peter F. R.; Hubbard, Tim; Campbell, R. Duncan; Rogers, Jane; Jackson, Ian J.

2006-01-01

Chromosome deletions in the mouse have proven invaluable in the dissection of gene function. The brown deletion complex comprises >28 independent genome rearrangements, which have been used to identify several functional loci on chromosome 4 required for normal embryonic and postnatal development. We have constructed a 172-bacterial artificial chromosome contig that spans this 22-megabase (Mb) interval and have produced a contiguous, finished, and manually annotated sequence from these clones. The deletion complex is strikingly gene-poor, containing only 52 protein-coding genes (of which only 39 are supported by human homologues) and has several further notable genomic features, including several segments of >1 Mb, apparently devoid of a coding sequence. We have used sequence polymorphisms to finely map the deletion breakpoints and identify strong candidate genes for the known phenotypes that map to this region, including three lethal loci (l4Rn1, l4Rn2, and l4Rn3) and the fitness mutant brown-associated fitness (baf). We have also characterized misexpression of the basonuclin homologue, Bnc2, associated with the inversion-mediated coat color mutant white-based brown (Bw). This study provides a molecular insight into the basis of several characterized mouse mutants, which will allow further dissection of this region by targeted or chemical mutagenesis. PMID:16505357

Comparative Sequence and X-Inactivation Analyses of a Domain of Escape in Human Xp11.2 and the Conserved Segment in Mouse

PubMed Central

Tsuchiya, Karen D.; Greally, John M.; Yi, Yajun; Noel, Kevin P.; Truong, Jean-Pierre; Disteche, Christine M.

2004-01-01

We have performed X-inactivation and sequence analyses on 350 kb of sequence from human Xp11.2, a region shown previously to contain a cluster of genes that escape X inactivation, and we compared this region with the region of conserved synteny in mouse. We identified several new transcripts from this region in human and in mouse, which defined the full extent of the domain escaping X inactivation in both species. In human, escape from X inactivation involves an uninterrupted 235-kb domain of multiple genes. Despite highly conserved gene content and order between the two species, Smcx is the only mouse gene from the conserved segment that escapes inactivation. As repetitive sequences are believed to facilitate spreading of X inactivation along the chromosome, we compared the repetitive sequence composition of this region between the two species. We found that long terminal repeats (LTRs) were decreased in the human domain of escape, but not in the majority of the conserved mouse region adjacent to Smcx in which genes were subject to X inactivation, suggesting that these repeats might be excluded from escape domains to prevent spreading of silencing. Our findings indicate that genomic context, as well as gene-specific regulatory elements, interact to determine expression of a gene from the inactive X-chromosome. PMID:15197169

The Role of microRNA miR-101 in Prostate Cancer Progression

DTIC Science & Technology

2012-09-01

genome -wide mapping of PcG binding in human fibroblasts, human ES cells, mouse ES cells, and Drosophila 37-41 . All of the studies demonstrated that...development. Mamm Genome 2002; 13(9): 493-503. 15. Simon J, Chiang A, Bender W, Shimell MJ, O’Connor M. Elements of the Drosophila bithorax complex... sequencing analysis of the miR-203 genomic region revealed cancer-specific DNA methylation in a region proximal to miR-203 in prostate cancer tissues

Fetal and Placental DNA Stimulation of TLR9: A Mechanism Possibly Contributing to the Pro-inflammatory Events During Parturition.

PubMed

Goldfarb, Ilona Telefus; Adeli, Sharareh; Berk, Tucker; Phillippe, Mark

2018-05-01

While there is evidence for a relationship between cell-free fetal DNA (cffDNA) and parturition, questions remain regarding whether cffDNA could trigger a pro-inflammatory response on the pathway to parturition. We hypothesized that placental and/or fetal DNA stimulates toll-like receptor 9 (TLR9) leading to secretion of pro-inflammatory cytokines by macrophage cells. Four in vitro DNA stimulation studies were performed using RAW 264.7 mouse peritoneal macrophage cells incubated in media containing the following DNA particles: an oligodeoxynucleotide (ODN2395), intact genomic DNA (from mouse placentas, fetuses and adult liver), mouse DNA complexed with DOTAP (a cationic liposome forming compound), and telomere-depleted mouse DNA. Interleukin 6 (IL6) secretion was measured in the media by enzyme-linked immunosorbent assay; and the cell pellet was homogenized for protein content (picograms IL6/mg protein). Robust IL6 secretion was observed in response to ODN2395 (a CpG-rich TLR9 agonist), mouse DNA-DOTAP complexes, and telomere-depleted mouse DNA in concentrations of 5 to 15 μg/mL. In contrast, ODN A151 (containing telomere sequence motifs), intact genomic mouse DNA, and restriction enzyme-digested DNA had no effect on IL6 secretion. The IL6 response was significantly inhibited by chloroquine (10 μg/mL), thereby confirming the important role for TLR9 in the response by macrophage cells. DNA derived from mouse placentas and fetuses, and depleted of telomeric sequences, stimulates a robust pro-inflammatory response by macrophage cells, thereby supporting the hypothesis that cffDNA is able to stimulate an innate immune response that could trigger the onset of parturition. These findings are of clinical importance, as we search for effective treatment/prevention of preterm parturition.

Genomic resources for wild populations of the house mouse, Mus musculus and its close relative Mus spretus

PubMed Central

Harr, Bettina; Karakoc, Emre; Neme, Rafik; Teschke, Meike; Pfeifle, Christine; Pezer, Željka; Babiker, Hiba; Linnenbrink, Miriam; Montero, Inka; Scavetta, Rick; Abai, Mohammad Reza; Molins, Marta Puente; Schlegel, Mathias; Ulrich, Rainer G.; Altmüller, Janine; Franitza, Marek; Büntge, Anna; Künzel, Sven; Tautz, Diethard

2016-01-01

Wild populations of the house mouse (Mus musculus) represent the raw genetic material for the classical inbred strains in biomedical research and are a major model system for evolutionary biology. We provide whole genome sequencing data of individuals representing natural populations of M. m. domesticus (24 individuals from 3 populations), M. m. helgolandicus (3 individuals), M. m. musculus (22 individuals from 3 populations) and M. spretus (8 individuals from one population). We use a single pipeline to map and call variants for these individuals and also include 10 additional individuals of M. m. castaneus for which genomic data are publically available. In addition, RNAseq data were obtained from 10 tissues of up to eight adult individuals from each of the three M. m. domesticus populations for which genomic data were collected. Data and analyses are presented via tracks viewable in the UCSC or IGV genome browsers. We also provide information on available outbred stocks and instructions on how to keep them in the laboratory. PMID:27622383

Identification of an active ID-like group of SINEs in the mouse

PubMed Central

Kass, David H; Jamison, Nicole

2007-01-01

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 M. musculus as well as other species of Mus using a PCR-based assay. All four analyzed elements were either identified 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 thus far has mainly been observed in M. musculus, we then refer to this family as MMIDL. PMID:17572061

Identification of an active ID-like group of SINEs in the mouse.

PubMed

Kass, David H; Jamison, Nicole

2007-09-01

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.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice.

PubMed

Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, Hongbin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

2007-04-17

The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice

PubMed Central

Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, HongBin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

2007-01-01

Background The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. Results A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. Conclusion A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia. PMID:17439653

Disrupting the male germ line to find infertility and contraception targets.

PubMed

Archambeault, Denise R; Matzuk, Martin M

2014-05-01

Genetically-manipulated mouse models have become indispensible for broadening our understanding of genes and pathways related to male germ cell development. Until suitable in vitro systems for studying spermatogenesis are perfected, in vivo models will remain the gold standard for inquiry into testicular function. Here, we discuss exciting advances that are allowing researchers faster, easier, and more customizable access to their mouse models of interest. Specifically, the trans-NIH Knockout Mouse Project (KOMP) is working to generate knockout mouse models of every gene in the mouse genome. The related Knockout Mouse Phenotyping Program (KOMP2) is performing systematic phenotypic analysis of this genome-wide collection of knockout mice, including fertility screening. Together, these programs will not only uncover new genes involved in male germ cell development but also provide the research community with the mouse models necessary for further investigations. In addition to KOMP/KOMP2, another promising development in the field of mouse models is the advent of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas technology. Utilizing 20 nucleotide guide sequences, CRISPR/Cas has the potential to introduce sequence-specific insertions, deletions, and point mutations to produce null, conditional, activated, or reporter-tagged alleles. CRISPR/Cas can also successfully target multiple genes in a single experimental step, forgoing the multiple generations of breeding traditionally required to produce mouse models with deletions, insertions, or mutations in multiple genes. In addition, CRISPR/Cas can be used to create mouse models carrying variants identical to those identified in infertile human patients, providing the opportunity to explore the effects of such mutations in an in vivo system. Both the KOMP/KOMP2 projects and the CRISPR/Cas system provide powerful, accessible genetic approaches to the study of male germ cell development in the mouse. A more complete understanding of male germ cell biology is critical for the identification of novel targets for potential non-hormonal contraceptive intervention. Copyright © 2014. Published by Elsevier Masson SAS.

Informatic and genomic analysis of melanocyte cDNA libraries as a resource for the study of melanocyte development and function.

PubMed

Baxter, Laura L; Hsu, Benjamin J; Umayam, Lowell; Wolfsberg, Tyra G; Larson, Denise M; Frith, Martin C; Kawai, Jun; Hayashizaki, Yoshihide; Carninci, Piero; Pavan, William J

2007-06-01

As part of the RIKEN mouse encyclopedia project, two cDNA libraries were prepared from melanocyte-derived cell lines, using techniques of full-length clone selection and subtraction/normalization to enrich for rare transcripts. End sequencing showed that these libraries display over 83% complete coding sequence at the 5' end and 96-97% complete coding sequence at the 3' end. Evaluation of the libraries, derived from B16F10Y tumor cells and melan-c cells, revealed that they contain clones for a majority of the genes previously demonstrated to function in melanocyte biology. Analysis of genomic locations for transcripts revealed that the distribution of melanocyte genes is non-random throughout the genome. Three genomic regions identified that showed significant clustering of melanocyte-expressed genes contain one or more genes previously shown to regulate melanocyte development or function. A catalog of genes expressed in these libraries is presented, providing a valuable resource of cDNA clones and sequence information that can be used for identification of new genes important for melanocyte development, function, and disease.

Finding the missing honey bee genes: lessons learned from a genome upgrade.

PubMed

Elsik, Christine G; Worley, Kim C; Bennett, Anna K; Beye, Martin; Camara, Francisco; Childers, Christopher P; de Graaf, Dirk C; Debyser, Griet; Deng, Jixin; Devreese, Bart; Elhaik, Eran; Evans, Jay D; Foster, Leonard J; Graur, Dan; Guigo, Roderic; Hoff, Katharina Jasmin; Holder, Michael E; Hudson, Matthew E; Hunt, Greg J; Jiang, Huaiyang; Joshi, Vandita; Khetani, Radhika S; Kosarev, Peter; Kovar, Christie L; Ma, Jian; Maleszka, Ryszard; Moritz, Robin F A; Munoz-Torres, Monica C; Murphy, Terence D; Muzny, Donna M; Newsham, Irene F; Reese, Justin T; Robertson, Hugh M; Robinson, Gene E; Rueppell, Olav; Solovyev, Victor; Stanke, Mario; Stolle, Eckart; Tsuruda, Jennifer M; Vaerenbergh, Matthias Van; Waterhouse, Robert M; Weaver, Daniel B; Whitfield, Charles W; Wu, Yuanqing; Zdobnov, Evgeny M; Zhang, Lan; Zhu, Dianhui; Gibbs, Richard A

2014-01-30

The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Finding the missing honey bee genes: lessons learned from a genome upgrade

PubMed Central

2014-01-01

Background The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. Results Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. Conclusions Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination. PMID:24479613

Astonishing advances in mouse genetic tools for biomedical research.

PubMed

Kaczmarczyk, Lech; Jackson, Walker S

2015-01-01

The humble house mouse has long been a workhorse model system in biomedical research. The technology for introducing site-specific genome modifications led to Nobel Prizes for its pioneers and opened a new era of mouse genetics. However, this technology was very time-consuming and technically demanding. As a result, many investigators continued to employ easier genome manipulation methods, though resulting models can suffer from overlooked or underestimated consequences. Another breakthrough, invaluable for the molecular dissection of disease mechanisms, was the invention of high-throughput methods to measure the expression of a plethora of genes in parallel. However, the use of samples containing material from multiple cell types could obfuscate data, and thus interpretations. In this review we highlight some important issues in experimental approaches using mouse models for biomedical research. We then discuss recent technological advances in mouse genetics that are revolutionising human disease research. Mouse genomes are now easily manipulated at precise locations thanks to guided endonucleases, such as transcription activator-like effector nucleases (TALENs) or the CRISPR/Cas9 system, both also having the potential to turn the dream of human gene therapy into reality. Newly developed methods of cell type-specific isolation of transcriptomes from crude tissue homogenates, followed by detection with next generation sequencing (NGS), are vastly improving gene regulation studies. Taken together, these amazing tools simplify the creation of much more accurate mouse models of human disease, and enable the extraction of hitherto unobtainable data.

UCbase 2.0: ultraconserved sequences database (2014 update).

PubMed

Lomonaco, Vincenzo; Martoglia, Riccardo; Mandreoli, Federica; Anderlucci, Laura; Emmett, Warren; Bicciato, Silvio; Taccioli, Cristian

2014-01-01

UCbase 2.0 (http://ucbase.unimore.it) is an update, extension and evolution of UCbase, a Web tool dedicated to the analysis of ultraconserved sequences (UCRs). UCRs are 481 sequences >200 bases sharing 100% identity among human, mouse and rat genomes. They are frequently located in genomic regions known to be involved in cancer or differentially expressed in human leukemias and carcinomas. UCbase 2.0 is a platform-independent Web resource that includes the updated version of the human genome annotation (hg19), information linking disorders to chromosomal coordinates based on the Systematized Nomenclature of Medicine classification, a query tool to search for Single Nucleotide Polymorphisms (SNPs) and a new text box to directly interrogate the database using a MySQL interface. To facilitate the interactive visual interpretation of UCR chromosomal positioning, UCbase 2.0 now includes a graph visualization interface directly linked to UCSC genome browser. Database URL: http://ucbase.unimore.it. © The Author(s) 2014. Published by Oxford University Press.

A Mouse Geneticist’s Practical Guide to CRISPR Applications

PubMed Central

Singh, Priti; Schimenti, John C.; Bolcun-Filas, Ewelina

2015-01-01

CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice. Moreover, it holds promise for other applications beyond genome editing. The crux of this system is the efficient and targeted introduction of DNA breaks that are repaired by any of several pathways in a predictable but not entirely controllable manner. Thus, further optimizations and improvements are being developed. Here, we summarize current applications and provide a practical guide to use the CRISPR/Cas9 system for mouse mutagenesis, based on published reports and our own experiences. We discuss critical points and suggest technical improvements to increase efficiency of RNA-guided genome editing in mouse embryos and address practical problems such as mosaicism in founders, which complicates genotyping and phenotyping. We describe a next-generation sequencing strategy for simultaneous characterization of on- and off-target editing in mice derived from multiple CRISPR experiments. Additionally, we report evidence that elevated frequency of precise, homology-directed editing can be achieved by transient inhibition of the Ligase IV-dependent nonhomologous end-joining pathway in one-celled mouse embryos. PMID:25271304

PolyA_DB 3 catalogs cleavage and polyadenylation sites identified by deep sequencing in multiple genomes

PubMed Central

Wang, Ruijia; Nambiar, Ram; Zheng, Dinghai

2018-01-01

Abstract PolyA_DB is a database cataloging cleavage and polyadenylation sites (PASs) in several genomes. Previous versions were based mainly on expressed sequence tags (ESTs), which had a limited amount and could lead to inaccurate PAS identification due to the presence of internal A-rich sequences in transcripts. Here, we present an updated version of the database based solely on deep sequencing data. First, PASs are mapped by the 3′ region extraction and deep sequencing (3′READS) method, ensuring unequivocal PAS identification. Second, a large volume of data based on diverse biological samples increases PAS coverage by 3.5-fold over the EST-based version and provides PAS usage information. Third, strand-specific RNA-seq data are used to extend annotated 3′ ends of genes to obtain more thorough annotations of alternative polyadenylation (APA) sites. Fourth, conservation information of PAS across mammals sheds light on significance of APA sites. The database (URL: http://www.polya-db.org/v3) currently holds PASs in human, mouse, rat and chicken, and has links to the UCSC genome browser for further visualization and for integration with other genomic data. PMID:29069441

Eimeria genomics: Where are we now and where are we going?

PubMed

Blake, Damer P

2015-08-15

The evolution of sequencing technologies, from Sanger to next generation (NGS) and now the emerging third generation, has prompted a radical frameshift moving genomics from the specialist to the mainstream. For parasitology, genomics has moved fastest for the protozoa with sequence assemblies becoming available for multiple genera including Babesia, Cryptosporidium, Eimeria, Giardia, Leishmania, Neospora, Plasmodium, Theileria, Toxoplasma and Trypanosoma. Progress has commonly been slower for parasites of animals which lack zoonotic potential, but the deficit is now being redressed with impact likely in the areas of drug and vaccine development, molecular diagnostics and population biology. Genomics studies with the apicomplexan Eimeria species clearly illustrate the approaches and opportunities available. Specifically, more than ten years after initiation of a genome sequencing project a sequence assembly was published for Eimeria tenella in 2014, complemented by assemblies for all other Eimeria species which infect the chicken and Eimeria falciformis, a parasite of the mouse. Public access to these and other coccidian genome assemblies through resources such as GeneDB and ToxoDB now promotes comparative analysis, encouraging better use of shared resources and enhancing opportunities for development of novel diagnostic and control strategies. In the short term genomics resources support development of targeted and genome-wide genetic markers such as single nucleotide polymorphisms (SNPs), with whole genome re-sequencing becoming viable in the near future. Experimental power will develop rapidly as additional species, strains and isolates are sampled with particular emphasis on population structure and allelic diversity. Copyright © 2015 Elsevier B.V. All rights reserved.

A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion

PubMed Central

Zhao, Guoyan; Penna, Vinay R.; Park, Eugene; Lauron, Elvin J.; Harvey, Ian B.; Beatty, Wandy L.; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H.; Wang, David

2017-01-01

ABSTRACT The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4+ T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties. PMID:28179532

A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion.

PubMed

Patel, Swapneel J; Zhao, Guoyan; Penna, Vinay R; Park, Eugene; Lauron, Elvin J; Harvey, Ian B; Beatty, Wandy L; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H; Wang, David; Yokoyama, Wayne M

2017-05-01

The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4 + T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties. Copyright © 2017 American Society for Microbiology.

Mapping cis-Regulatory Domains in the Human Genome UsingMulti-Species Conservation of Synteny

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahituv, Nadav; Prabhakar, Shyam; Poulin, Francis

2005-06-13

Our inability to associate distant regulatory elements with the genes that they regulate has largely precluded their examination for sequence alterations contributing to human disease. One major obstacle is the large genomic space surrounding targeted genes in which such elements could potentially reside. In order to delineate gene regulatory boundaries we used whole-genome human-mouse-chicken (HMC) and human-mouse-frog (HMF) multiple alignments to compile conserved blocks of synteny (CBS), under the hypothesis that these blocks have been kept intact throughout evolution at least in part by the requirement of regulatory elements to stay linked to the genes that they regulate. A totalmore » of 2,116 and 1,942 CBS>200 kb were assembled for HMC and HMF respectively, encompassing 1.53 and 0.86 Gb of human sequence. To support the existence of complex long-range regulatory domains within these CBS we analyzed the prevalence and distribution of chromosomal aberrations leading to position effects (disruption of a genes regulatory environment), observing a clear bias not only for mapping onto CBS but also for longer CBS size. Our results provide a genome wide data set characterizing the regulatory domains of genes and the conserved regulatory elements within them.« less

The UCSC Genome Browser database: extensions and updates 2013.

PubMed

Meyer, Laurence R; Zweig, Ann S; Hinrichs, Angie S; Karolchik, Donna; Kuhn, Robert M; Wong, Matthew; Sloan, Cricket A; Rosenbloom, Kate R; Roe, Greg; Rhead, Brooke; Raney, Brian J; Pohl, Andy; Malladi, Venkat S; Li, Chin H; Lee, Brian T; Learned, Katrina; Kirkup, Vanessa; Hsu, Fan; Heitner, Steve; Harte, Rachel A; Haeussler, Maximilian; Guruvadoo, Luvina; Goldman, Mary; Giardine, Belinda M; Fujita, Pauline A; Dreszer, Timothy R; Diekhans, Mark; Cline, Melissa S; Clawson, Hiram; Barber, Galt P; Haussler, David; Kent, W James

2013-01-01

The University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analysing and sharing both publicly available and user-generated genomic datasets. As of September 2012, genomic sequence and a basic set of annotation 'tracks' are provided for 63 organisms, including 26 mammals, 13 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms, yeast and sea hare. In the past year 19 new genome assemblies have been added, and we anticipate releasing another 28 in early 2013. Further, a large number of annotation tracks have been either added, updated by contributors or remapped to the latest human reference genome. Among these are an updated UCSC Genes track for human and mouse assemblies. We have also introduced several features to improve usability, including new navigation menus. This article provides an update to the UCSC Genome Browser database, which has been previously featured in the Database issue of this journal.

Library Resources for Bac End Sequencing. Final Technical Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pieter J. de Jong

2000-10-01

Studies directed towards the specific aims outlined for this research award are summarized. The RPCI II Human Bac Library has been expanded by the addition of 6.9-fold genomic coverage. This segment has been generated from a MBOI partial digest of the same anonymous donor DNA used for the rest of the library. A new cloning vector, pTARBAC1, has been constructed and used in the construction of RPCI-II segment 5. This new cloning vector provides a new strategy in identifying targeted genomic regions and will greatly facilitate a large-scale analysis for positional cloning. A new maleCS7BC/6J mouse BAC library has beenmore » constructed. RPCI-23 contain 576 plates (approx 210,000 clones) and represents approximately 11-fold coverage of the mouse genome.« less

Discovery of the "RNA continent" through a contrarian's research strategy.

PubMed

Hayashizaki, Yoshihide

2011-01-01

The International Human Genome Sequencing Consortium completed the decoding of the human genome sequence in 2003. Readers will be aware of the paradigm shift which has occurred since then in the field of life science research. At last, mankind has been able to focus on a complete picture of the full extent of the genome, on which is recorded the basic information that controls all life. Meanwhile, another genome project, centered on Japan and known as the mouse genome encyclopedia project, was progressing with participation from around the world. Led by our research group at RIKEN, it was a full-length cDNA project which aimed to decode the whole RNA (transcriptome) using the mouse as a model. The basic information that controls all life is recorded on the genome, but in order to obtain a complete picture of this extensive information, the decoding of the genome alone is far from sufficient. These two genome projects established that the number of letters in the genome, which is the blueprint of life, is finite, that the number of RNA molecules derived from it is also finite, and that the number of protein molecules derived from the RNA is probably finite too. A massive number of combinations is still involved, but we are now able to understand one section of the network formed by these data. Once an object of study has been understood to be finite, establishing an image of the whole is certain to lead us to an understanding of the whole. Omics is an approach that views the information controlling life as finite and seeks to assemble and analyze it as a whole. Here, I would like to present our transcriptome research while making reference to our unique research strategy.

Identification of two novel mammalian genes establishes a subfamily of KH-domain RNA-binding proteins.

PubMed

Makeyev, A V; Liebhaber, S A

2000-08-01

We have identified two novel human genes encoding proteins with a high level of sequence identity to two previously characterized RNA-binding proteins, alphaCP-1 and alphaCP-2. Both of these novel genes, alphaCP-3 and alphaCP-4, are predicted to encode proteins with triplicated KH domains. The number and organization of the KH domains, their sequences, and the sequences of the contiguous regions are conserved among all four alphaCP proteins. The common evolutionary origin of these proteins is substantiated by conservation of exon-intron organization in the corresponding genes. The map positions of alphaCP-1 and alphaCP-2 (previously reported) and those of alphaCP-3 and alphaCP-4 (present report) reveal that the four alphaCP loci are dispersed in the human genome; alphaCP-3 and alphaCP-4 mapped to 21q22.3 and 3p21, and the respective mouse orthologues mapped to syntenic regions of the mouse genome, 10B5 and 9F1-F2, respectively. Two additional loci in the human genome were identified as alphaCP-2 processed pseudogenes (PCBP2P1, 21q22.3, and PCBP2P2, 8q21-q22). Although the overall levels of alphaCP-3 and alphaCP-4 mRNAs are substantially lower than those of alphaCP-1 and alphaCP-2, transcripts of alphaCP-3 and alphaCP-4 were found in all mouse tissues tested. These data establish a new subfamily of genes predicted to encode closely related KH-containing RNA-binding proteins with potential functions in posttranscriptional controls. Copyright 2000 Academic Press.

Whole-Genome-Sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374.

PubMed

Wang, Xiaoli; Xie, Yingzhou; Li, Gang; Liu, Jialin; Li, Xiaobin; Tian, Lijun; Sun, Jingyong; Ou, Hong-Yu; Qu, Hongping

2018-01-01

Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 10 2 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance.

Whole-Genome-Sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374

PubMed Central

Wang, Xiaoli; Xie, Yingzhou; Li, Gang; Liu, Jialin; Li, Xiaobin; Tian, Lijun; Sun, Jingyong; Qu, Hongping

2018-01-01

ABSTRACT Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 102 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance. PMID:29338592

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

PubMed

Agrotis, Alexander; Ketteler, Robin

2015-01-01

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

A genome sequence resource for the aye-aye (Daubentonia madagascariensis), a nocturnal lemur from Madagascar.

PubMed

Perry, George H; Reeves, Darryl; Melsted, Páll; Ratan, Aakrosh; Miller, Webb; Michelini, Katelyn; Louis, Edward E; Pritchard, Jonathan K; Mason, Christopher E; Gilad, Yoav

2012-01-01

We present a high-coverage draft genome assembly of the aye-aye (Daubentonia madagascariensis), a highly unusual nocturnal primate from Madagascar. Our assembly totals ~3.0 billion bp (3.0 Gb), roughly the size of the human genome, comprised of ~2.6 million scaffolds (N50 scaffold size = 13,597 bp) based on short paired-end sequencing reads. We compared the aye-aye genome sequence data with four other published primate genomes (human, chimpanzee, orangutan, and rhesus macaque) as well as with the mouse and dog genomes as nonprimate outgroups. Unexpectedly, we observed strong evidence for a relatively slow substitution rate in the aye-aye lineage compared with these and other primates. In fact, the aye-aye branch length is estimated to be ~10% shorter than that of the human lineage, which is known for its low substitution rate. This finding may be explained, in part, by the protracted aye-aye life-history pattern, including late weaning and age of first reproduction relative to other lemurs. Additionally, the availability of this draft lemur genome sequence allowed us to polarize nucleotide and protein sequence changes to the ancestral primate lineage-a critical period in primate evolution, for which the relevant fossil record is sparse. Finally, we identified 293,800 high-confidence single nucleotide polymorphisms in the donor individual for our aye-aye genome sequence, a captive-born individual from two wild-born parents. The resulting heterozygosity estimate of 0.051% is the lowest of any primate studied to date, which is understandable considering the aye-aye's extensive home-range size and relatively low population densities. Yet this level of genetic diversity also suggests that conservation efforts benefiting this unusual species should be prioritized, especially in the face of the accelerating degradation and fragmentation of Madagascar's forests.

Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease

PubMed Central

Rusniok, Christophe; Lomma, Mariella; Dervins-Ravault, Delphine; Newton, Hayley J.; Sansom, Fiona M.; Jarraud, Sophie; Zidane, Nora; Ma, Laurence; Bouchier, Christiane; Etienne, Jerôme; Hartland, Elizabeth L.; Buchrieser, Carmen

2010-01-01

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species. PMID:20174605

Genomes as geography: using GIS technology to build interactive genome feature maps

PubMed Central

Dolan, Mary E; Holden, Constance C; Beard, M Kate; Bult, Carol J

2006-01-01

Background Many commonly used genome browsers display sequence annotations and related attributes as horizontal data tracks that can be toggled on and off according to user preferences. Most genome browsers use only simple keyword searches and limit the display of detailed annotations to one chromosomal region of the genome at a time. We have employed concepts, methodologies, and tools that were developed for the display of geographic data to develop a Genome Spatial Information System (GenoSIS) for displaying genomes spatially, and interacting with genome annotations and related attribute data. In contrast to the paradigm of horizontally stacked data tracks used by most genome browsers, GenoSIS uses the concept of registered spatial layers composed of spatial objects for integrated display of diverse data. In addition to basic keyword searches, GenoSIS supports complex queries, including spatial queries, and dynamically generates genome maps. Our adaptation of the geographic information system (GIS) model in a genome context supports spatial representation of genome features at multiple scales with a versatile and expressive query capability beyond that supported by existing genome browsers. Results We implemented an interactive genome sequence feature map for the mouse genome in GenoSIS, an application that uses ArcGIS, a commercially available GIS software system. The genome features and their attributes are represented as spatial objects and data layers that can be toggled on and off according to user preferences or displayed selectively in response to user queries. GenoSIS supports the generation of custom genome maps in response to complex queries about genome features based on both their attributes and locations. Our example application of GenoSIS to the mouse genome demonstrates the powerful visualization and query capability of mature GIS technology applied in a novel domain. Conclusion Mapping tools developed specifically for geographic data can be exploited to display, explore and interact with genome data. The approach we describe here is organism independent and is equally useful for linear and circular chromosomes. One of the unique capabilities of GenoSIS compared to existing genome browsers is the capacity to generate genome feature maps dynamically in response to complex attribute and spatial queries. PMID:16984652

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells.

PubMed

Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

2017-08-01

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process.

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells

PubMed Central

Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

2017-01-01

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process. PMID:28621329

Mouse chromosomal mapping of a murine leukemia virus integration region (Mis-1) first identified in rat thymic leukemia.

PubMed Central

Jolicoeur, P; Villeneuve, L; Rassart, E; Kozak, C

1985-01-01

We have previously identified a region of genomic DNA which constitutes the site of frequent provirus integration in rat thymomas induced by Moloney murine leukemia virus (Lemay and Jolicoeur, Proc. Natl. Acad. Sci. USA 81:38-42, 1984). This genetic locus is now designated Mis-1 (Moloney integration site). Cellular sequences homologous to Mis-1 are present in mouse DNA. Using a series of hamster-mouse somatic cell hybrids, we mapped the Mis-1 locus to mouse chromosome 15. Frequent chromosome 15 aberrations have been described in mouse thymomas. Mis-1 represents a putative new oncogene which might be involved in the initiation or maintenance or both of these neoplasms. Images PMID:4068142

Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity

NASA Astrophysics Data System (ADS)

Corcoran, Martin M.; Phad, Ganesh E.; Bernat, Néstor Vázquez; Stahl-Hennig, Christiane; Sumida, Noriyuki; Persson, Mats A. A.; Martin, Marcel; Hedestam, Gunilla B. Karlsson

2016-12-01

Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool.

Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity

PubMed Central

Corcoran, Martin M.; Phad, Ganesh E.; Bernat, Néstor Vázquez; Stahl-Hennig, Christiane; Sumida, Noriyuki; Persson, Mats A.A.; Martin, Marcel; Hedestam, Gunilla B. Karlsson

2016-01-01

Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool. PMID:27995928

J Genes for Heavy Chain Immunoglobulins of Mouse

NASA Astrophysics Data System (ADS)

Newell, Nanette; Richards, Julia E.; Tucker, Philip W.; Blattner, Frederick R.

1980-09-01

A 15.8-kilobase pair fragment of BALB/c mouse liver DNA, cloned in the Charon 4Aλ phage vector system, was shown to contain the μ heavy chain constant region (CHμ ) gene for the mouse immunoglobulin M. In addition, this fragment of DNA contains at least two J genes, used to code for the carboxyl terminal portion of heavy chain variable regions. These genes are located in genomic DNA about eight kilobase pairs to the 5' side of the CHμ gene. The complete nucleotide sequence of a 1120-base pair stretch of DNA that includes the two J genes has been determined.

Statistical methods for detecting periodic fragments in DNA sequence data

PubMed Central

2011-01-01

Background Period 10 dinucleotides are structurally and functionally validated factors that influence the ability of DNA to form nucleosomes, histone core octamers. Robust identification of periodic signals in DNA sequences is therefore required to understand nucleosome organisation in genomes. While various techniques for identifying periodic components in genomic sequences have been proposed or adopted, the requirements for such techniques have not been considered in detail and confirmatory testing for a priori specified periods has not been developed. Results We compared the estimation accuracy and suitability for confirmatory testing of autocorrelation, discrete Fourier transform (DFT), integer period discrete Fourier transform (IPDFT) and a previously proposed Hybrid measure. A number of different statistical significance procedures were evaluated but a blockwise bootstrap proved superior. When applied to synthetic data whose period-10 signal had been eroded, or for which the signal was approximately period-10, the Hybrid technique exhibited superior properties during exploratory period estimation. In contrast, confirmatory testing using the blockwise bootstrap procedure identified IPDFT as having the greatest statistical power. These properties were validated on yeast sequences defined from a ChIP-chip study where the Hybrid metric confirmed the expected dominance of period-10 in nucleosome associated DNA but IPDFT identified more significant occurrences of period-10. Application to the whole genomes of yeast and mouse identified ~ 21% and ~ 19% respectively of these genomes as spanned by period-10 nucleosome positioning sequences (NPS). Conclusions For estimating the dominant period, we find the Hybrid period estimation method empirically to be the most effective for both eroded and approximate periodicity. The blockwise bootstrap was found to be effective as a significance measure, performing particularly well in the problem of period detection in the presence of eroded periodicity. The autocorrelation method was identified as poorly suited for use with the blockwise bootstrap. Application of our methods to the genomes of two model organisms revealed a striking proportion of the yeast and mouse genomes are spanned by NPS. Despite their markedly different sizes, roughly equivalent proportions (19-21%) of the genomes lie within period-10 spans of the NPS dinucleotides {AA, TT, TA}. The biological significance of these regions remains to be demonstrated. To facilitate this, the genomic coordinates are available as Additional files 1, 2, and 3 in a format suitable for visualisation as tracks on popular genome browsers. Reviewers This article was reviewed by Prof Tomas Radivoyevitch, Dr Vsevolod Makeev (nominated by Dr Mikhail Gelfand), and Dr Rob D Knight. PMID:21527008

CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli.

PubMed Central

Kipling, D; Mitchell, A R; Masumoto, H; Wilson, H E; Nicol, L; Cooke, H J

1995-01-01

Minor satellite DNA, found at Mus musculus centromeres, is not present in the genome of the Asian mouse Mus caroli. This repetitive sequence family is speculated to have a role in centromere function by providing an array of binding sites for the centromere-associated protein CENP-B. The apparent absence of CENP-B binding sites in the M. caroli genome poses a major challenge to this hypothesis. Here we describe two abundant satellite DNA sequences present at M. caroli centromeres. These satellites are organized as tandem repeat arrays, over 1 Mb in size, of either 60- or 79-bp monomers. All autosomes carry both satellites and small amounts of a sequence related to the M. musculus major satellite. The Y chromosome contains small amounts of both major satellite and the 60-bp satellite, whereas the X chromosome carries only major satellite sequences. M. caroli chromosomes segregate in M. caroli x M. musculus interspecific hybrid cell lines, indicating that the two sets of chromosomes can interact with the same mitotic spindle. Using a polyclonal CENP-B antiserum, we demonstrate that M. caroli centromeres can bind murine CENP-B in such an interspecific cell line, despite the absence of canonical 17-bp CENP-B binding sites in the M. caroli genome. Sequence analysis of the 79-bp M. caroli satellite reveals a 17-bp motif that contains all nine bases previously shown to be necessary for in vitro binding of CENP-B. This M. caroli motif binds CENP-B from HeLa cell nuclear extract in vitro, as indicated by gel mobility shift analysis. We therefore suggest that this motif also causes CENP-B to associate with M. caroli centromeres in vivo. Despite the sequence differences, M. caroli presents a third, novel mammalian centromeric sequence producing an array of binding sites for CENP-B. PMID:7623797

Sequencing, Analysis, and Annotation of Expressed Sequence Tags for Camelus dromedarius

PubMed Central

Al-Swailem, Abdulaziz M.; Shehata, Maher M.; Abu-Duhier, Faisel M.; Al-Yamani, Essam J.; Al-Busadah, Khalid A.; Al-Arawi, Mohammed S.; Al-Khider, Ali Y.; Al-Muhaimeed, Abdullah N.; Al-Qahtani, Fahad H.; Manee, Manee M.; Al-Shomrani, Badr M.; Al-Qhtani, Saad M.; Al-Harthi, Amer S.; Akdemir, Kadir C.; Otu, Hasan H.

2010-01-01

Despite its economical, cultural, and biological importance, there has not been a large scale sequencing project to date for Camelus dromedarius. With the goal of sequencing complete DNA of the organism, we first established and sequenced camel EST libraries, generating 70,272 reads. Following trimming, chimera check, repeat masking, cluster and assembly, we obtained 23,602 putative gene sequences, out of which over 4,500 potentially novel or fast evolving gene sequences do not carry any homology to other available genomes. Functional annotation of sequences with similarities in nucleotide and protein databases has been obtained using Gene Ontology classification. Comparison to available full length cDNA sequences and Open Reading Frame (ORF) analysis of camel sequences that exhibit homology to known genes show more than 80% of the contigs with an ORF>300 bp and ∼40% hits extending to the start codons of full length cDNAs suggesting successful characterization of camel genes. Similarity analyses are done separately for different organisms including human, mouse, bovine, and rat. Accompanying web portal, CAGBASE (http://camel.kacst.edu.sa/), hosts a relational database containing annotated EST sequences and analysis tools with possibility to add sequences from public domain. We anticipate our results to provide a home base for genomic studies of camel and other comparative studies enabling a starting point for whole genome sequencing of the organism. PMID:20502665

Human cDNA mapping using fluorescence in situ hybridization. Final progress report, April 1, 1994--July 31, 1997

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korenberg, J.R.

The ultimate goal of this research is to generate and apply novel technologies to speed completion and integration of the human genome map and sequence with biomedical problems. To do this, techniques were developed and genome-wide resources generated. This includes a genome-wide Mapped and Integrated BAC/PAC Resource that has been used for gene finding, map completion and anchoring, breakpoint definition and sequencing. In the last period of the grant, the Human Mapped BAC/PAC Resource was also applied to determine regions of human variation and to develop a novel paradigm of primate evolution through to humans. Further, in order to moremore » rapidly evaluate animal models of human disease, a BAC Map of the mouse was generated in collaboration with the MTI Genome Center, Dr. Bruce Birren.« less

Characterization and mapping of the mouse NDP (Norrie disease) locus (Ndp).

PubMed

Battinelli, E M; Boyd, Y; Craig, I W; Breakefield, X O; Chen, Z Y

1996-02-01

Norrie disease is a severe X-linked recessive neurological disorder characterized by congenital blindness with progressive loss of hearing. Over half of Norrie patients also manifest different degrees of mental retardation. The gene for Norrie disease (NDP) has recently been cloned and characterized. With the human NDP cDNA, mouse genomic phage libraries were screened for the homolog of the gene. Comparison between mouse and human genomic DNA blots hybridized with the NDP cDNA, as well as analysis of phage clones, shows that the mouse NDP gene is 29 kb in size (28 kb for the human gene). The organization in the two species is very similar. Both have three exons with similar-sized introns and identical exon-intron boundaries between exon 2 and 3. The mouse open reading frame is 393 bp and, like the human coding sequence, is encoded in exons 2 and 3. The absence of six nucleotides in the second mouse exon results in the encoded protein being two amino acids smaller than its human counterpart. The overall homology between the human and mouse NDP protein is 95% and is particularly high (99%) in exon 3, consistent with the apparent functional importance of this region. Analysis of transcription initiation sites suggests the presence of multiple start sites associated with expression of the mouse NDP gene. Pedigree analysis of an interspecific mouse backcross localizes the mouse NDP gene close to Maoa in the conserved segment, which runs from CYBB to PFC in both human and mouse.

HnRNP A3 genes and pseudogenes in the vertebrate genomes.

PubMed

Makeyev, Aleksandr V; Kim, Chang Bae; Ruddle, Frank H; Enkhmandakh, Badam; Erdenechimeg, Lkhamsuren; Bayarsaihan, Dashzeveg

2005-04-01

The hnRNP A/B type proteins are abundant nuclear factors that bind to Pol II transcripts and are involved in numerous RNA-related activities. To date most data on the hnRNP A/B family have been obtained with recombinant proteins and cell cultures. Further characterization can result from an examination of the impact of various modifications in intact functional loci; however, such characterization is hampered by the presence of numerous and widely dispersed hnRNP A/B-related sequences in the mammalian genome. We have found hnRNP A3, a poorly recognized member of the hnRNP A/B family, among candidate transcription factors that interact with the regulatory region of the Hoxc8 gene and screened the human and mouse genomes for genes that encode hnRNP A3. We demonstrate that the sequence reported previously as the human hnRNP A3 gene (Accession number S63912) and located on 10p11.1 belongs to a processed pseudogene of the functional intron-containing locus HNRPA3, which we have identified on 2q31.2. We have also identified its murine orthologs on mouse chromosome 2D and rat chromosome 3q23. Alternative splices were revealed at the N-terminus and in the middle of hnRNP A3. 14 and 28 additional loci in the human and mouse genome, respectively, were mapped and identified as hnRNP A3 processed pseudogenes. In addition, we have found and compared hnRNP A3 orthologous genes in Gallus gallus, Xenopus tropicalis, and Danio rerio. The present in silico analysis serves as a necessary step toward a further functional characterization of hnRNP A3. (c) 2005 Wiley-Liss, Inc.

Genome characterization of the selected long- and short-sleep mouse lines.

PubMed

Dowell, Robin; Odell, Aaron; Richmond, Phillip; Malmer, Daniel; Halper-Stromberg, Eitan; Bennett, Beth; Larson, Colin; Leach, Sonia; Radcliffe, Richard A

2016-12-01

The Inbred Long- and Short-Sleep (ILS, ISS) mouse lines were selected for differences in acute ethanol sensitivity using the loss of righting response (LORR) as the selection trait. The lines show an over tenfold difference in LORR and, along with a recombinant inbred panel derived from them (the LXS), have been widely used to dissect the genetic underpinnings of acute ethanol sensitivity. Here we have sequenced the genomes of the ILS and ISS to investigate the DNA variants that contribute to their sensitivity difference. We identified ~2.7 million high-confidence SNPs and small indels and ~7000 structural variants between the lines; variants were found to occur in 6382 annotated genes. Using a hidden Markov model, we were able to reconstruct the genome-wide ancestry patterns of the eight inbred progenitor strains from which the ILS and ISS were derived, and found that quantitative trait loci that have been mapped for LORR were slightly enriched for DNA variants. Finally, by mapping and quantifying RNA-seq reads from the ILS and ISS to their strain-specific genomes rather than to the reference genome, we found a substantial improvement in a differential expression analysis between the lines. This work will help in identifying and characterizing the DNA sequence variants that contribute to the difference in ethanol sensitivity between the ILS and ISS and will also aid in accurate quantification of RNA-seq data generated from the LXS RIs.

Expression profiling of the mouse early embryo: Reflections and Perspectives

PubMed Central

Ko, Minoru S. H.

2008-01-01

Laboratory mouse plays important role in our understanding of early mammalian development and provides invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. Comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over last two decades, have provided even more advantages to mouse models. Here the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells are reviewed and the future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, which include: bird’s-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks. PMID:16739220

An integrated expression atlas of miRNAs and their promoters in human and mouse

PubMed Central

de Rie, Derek; Abugessaisa, Imad; Alam, Tanvir; Arner, Erik; Arner, Peter; Ashoor, Haitham; Åström, Gaby; Babina, Magda; Bertin, Nicolas; Burroughs, A. Maxwell; Carlisle, Ailsa J.; Daub, Carsten O.; Detmar, Michael; Deviatiiarov, Ruslan; Fort, Alexandre; Gebhard, Claudia; Goldowitz, Daniel; Guhl, Sven; Ha, Thomas J.; Harshbarger, Jayson; Hasegawa, Akira; Hashimoto, Kosuke; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J.; Hon, Chung Chau; Huang, Edward; Ishizu, Yuri; Kai, Chieko; Kasukawa, Takeya; Klinken, Peter; Lassmann, Timo; Lecellier, Charles-Henri; Lee, Weonju; Lizio, Marina; Makeev, Vsevolod; Mathelier, Anthony; Medvedeva, Yulia A.; Mejhert, Niklas; Mungall, Christopher J.; Noma, Shohei; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Persson, Helena; Rizzu, Patrizia; Roudnicky, Filip; Sætrom, Pål; Sato, Hiroki; Severin, Jessica; Shin, Jay W.; Swoboda, Rolf K.; Tarui, Hiroshi; Toyoda, Hiroo; Vitting-Seerup, Kristoffer; Winteringham, Louise; Yamaguchi, Yoko; Yasuzawa, Kayoko; Yoneda, Misako; Yumoto, Noriko; Zabierowski, Susan; Zhang, Peter G.; Wells, Christine A.; Summers, Kim M.; Kawaji, Hideya; Sandelin, Albin; Rehli, Michael; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R. R.; de Hoon, Michiel J. L.

2018-01-01

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions. PMID:28829439

The identification and functional annotation of RNA structures conserved in vertebrates

PubMed Central

Seemann, Stefan E.; Mirza, Aashiq H.; Hansen, Claus; Bang-Berthelsen, Claus H.; Garde, Christian; Christensen-Dalsgaard, Mikkel; Torarinsson, Elfar; Yao, Zizhen; Workman, Christopher T.; Pociot, Flemming; Nielsen, Henrik; Tommerup, Niels; Ruzzo, Walter L.; Gorodkin, Jan

2017-01-01

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human–mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3′ ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality. PMID:28487280

Genetic exchange between endogenous and exogenous LINE-1 repetitive elements in mouse cells.

PubMed Central

Belmaaza, A; Wallenburg, J C; Brouillette, S; Gusew, N; Chartrand, P

1990-01-01

The repetitive LINE (L1) elements of the mouse, which are present at about 10(5) copies per genome and share over 80% of sequence homology, were examined for their ability to undergo genetic exchange with exogenous L1 sequences. The exogenous L1 sequences, carried by a shuttle vector, consisted of an internal fragment from L1Md-A2, a previously described member of the L1 family of the mouse. Using an assay that does not require the reconstitution of a selectable marker we found that this vector, in either circular or linear form, acquired DNA sequences from endogenous L1 elements at a frequency of 10(-3) to 10(-4) per rescued vector. Physical analysis of the acquired L1 sequences revealed that distinct endogenous L1 elements acted as donors and that different subfamilies participated. These results demonstrate that L1 elements are readily capable of genetic exchange. Apart from gene conversion events, the acquisition of L1 sequences outside the region of homology suggested that a second mechanism was also involved in the genetic exchange. A model which accounts for this mechanism is presented and its potential implication on the rearrangement of L1 elements is discussed. Images PMID:1978749

Ab initio gene identification in metagenomic sequences

PubMed Central

Zhu, Wenhan; Lomsadze, Alexandre; Borodovsky, Mark

2010-01-01

We describe an algorithm for gene identification in DNA sequences derived from shotgun sequencing of microbial communities. Accurate ab initio gene prediction in a short nucleotide sequence of anonymous origin is hampered by uncertainty in model parameters. While several machine learning approaches could be proposed to bypass this difficulty, one effective method is to estimate parameters from dependencies, formed in evolution, between frequencies of oligonucleotides in protein-coding regions and genome nucleotide composition. Original version of the method was proposed in 1999 and has been used since for (i) reconstructing codon frequency vector needed for gene finding in viral genomes and (ii) initializing parameters of self-training gene finding algorithms. With advent of new prokaryotic genomes en masse it became possible to enhance the original approach by using direct polynomial and logistic approximations of oligonucleotide frequencies, as well as by separating models for bacteria and archaea. These advances have increased the accuracy of model reconstruction and, subsequently, gene prediction. We describe the refined method and assess its accuracy on known prokaryotic genomes split into short sequences. Also, we show that as a result of application of the new method, several thousands of new genes could be added to existing annotations of several human and mouse gut metagenomes. PMID:20403810

Theory on the mechanism of site-specific DNA-protein interactions in the presence of traps

NASA Astrophysics Data System (ADS)

Niranjani, G.; Murugan, R.

2016-08-01

The speed of site-specific binding of transcription factor (TFs) proteins with genomic DNA seems to be strongly retarded by the randomly occurring sequence traps. Traps are those DNA sequences sharing significant similarity with the original specific binding sites (SBSs). It is an intriguing question how the naturally occurring TFs and their SBSs are designed to manage the retarding effects of such randomly occurring traps. We develop a simple random walk model on the site-specific binding of TFs with genomic DNA in the presence of sequence traps. Our dynamical model predicts that (a) the retarding effects of traps will be minimum when the traps are arranged around the SBS such that there is a negative correlation between the binding strength of TFs with traps and the distance of traps from the SBS and (b) the retarding effects of sequence traps can be appeased by the condensed conformational state of DNA. Our computational analysis results on the distribution of sequence traps around the putative binding sites of various TFs in mouse and human genome clearly agree well the theoretical predictions. We propose that the distribution of traps can be used as an additional metric to efficiently identify the SBSs of TFs on genomic DNA.

SGP-1: Prediction and Validation of Homologous Genes Based on Sequence Alignments

PubMed Central

Wiehe, Thomas; Gebauer-Jung, Steffi; Mitchell-Olds, Thomas; Guigó, Roderic

2001-01-01

Conventional methods of gene prediction rely on the recognition of DNA-sequence signals, the coding potential or the comparison of a genomic sequence with a cDNA, EST, or protein database. Reasons for limited accuracy in many circumstances are species-specific training and the incompleteness of reference databases. Lately, comparative genome analysis has attracted increasing attention. Several analysis tools that are based on human/mouse comparisons are already available. Here, we present a program for the prediction of protein-coding genes, termed SGP-1 (Syntenic Gene Prediction), which is based on the similarity of homologous genomic sequences. In contrast to most existing tools, the accuracy of SGP-1 depends little on species-specific properties such as codon usage or the nucleotide distribution. SGP-1 may therefore be applied to nonstandard model organisms in vertebrates as well as in plants, without the need for extensive parameter training. In addition to predicting genes in large-scale genomic sequences, the program may be useful to validate gene structure annotations from databases. To this end, SGP-1 output also contains comparisons between predicted and annotated gene structures in HTML format. The program can be accessed via a Web server at http://soft.ice.mpg.de/sgp-1. The source code, written in ANSI C, is available on request from the authors. PMID:11544202

Coval: Improving Alignment Quality and Variant Calling Accuracy for Next-Generation Sequencing Data

PubMed Central

Kosugi, Shunichi; Natsume, Satoshi; Yoshida, Kentaro; MacLean, Daniel; Cano, Liliana; Kamoun, Sophien; Terauchi, Ryohei

2013-01-01

Accurate identification of DNA polymorphisms using next-generation sequencing technology is challenging because of a high rate of sequencing error and incorrect mapping of reads to reference genomes. Currently available short read aligners and DNA variant callers suffer from these problems. We developed the Coval software to improve the quality of short read alignments. Coval is designed to minimize the incidence of spurious alignment of short reads, by filtering mismatched reads that remained in alignments after local realignment and error correction of mismatched reads. The error correction is executed based on the base quality and allele frequency at the non-reference positions for an individual or pooled sample. We demonstrated the utility of Coval by applying it to simulated genomes and experimentally obtained short-read data of rice, nematode, and mouse. Moreover, we found an unexpectedly large number of incorrectly mapped reads in ‘targeted’ alignments, where the whole genome sequencing reads had been aligned to a local genomic segment, and showed that Coval effectively eliminated such spurious alignments. We conclude that Coval significantly improves the quality of short-read sequence alignments, thereby increasing the calling accuracy of currently available tools for SNP and indel identification. Coval is available at http://sourceforge.net/projects/coval105/. PMID:24116042

A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly

PubMed Central

2013-01-01

Background The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes. Results We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters. Conclusions These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies. PMID:23496952

A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly.

PubMed

Francis, Warren R; Christianson, Lynne M; Kiko, Rainer; Powers, Meghan L; Shaner, Nathan C; Haddock, Steven H D

2013-03-12

The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes. We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters. These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies.

Genomic Identification and Analysis of Shared Cis-regulator Elements in a Developmentally Critical homeobox Cluster

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chris Amemiya

2003-04-01

The goals of this project were to isolate, characterize, and sequence the Dlx3/Dlx7 bigene cluster from twelve different species of mammals. The Dlx3 and Dlx7 genes are known to encode homeobox transcription factors involved in patterning of structures in the vertebrate jaw as well as vertebrate limbs. Genomic sequences from the respective taxa will subsequently be compared in order to identify conserved non-coding sequences that are potential cis-regulatory elements. Based on the comparisons they will fashion transgenic mouse experiments to functionally test the strength of the potential cis-regulatory elements. A goal of the project is to attempt to identify thosemore » elements that may function in coordinately regulating both Dlx3 and Dlx7 functions.« less

Comparative Sequence Analysis of the X-Inactivation Center Region in Mouse, Human, and Bovine

PubMed Central

Chureau, Corinne; Prissette, Marine; Bourdet, Agnès; Barbe, Valérie; Cattolico, Laurence; Jones, Louis; Eggen, André; Avner, Philip; Duret, Laurent

2002-01-01

We have sequenced to high levels of accuracy 714-kb and 233-kb regions of the mouse and bovine X-inactivation centers (Xic), respectively, centered on the Xist gene. This has provided the basis for a fully annotated comparative analysis of the mouse Xic with the 2.3-Mb orthologous region in human and has allowed a three-way species comparison of the core central region, including the Xist gene. These comparisons have revealed conserved genes, both coding and noncoding, conserved CpG islands and, more surprisingly, conserved pseudogenes. The distribution of repeated elements, especially LINE repeats, in the mouse Xic region when compared to the rest of the genome does not support the hypothesis of a role for these repeat elements in the spreading of X inactivation. Interestingly, an asymmetric distribution of LINE elements on the two DNA strands was observed in the three species, not only within introns but also in intergenic regions. This feature is suggestive of important transcriptional activity within these intergenic regions. In silico prediction followed by experimental analysis has allowed four new genes, Cnbp2, Ftx, Jpx, and Ppnx, to be identified and novel, widespread, complex, and apparently noncoding transcriptional activity to be characterized in a region 5′ of Xist that was recently shown to attract histone modification early after the onset of X inactivation. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ421478, AJ421479, AJ421480, and AJ421481. Online supplemental data are available at http://pbil.univ-lyon1.fr/datasets/Xic2002/data.html and www.genome.org.] PMID:12045143

The Mouse Lemur, a Genetic Model Organism for Primate Biology, Behavior, and Health

PubMed Central

Ezran, Camille; Karanewsky, Caitlin J.; Pendleton, Jozeph L.; Sholtz, Alex; Krasnow, Maya R.; Willick, Jason; Razafindrakoto, Andriamahery; Zohdy, Sarah; Albertelli, Megan A.; Krasnow, Mark A.

2017-01-01

Systematic genetic studies of a handful of diverse organisms over the past 50 years have transformed our understanding of biology. However, many aspects of primate biology, behavior, and disease are absent or poorly modeled in any of the current genetic model organisms including mice. We surveyed the animal kingdom to find other animals with advantages similar to mice that might better exemplify primate biology, and identified mouse lemurs (Microcebus spp.) as the outstanding candidate. Mouse lemurs are prosimian primates, roughly half the genetic distance between mice and humans. They are the smallest, fastest developing, and among the most prolific and abundant primates in the world, distributed throughout the island of Madagascar, many in separate breeding populations due to habitat destruction. Their physiology, behavior, and phylogeny have been studied for decades in laboratory colonies in Europe and in field studies in Malagasy rainforests, and a high quality reference genome sequence has recently been completed. To initiate a classical genetic approach, we developed a deep phenotyping protocol and have screened hundreds of laboratory and wild mouse lemurs for interesting phenotypes and begun mapping the underlying mutations, in collaboration with leading mouse lemur biologists. We also seek to establish a mouse lemur gene “knockout” library by sequencing the genomes of thousands of mouse lemurs to identify null alleles in most genes from the large pool of natural genetic variants. As part of this effort, we have begun a citizen science project in which students across Madagascar explore the remarkable biology around their schools, including longitudinal studies of the local mouse lemurs. We hope this work spawns a new model organism and cultivates a deep genetic understanding of primate biology and health. We also hope it establishes a new and ethical method of genetics that bridges biological, behavioral, medical, and conservation disciplines, while providing an example of how hands-on science education can help transform developing countries. PMID:28592502

The Mouse Lemur, a Genetic Model Organism for Primate Biology, Behavior, and Health.

PubMed

Ezran, Camille; Karanewsky, Caitlin J; Pendleton, Jozeph L; Sholtz, Alex; Krasnow, Maya R; Willick, Jason; Razafindrakoto, Andriamahery; Zohdy, Sarah; Albertelli, Megan A; Krasnow, Mark A

2017-06-01

Systematic genetic studies of a handful of diverse organisms over the past 50 years have transformed our understanding of biology. However, many aspects of primate biology, behavior, and disease are absent or poorly modeled in any of the current genetic model organisms including mice. We surveyed the animal kingdom to find other animals with advantages similar to mice that might better exemplify primate biology, and identified mouse lemurs ( Microcebus spp.) as the outstanding candidate. Mouse lemurs are prosimian primates, roughly half the genetic distance between mice and humans. They are the smallest, fastest developing, and among the most prolific and abundant primates in the world, distributed throughout the island of Madagascar, many in separate breeding populations due to habitat destruction. Their physiology, behavior, and phylogeny have been studied for decades in laboratory colonies in Europe and in field studies in Malagasy rainforests, and a high quality reference genome sequence has recently been completed. To initiate a classical genetic approach, we developed a deep phenotyping protocol and have screened hundreds of laboratory and wild mouse lemurs for interesting phenotypes and begun mapping the underlying mutations, in collaboration with leading mouse lemur biologists. We also seek to establish a mouse lemur gene "knockout" library by sequencing the genomes of thousands of mouse lemurs to identify null alleles in most genes from the large pool of natural genetic variants. As part of this effort, we have begun a citizen science project in which students across Madagascar explore the remarkable biology around their schools, including longitudinal studies of the local mouse lemurs. We hope this work spawns a new model organism and cultivates a deep genetic understanding of primate biology and health. We also hope it establishes a new and ethical method of genetics that bridges biological, behavioral, medical, and conservation disciplines, while providing an example of how hands-on science education can help transform developing countries. Copyright © 2017 by the Genetics Society of America.

Comparative and evolutionary studies of vertebrate ALDH1A-like genes and proteins.

PubMed

Holmes, Roger S

2015-06-05

Vertebrate ALDH1A-like genes encode cytosolic enzymes capable of metabolizing all-trans-retinaldehyde to retinoic acid which is a molecular 'signal' guiding vertebrate development and adipogenesis. Bioinformatic analyses of vertebrate and invertebrate genomes were undertaken using known ALDH1A1, ALDH1A2 and ALDH1A3 amino acid sequences. Comparative analyses of the corresponding human genes provided evidence for distinct modes of gene regulation and expression with putative transcription factor binding sites (TFBS), CpG islands and micro-RNA binding sites identified for the human genes. ALDH1A-like sequences were identified for all mammalian, bird, lizard and frog genomes examined, whereas fish genomes displayed a more restricted distribution pattern for ALDH1A1 and ALDH1A3 genes. The ALDH1A1 gene was absent in many bony fish genomes examined, with the ALDH1A3 gene also absent in the medaka and tilapia genomes. Multiple ALDH1A1-like genes were identified in mouse, rat and marsupial genomes. Vertebrate ALDH1A1, ALDH1A2 and ALDH1A3 subunit sequences were highly conserved throughout vertebrate evolution. Comparative amino acid substitution rates showed that mammalian ALDH1A2 sequences were more highly conserved than for the ALDH1A1 and ALDH1A3 sequences. Phylogenetic studies supported an hypothesis for ALDH1A2 as a likely primordial gene originating in invertebrate genomes and undergoing sequential gene duplication to generate two additional genes, ALDH1A1 and ALDH1A3, in most vertebrate genomes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome

USDA-ARS?s Scientific Manuscript database

DNA methylation is an epigenetic mechanism central to the development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited. We used whole genome bisulfite sequencing, and found that differentiation of mouse colonic intestinal stem cell...

CpG methylation differences between neurons and glia are highly conserved from mouse to human

USDA-ARS?s Scientific Manuscript database

Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study used genome-wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That stud...

Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing.

PubMed

Liu, Zhen; Lu, Zongyang; Yang, Guang; Huang, Shisheng; Li, Guanglei; Feng, Songjie; Liu, Yajing; Li, Jianan; Yu, Wenxia; Zhang, Yu; Chen, Jia; Sun, Qiang; Huang, Xingxu

2018-06-14

A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.

Genome-wide identification of conserved intronic non-coding sequences using a Bayesian segmentation approach.

PubMed

Algama, Manjula; Tasker, Edward; Williams, Caitlin; Parslow, Adam C; Bryson-Richardson, Robert J; Keith, Jonathan M

2017-03-27

Computational identification of non-coding RNAs (ncRNAs) is a challenging problem. We describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species: human, mouse and zebrafish. We investigate the extent to which these elements include ncRNAs (or conserved domains of ncRNAs) and regulatory sequences. We identified 655 deeply conserved intronic sequences in a genome-wide analysis. We also performed a pathway-focussed analysis on genes involved in muscle development, detecting 27 intronic elements, of which 22 were not detected in the genome-wide analysis. At least 87% of the genome-wide and 70% of the pathway-focussed elements have existing annotations indicative of conserved RNA secondary structure. The expression of 26 of the pathway-focused elements was examined using RT-PCR, providing confirmation that they include expressed ncRNAs. Consistent with previous studies, these elements are significantly over-represented in the introns of transcription factors. This study demonstrates a novel, highly effective, Bayesian approach to identifying conserved non-coding sequences. Our results complement previous findings that these sequences are enriched in transcription factors. However, in contrast to previous studies which suggest the majority of conserved sequences are regulatory factor binding sites, the majority of conserved sequences identified using our approach contain evidence of conserved RNA secondary structures, and our laboratory results suggest most are expressed. Functional roles at DNA and RNA levels are not mutually exclusive, and many of our elements possess evidence of both. Moreover, ncRNAs play roles in transcriptional and post-transcriptional regulation, and this may contribute to the over-representation of these elements in introns of transcription factors. We attribute the higher sensitivity of the pathway-focussed analysis compared to the genome-wide analysis to improved alignment quality, suggesting that enhanced genomic alignments may reveal many more conserved intronic sequences.

Mouse Models for Drug Discovery. Can New Tools and Technology Improve Translational Power?

PubMed Central

Zuberi, Aamir; Lutz, Cathleen

2016-01-01

Abstract The use of mouse models in biomedical research and preclinical drug evaluation is on the rise. The advent of new molecular genome-altering technologies such as CRISPR/Cas9 allows for genetic mutations to be introduced into the germ line of a mouse faster and less expensively than previous methods. In addition, the rapid progress in the development and use of somatic transgenesis using viral vectors, as well as manipulations of gene expression with siRNAs and antisense oligonucleotides, allow for even greater exploration into genomics and systems biology. These technological advances come at a time when cost reductions in genome sequencing have led to the identification of pathogenic mutations in patient populations, providing unprecedented opportunities in the use of mice to model human disease. The ease of genetic engineering in mice also offers a potential paradigm shift in resource sharing and the speed by which models are made available in the public domain. Predictively, the knowledge alone that a model can be quickly remade will provide relief to resources encumbered by licensing and Material Transfer Agreements. For decades, mouse strains have provided an exquisite experimental tool to study the pathophysiology of the disease and assess therapeutic options in a genetically defined system. However, a major limitation of the mouse has been the limited genetic diversity associated with common laboratory mice. This has been overcome with the recent development of the Collaborative Cross and Diversity Outbred mice. These strains provide new tools capable of replicating genetic diversity to that approaching the diversity found in human populations. The Collaborative Cross and Diversity Outbred strains thus provide a means to observe and characterize toxicity or efficacy of new therapeutic drugs for a given population. The combination of traditional and contemporary mouse genome editing tools, along with the addition of genetic diversity in new modeling systems, are synergistic and serve to make the mouse a better model for biomedical research, enhancing the potential for preclinical drug discovery and personalized medicine. PMID:28053071

Mutational landscape of a chemically-induced mouse model of liver cancer.

PubMed

Connor, Frances; Rayner, Tim F; Aitken, Sarah J; Feig, Christine; Lukk, Margus; Santoyo-Lopez, Javier; Odom, Duncan T

2018-06-26

Carcinogen-induced mouse models of liver cancer are used extensively to study pathogenesis of the disease and have a critical role in validating candidate therapeutics. These models can recapitulate molecular and histological features of human disease. However, it is not known if the genomic alterations driving these mouse tumour genomes are comparable to those found in human tumours. Here, we provide a detailed genomic characterisation of tumours from a commonly used mouse model of hepatocellular carcinoma (HCC). We analysed whole exome sequences of liver tumours arising in mice exposed to diethylnitrosamine (DEN). DEN-initiated tumours had a high, uniform number of somatic single nucleotide variants (SNVs), with few insertions, deletions or copy number alterations, consistent with the known genotoxic action of DEN. Exposure of hepatocytes to DEN left a reproducible mutational imprint in resulting tumour exomes which we could computationally reconstruct using six known COSMIC mutational signatures. The tumours carried a high diversity of low-incidence, non-synonymous point mutations in many oncogenes and tumour suppressors, reflecting the stochastic introduction of SNVs into the hepatocyte genome by the carcinogen. We identified four recurrently mutated genes that were putative oncogenic drivers of HCC in this model. Every neoplasm carried activating hotspot mutations either in codon 61 of Hras, in codon 584 of Braf or in codon 254 of Egfr. Truncating mutations of Apc occurred in 21% of neoplasms, which were exclusively carcinomas supporting a role for deregulation of Wnt/β-catenin signalling in cancer progression. Our study provides detailed insight into the mutational landscape of tumours arising in a commonly-used carcinogen model of HCC, facilitating the future use of this model to understand the human disease. Mouse models are widely used to study the biology of cancer and to test potential therapies. Here, we have described the mutational landscape of tumours arising in a carcinogen-induced mouse model of liver cancer. Since cancer is a disease caused by genomic alterations, information about the patterns and types of mutations in the tumours in this mouse model should facilitate its use to study human liver cancer. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Selective Amplification of the Genome Surrounding Key Placental Genes in Trophoblast Giant Cells.

PubMed

Hannibal, Roberta L; Baker, Julie C

2016-01-25

While most cells maintain a diploid state, polyploid cells exist in many organisms and are particularly prevalent within the mammalian placenta [1], where they can generate more than 900 copies of the genome [2]. Polyploidy is thought to be an efficient method of increasing the content of the genome by avoiding the costly and slow process of cytokinesis [1, 3, 4]. Polyploidy can also affect gene regulation by amplifying a subset of genomic regions required for specific cellular function [1, 3, 4]. This mechanism is found in the fruit fly Drosophila melanogaster, where polyploid ovarian follicle cells amplify genomic regions containing chorion genes, which facilitate secretion of eggshell proteins [5]. Here, we report that genomic amplification also occurs in mammals at selective regions of the genome in parietal trophoblast giant cells (p-TGCs) of the mouse placenta. Using whole-genome sequencing (WGS) and digital droplet PCR (ddPCR) of mouse p-TGCs, we identified five amplified regions, each containing a gene family known to be involved in mammalian placentation: the prolactins (two clusters), serpins, cathepsins, and the natural killer (NK)/C-type lectin (CLEC) complex [6-12]. We report here the first description of amplification at selective genomic regions in mammals and present evidence that this is an important mode of genome regulation in placental TGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fox gene loci in Takifugu rubripes and Tetraodon nigroviridis genomes and comparison with those of medaka and zebrafish genomes.

PubMed

Shen, Xueyan; Cui, Jianzhou; Gong, Qingli

2011-12-01

Members of the Fox gene family of transcriptional regulators are essential for animal development and have been extensively studied in vertebrates. The mouse and human genomes contain at least 40 FOX genes which are divided into 19 subclasses based on the sequence similarity of the highly conserved forkhead domain. Using the genome sequence of the Takifugu rubripes and Tetraodon nigroviridis , we examined the genomic complement of fox genes in these organisms to gain insight into the evolutionary relationship of this gene family. We identified 53 fox genes in Tetraodon nigroviridis and Takifugu rubripes genome by searching the forkhead domain. These genes are divided into 18 subclasses as follows: 8 fox genes in subclass O; 6 in subclass P ; 4 in subclasses D, J, and N; 3 in subclasses A, B, C, E, F, and I; 2 in subclasses K, L, and Q; and 1 in subclasses G, H, M, and R. Together with the forkhead domain sequences of human, chicken, frog, zebrafish, medaka, and Caenorhabditis elegans, the phylogenetic relationship of the fox genes in Takifugu rubripes and Tetraodon nigroviridis were analyzed and compared. The genes structure, general features, and the three-dimensional model of these genes were also discussed.

Organization and roles of nucleosomes at mouse meiotic recombination hotspots

PubMed Central

Getun, Irina V.; Wu, Zhen K.; Bois, Philippe R.J.

2012-01-01

Meiotic double strand breaks (DSBs) occur at discrete regions in the genome coined hotspots. Precisely what directs site selection of these DSBs is hotly debated and in particular it is unclear which chromatin features, and regulatory factors are necessary for a genomic region to initiate and resolve DSBs as a crossover (CO) event. In human and mouse, one layer of hotspot selection control is a recognition sequence element present at these sites that is bound by the Prdm9 zinc-finger protein. Furthermore, an overall open chromatin structure is thought to be required to allow access of the recombination machinery, and this is often dictated by the packaging of DNA around nucleosomes. We recently defined the nucleosome occupancy maps of four mouse recombination hotspots throughout meiosis. These analyses revealed no obvious dynamic changes in nucleosome occupancy, suggesting an intrinsic nature of recombinogenic sites, yet they also revealed that nucleosomes define zones of exclusion for CO resolution. Here, we discuss new evidence implicating nucleosome occupancy in recombinogenic repair and its potential roles in controlling chromatin structure at mouse meiotic hotspots. PMID:22572955

In vivo gene correction with targeted sequence substitution through microhomology-mediated end joining.

PubMed

Shin, Jeong Hong; Jung, Soobin; Ramakrishna, Suresh; Kim, Hyongbum Henry; Lee, Junwon

2018-07-07

Genome editing technology using programmable nucleases has rapidly evolved in recent years. The primary mechanism to achieve precise integration of a transgene is mainly based on homology-directed repair (HDR). However, an HDR-based genome-editing approach is less efficient than non-homologous end-joining (NHEJ). Recently, a microhomology-mediated end-joining (MMEJ)-based transgene integration approach was developed, showing feasibility both in vitro and in vivo. We expanded this method to achieve targeted sequence substitution (TSS) of mutated sequences with normal sequences using double-guide RNAs (gRNAs), and a donor template flanking the microhomologies and target sequence of the gRNAs in vitro and in vivo. Our method could realize more efficient sequence substitution than the HDR-based method in vitro using a reporter cell line, and led to the survival of a hereditary tyrosinemia mouse model in vivo. The proposed MMEJ-based TSS approach could provide a novel therapeutic strategy, in addition to HDR, to achieve gene correction from a mutated sequence to a normal sequence. Copyright © 2018 Elsevier Inc. All rights reserved.

Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved

PubMed Central

Long, Hannah K.; King, Hamish W.; Patient, Roger K.; Odom, Duncan T.; Klose, Robert J.

2016-01-01

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. PMID:27084945

Discriminative prediction of mammalian enhancers from DNA sequence

PubMed Central

Lee, Dongwon; Karchin, Rachel; Beer, Michael A.

2011-01-01

Accurately predicting regulatory sequences and enhancers in entire genomes is an important but difficult problem, especially in large vertebrate genomes. With the advent of ChIP-seq technology, experimental detection of genome-wide EP300/CREBBP bound regions provides a powerful platform to develop predictive tools for regulatory sequences and to study their sequence properties. Here, we develop a support vector machine (SVM) framework which can accurately identify EP300-bound enhancers using only genomic sequence and an unbiased set of general sequence features. Moreover, we find that the predictive sequence features identified by the SVM classifier reveal biologically relevant sequence elements enriched in the enhancers, but we also identify other features that are significantly depleted in enhancers. The predictive sequence features are evolutionarily conserved and spatially clustered, providing further support of their functional significance. Although our SVM is trained on experimental data, we also predict novel enhancers and show that these putative enhancers are significantly enriched in both ChIP-seq signal and DNase I hypersensitivity signal in the mouse brain and are located near relevant genes. Finally, we present results of comparisons between other EP300/CREBBP data sets using our SVM and uncover sequence elements enriched and/or depleted in the different classes of enhancers. Many of these sequence features play a role in specifying tissue-specific or developmental-stage-specific enhancer activity, but our results indicate that some features operate in a general or tissue-independent manner. In addition to providing a high confidence list of enhancer targets for subsequent experimental investigation, these results contribute to our understanding of the general sequence structure of vertebrate enhancers. PMID:21875935

Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

PubMed Central

Michaud, Edward J; Culiat, Cymbeline T; Klebig, Mitchell L; Barker, Paul E; Cain, KT; Carpenter, Debra J; Easter, Lori L; Foster, Carmen M; Gardner, Alysyn W; Guo, ZY; Houser, Kay J; Hughes, Lori A; Kerley, Marilyn K; Liu, Zhaowei; Olszewski, Robert E; Pinn, Irina; Shaw, Ginger D; Shinpock, Sarah G; Wymore, Ann M; Rinchik, Eugene M; Johnson, Dabney K

2005-01-01

Background Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results We produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations. PMID:16300676

Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma

PubMed Central

Weiser, Keith C.; Liu, Bin; Hansen, Gwenn M.; Skapura, Darlene; Hentges, Kathryn E.; Yarlagadda, Sujatha; Morse III, Herbert C.

2007-01-01

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFκB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision. PMID:17926094

Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma.

PubMed

Weiser, Keith C; Liu, Bin; Hansen, Gwenn M; Skapura, Darlene; Hentges, Kathryn E; Yarlagadda, Sujatha; Morse Iii, Herbert C; Justice, Monica J

2007-10-01

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFkappaB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision .

Mining biological databases for candidate disease genes

NASA Astrophysics Data System (ADS)

Braun, Terry A.; Scheetz, Todd; Webster, Gregg L.; Casavant, Thomas L.

2001-07-01

The publicly-funded effort to sequence the complete nucleotide sequence of the human genome, the Human Genome Project (HGP), has currently produced more than 93% of the 3 billion nucleotides of the human genome into a preliminary `draft' format. In addition, several valuable sources of information have been developed as direct and indirect results of the HGP. These include the sequencing of model organisms (rat, mouse, fly, and others), gene discovery projects (ESTs and full-length), and new technologies such as expression analysis and resources (micro-arrays or gene chips). These resources are invaluable for the researchers identifying the functional genes of the genome that transcribe and translate into the transcriptome and proteome, both of which potentially contain orders of magnitude more complexity than the genome itself. Preliminary analyses of this data identified approximately 30,000 - 40,000 human `genes.' However, the bulk of the effort still remains -- to identify the functional and structural elements contained within the transcriptome and proteome, and to associate function in the transcriptome and proteome to genes. A fortuitous consequence of the HGP is the existence of hundreds of databases containing biological information that may contain relevant data pertaining to the identification of disease-causing genes. The task of mining these databases for information on candidate genes is a commercial application of enormous potential. We are developing a system to acquire and mine data from specific databases to aid our efforts to identify disease genes. A high speed cluster of Linux of workstations is used to analyze sequence and perform distributed sequence alignments as part of our data mining and processing. This system has been used to mine GeneMap99 sequences within specific genomic intervals to identify potential candidate disease genes associated with Bardet-Biedle Syndrome (BBS).

Chromatin immunoprecipitation of mouse embryos.

PubMed

Voss, Anne K; Dixon, Mathew P; McLennan, Tamara; Kueh, Andrew J; Thomas, Tim

2012-01-01

During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos.

A Genome Sequence Resource for the Aye-Aye (Daubentonia madagascariensis), a Nocturnal Lemur from Madagascar

PubMed Central

Perry, George H.; Reeves, Darryl; Melsted, Páll; Ratan, Aakrosh; Miller, Webb; Michelini, Katelyn; Louis, Edward E.; Pritchard, Jonathan K.; Mason, Christopher E.; Gilad, Yoav

2012-01-01

We present a high-coverage draft genome assembly of the aye-aye (Daubentonia madagascariensis), a highly unusual nocturnal primate from Madagascar. Our assembly totals ∼3.0 billion bp (3.0 Gb), roughly the size of the human genome, comprised of ∼2.6 million scaffolds (N50 scaffold size = 13,597 bp) based on short paired-end sequencing reads. We compared the aye-aye genome sequence data with four other published primate genomes (human, chimpanzee, orangutan, and rhesus macaque) as well as with the mouse and dog genomes as nonprimate outgroups. Unexpectedly, we observed strong evidence for a relatively slow substitution rate in the aye-aye lineage compared with these and other primates. In fact, the aye-aye branch length is estimated to be ∼10% shorter than that of the human lineage, which is known for its low substitution rate. This finding may be explained, in part, by the protracted aye-aye life-history pattern, including late weaning and age of first reproduction relative to other lemurs. Additionally, the availability of this draft lemur genome sequence allowed us to polarize nucleotide and protein sequence changes to the ancestral primate lineage—a critical period in primate evolution, for which the relevant fossil record is sparse. Finally, we identified 293,800 high-confidence single nucleotide polymorphisms in the donor individual for our aye-aye genome sequence, a captive-born individual from two wild-born parents. The resulting heterozygosity estimate of 0.051% is the lowest of any primate studied to date, which is understandable considering the aye-aye's extensive home-range size and relatively low population densities. Yet this level of genetic diversity also suggests that conservation efforts benefiting this unusual species should be prioritized, especially in the face of the accelerating degradation and fragmentation of Madagascar's forests. PMID:22155688

Cloning, structure, and chromosome localization of the mouse glutaryl-CoA dehydrogenase gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koeller, D.M.; DiGiulio, A.; Frerman, F.E.

Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, and inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains and open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdhmore » was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped. 14 refs., 3 figs.« less

Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

ERIC Educational Resources Information Center

Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

2006-01-01

We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

Introductions of West Nile Virus Strains to Mexico

PubMed Central

Deardorff, Eleanor; Estrada-Franco, José G.; Brault, Aaron C.; Navarro-Lopez, Roberto; Campomanes-Cortes, Arturo; Paz-Ramirez, Pedro; Solis-Hernandez, Mario; Ramey, Wanichaya N.; Davis, C. Todd; Beasley, David W.C.; Tesh, Robert B.; Barrett, Alan D.T.

2006-01-01

Complete genome sequencing of 22 West Nile virus isolates suggested 2 independent introductions into Mexico. A previously identified mouse-attenuated glycosylation variant was introduced into southern Mexico through the southeastern United States, while a common US genotype appears to have been introduced incrementally into northern Mexico through the southwestern United States. PMID:16494762

MutSpec: a Galaxy toolbox for streamlined analyses of somatic mutation spectra in human and mouse cancer genomes.

PubMed

Ardin, Maude; Cahais, Vincent; Castells, Xavier; Bouaoun, Liacine; Byrnes, Graham; Herceg, Zdenko; Zavadil, Jiri; Olivier, Magali

2016-04-18

The nature of somatic mutations observed in human tumors at single gene or genome-wide levels can reveal information on past carcinogenic exposures and mutational processes contributing to tumor development. While large amounts of sequencing data are being generated, the associated analysis and interpretation of mutation patterns that may reveal clues about the natural history of cancer present complex and challenging tasks that require advanced bioinformatics skills. To make such analyses accessible to a wider community of researchers with no programming expertise, we have developed within the web-based user-friendly platform Galaxy a first-of-its-kind package called MutSpec. MutSpec includes a set of tools that perform variant annotation and use advanced statistics for the identification of mutation signatures present in cancer genomes and for comparing the obtained signatures with those published in the COSMIC database and other sources. MutSpec offers an accessible framework for building reproducible analysis pipelines, integrating existing methods and scripts developed in-house with publicly available R packages. MutSpec may be used to analyse data from whole-exome, whole-genome or targeted sequencing experiments performed on human or mouse genomes. Results are provided in various formats including rich graphical outputs. An example is presented to illustrate the package functionalities, the straightforward workflow analysis and the richness of the statistics and publication-grade graphics produced by the tool. MutSpec offers an easy-to-use graphical interface embedded in the popular Galaxy platform that can be used by researchers with limited programming or bioinformatics expertise to analyse mutation signatures present in cancer genomes. MutSpec can thus effectively assist in the discovery of complex mutational processes resulting from exogenous and endogenous carcinogenic insults.

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.

PubMed

Miura, Hiromi; Quadros, Rolen M; Gurumurthy, Channabasavaiah B; Ohtsuka, Masato

2018-01-01

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

Disease Model Discovery from 3,328 Gene Knockouts by The International Mouse Phenotyping Consortium

PubMed Central

Meehan, Terrence F.; Conte, Nathalie; West, David B.; Jacobsen, Julius O.; Mason, Jeremy; Warren, Jonathan; Chen, Chao-Kung; Tudose, Ilinca; Relac, Mike; Matthews, Peter; Karp, Natasha; Santos, Luis; Fiegel, Tanja; Ring, Natalie; Westerberg, Henrik; Greenaway, Simon; Sneddon, Duncan; Morgan, Hugh; Codner, Gemma F; Stewart, Michelle E; Brown, James; Horner, Neil; Haendel, Melissa; Washington, Nicole; Mungall, Christopher J.; Reynolds, Corey L; Gallegos, Juan; Gailus-Durner, Valerie; Sorg, Tania; Pavlovic, Guillaume; Bower, Lynette R; Moore, Mark; Morse, Iva; Gao, Xiang; Tocchini-Valentini, Glauco P; Obata, Yuichi; Cho, Soo Young; Seong, Je Kyung; Seavitt, John; Beaudet, Arthur L.; Dickinson, Mary E.; Herault, Yann; Wurst, Wolfgang; de Angelis, Martin Hrabe; Lloyd, K.C. Kent; Flenniken, Ann M; Nutter, Lauryl MJ; Newbigging, Susan; McKerlie, Colin; Justice, Monica J.; Murray, Stephen A.; Svenson, Karen L.; Braun, Robert E.; White, Jacqueline K.; Bradley, Allan; Flicek, Paul; Wells, Sara; Skarnes, William C.; Adams, David J.; Parkinson, Helen; Mallon, Ann-Marie; Brown, Steve D.M.; Smedley, Damian

2017-01-01

Although next generation sequencing has revolutionised the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by our lack of knowledge of function and pathobiological mechanism for most genes. To address this challenge, the International Mouse Phenotyping Consortium (IMPC) is creating a genome- and phenome-wide catalogue of gene function by characterizing new knockout mouse strains across diverse biological systems through a broad set of standardised phenotyping tests, with all mice made readily available to the biomedical community. Analysing the first 3328 genes reveals models for 360 diseases including the first for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations are novel, providing the first functional evidence for 1092 genes and candidates in unsolved diseases such as Arrhythmogenic Right Ventricular Dysplasia 3. Finally, we describe our role in variant functional validation with the 100,000 Genomes and other projects. PMID:28650483

Systematic Analysis of Long Noncoding RNAs in the Senescence-accelerated Mouse Prone 8 Brain Using RNA Sequencing.

PubMed

Zhang, Shuai; Qin, Chunxia; Cao, Guoqiong; Xin, Wenfeng; Feng, Chengqiang; Zhang, Wensheng

2016-08-02

Long noncoding RNAs (lncRNAs) may play an important role in Alzheimer's disease (AD) pathogenesis. However, despite considerable research in this area, the comprehensive and systematic understanding of lncRNAs in AD is still limited. The emergence of RNA sequencing provides a predictor and has incomparable advantage compared with other methods, including microarray. In this study, we identified lncRNAs in a 7-month-old mouse brain through deep RNA sequencing using the senescence-accelerated mouse prone 8 (SAMP8) and senescence-accelerated mouse resistant 1 (SAMR1) models. A total of 599,985,802 clean reads and 23,334 lncRNA transcripts were obtained. Then, we identified 97 significantly upregulated and 114 significantly downregulated lncRNA transcripts from all cases in SAMP8 mice relative to SAMR1 mice. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these significantly dysregulated lncRNAs were involved in regulating the development of AD from various angles, such as nerve growth factor term (GO: 1990089), mitogen-activated protein kinase signaling pathway, and AD pathway. Furthermore, the most probable AD-associated lncRNAs were predicted and listed in detail. Our study provided the systematic dissection of lncRNA profiling in SAMP8 mouse brain and accelerated the development of lncRNA biomarkers in AD. These attracting biomarkers could provide significant insights into AD therapy in the future.

DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

PubMed

Koike, Tasuku; Wakai, Takuya; Jincho, Yuko; Sakashita, Akihiko; Kobayashi, Hisato; Mizutani, Eiji; Wakayama, Sayaka; Miura, Fumihito; Ito, Takashi; Kono, Tomohiro

2016-06-01

The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier. © 2016 by the Society for the Study of Reproduction, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kvon, Evgeny Z.; Kamneva, Olga K.; Melo, Uirá S.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. In this paper, we identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologsmore » caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution.« less

The Status, Quality, and Expansion of the NIH Full-Length cDNA Project: The Mammalian Gene Collection (MGC)

PubMed Central

2004-01-01

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5′-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID:15489334

High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm.

PubMed

Carone, Benjamin R; Hung, Jui-Hung; Hainer, Sarah J; Chou, Min-Te; Carone, Dawn M; Weng, Zhiping; Fazzio, Thomas G; Rando, Oliver J

2014-07-14

Mammalian embryonic stem cells (ESCs) and sperm exhibit unusual chromatin packaging that plays important roles in cellular function. Here, we extend a recently developed technique, based on deep paired-end sequencing of lightly digested chromatin, to assess footprints of nucleosomes and other DNA-binding proteins genome-wide in murine ESCs and sperm. In ESCs, we recover well-characterized features of chromatin such as promoter nucleosome depletion and further identify widespread footprints of sequence-specific DNA-binding proteins such as CTCF, which we validate in knockdown studies. We document global differences in nuclease accessibility between ESCs and sperm, finding that the majority of histone retention in sperm preferentially occurs in large gene-poor genomic regions, with only a small subset of nucleosomes being retained over promoters of developmental regulators. Finally, we describe evidence that CTCF remains associated with the genome in mature sperm, where it could play a role in organizing the sperm genome. Copyright © 2014 Elsevier Inc. All rights reserved.

Genomic and chromatin features shaping meiotic double-strand break formation and repair in mice

PubMed Central

Jasin, Maria; Lange, Julian

2017-01-01

ABSTRACT The SPO11-generated DNA double-strand breaks (DSBs) that initiate meiotic recombination occur non-randomly across genomes, but mechanisms shaping their distribution and repair remain incompletely understood. Here, we expand on recent studies of nucleotide-resolution DSB maps in mouse spermatocytes. We find that trimethylation of histone H3 lysine 36 around DSB hotspots is highly correlated, both spatially and quantitatively, with trimethylation of H3 lysine 4, consistent with coordinated formation and action of both PRDM9-dependent histone modifications. In contrast, the DSB-responsive kinase ATM contributes independently of PRDM9 to controlling hotspot activity, and combined action of ATM and PRDM9 can explain nearly two-thirds of the variation in DSB frequency between hotspots. DSBs were modestly underrepresented in most repetitive sequences such as segmental duplications and transposons. Nonetheless, numerous DSBs form within repetitive sequences in each meiosis and some classes of repeats are preferentially targeted. Implications of these findings are discussed for evolution of PRDM9 and its role in hybrid strain sterility in mice. Finally, we document the relationship between mouse strain-specific DNA sequence variants within PRDM9 recognition motifs and attendant differences in recombination outcomes. Our results provide further insights into the complex web of factors that influence meiotic recombination patterns. PMID:28820351

The identification and functional annotation of RNA structures conserved in vertebrates.

PubMed

Seemann, Stefan E; Mirza, Aashiq H; Hansen, Claus; Bang-Berthelsen, Claus H; Garde, Christian; Christensen-Dalsgaard, Mikkel; Torarinsson, Elfar; Yao, Zizhen; Workman, Christopher T; Pociot, Flemming; Nielsen, Henrik; Tommerup, Niels; Ruzzo, Walter L; Gorodkin, Jan

2017-08-01

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3' ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality. © 2017 Seemann et al.; Published by Cold Spring Harbor Laboratory Press.

Manual Gene Ontology annotation workflow at the Mouse Genome Informatics Database

PubMed Central

Drabkin, Harold J.; Blake, Judith A.

2012-01-01

The Mouse Genome Database, the Gene Expression Database and the Mouse Tumor Biology database are integrated components of the Mouse Genome Informatics (MGI) resource (http://www.informatics.jax.org). The MGI system presents both a consensus view and an experimental view of the knowledge concerning the genetics and genomics of the laboratory mouse. From genotype to phenotype, this information resource integrates information about genes, sequences, maps, expression analyses, alleles, strains and mutant phenotypes. Comparative mammalian data are also presented particularly in regards to the use of the mouse as a model for the investigation of molecular and genetic components of human diseases. These data are collected from literature curation as well as downloads of large datasets (SwissProt, LocusLink, etc.). MGI is one of the founding members of the Gene Ontology (GO) and uses the GO for functional annotation of genes. Here, we discuss the workflow associated with manual GO annotation at MGI, from literature collection to display of the annotations. Peer-reviewed literature is collected mostly from a set of journals available electronically. Selected articles are entered into a master bibliography and indexed to one of eight areas of interest such as ‘GO’ or ‘homology’ or ‘phenotype’. Each article is then either indexed to a gene already contained in the database or funneled through a separate nomenclature database to add genes. The master bibliography and associated indexing provide information for various curator-reports such as ‘papers selected for GO that refer to genes with NO GO annotation’. Once indexed, curators who have expertise in appropriate disciplines enter pertinent information. MGI makes use of several controlled vocabularies that ensure uniform data encoding, enable robust analysis and support the construction of complex queries. These vocabularies range from pick-lists to structured vocabularies such as the GO. All data associations are supported with statements of evidence as well as access to source publications. PMID:23110975

Manual Gene Ontology annotation workflow at the Mouse Genome Informatics Database.

PubMed

Drabkin, Harold J; Blake, Judith A

2012-01-01

The Mouse Genome Database, the Gene Expression Database and the Mouse Tumor Biology database are integrated components of the Mouse Genome Informatics (MGI) resource (http://www.informatics.jax.org). The MGI system presents both a consensus view and an experimental view of the knowledge concerning the genetics and genomics of the laboratory mouse. From genotype to phenotype, this information resource integrates information about genes, sequences, maps, expression analyses, alleles, strains and mutant phenotypes. Comparative mammalian data are also presented particularly in regards to the use of the mouse as a model for the investigation of molecular and genetic components of human diseases. These data are collected from literature curation as well as downloads of large datasets (SwissProt, LocusLink, etc.). MGI is one of the founding members of the Gene Ontology (GO) and uses the GO for functional annotation of genes. Here, we discuss the workflow associated with manual GO annotation at MGI, from literature collection to display of the annotations. Peer-reviewed literature is collected mostly from a set of journals available electronically. Selected articles are entered into a master bibliography and indexed to one of eight areas of interest such as 'GO' or 'homology' or 'phenotype'. Each article is then either indexed to a gene already contained in the database or funneled through a separate nomenclature database to add genes. The master bibliography and associated indexing provide information for various curator-reports such as 'papers selected for GO that refer to genes with NO GO annotation'. Once indexed, curators who have expertise in appropriate disciplines enter pertinent information. MGI makes use of several controlled vocabularies that ensure uniform data encoding, enable robust analysis and support the construction of complex queries. These vocabularies range from pick-lists to structured vocabularies such as the GO. All data associations are supported with statements of evidence as well as access to source publications.

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

PubMed Central

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-01-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents. PMID:22735701

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

PubMed

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-08-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates

PubMed Central

Chitsazzadeh, Vida; Coarfa, Cristian; Drummond, Jennifer A.; Nguyen, Tri; Joseph, Aaron; Chilukuri, Suneel; Charpiot, Elizabeth; Adelmann, Charles H.; Ching, Grace; Nguyen, Tran N.; Nicholas, Courtney; Thomas, Valencia D.; Migden, Michael; MacFarlane, Deborah; Thompson, Erika; Shen, Jianjun; Takata, Yoko; McNiece, Kayla; Polansky, Maxim A.; Abbas, Hussein A.; Rajapakshe, Kimal; Gower, Adam; Spira, Avrum; Covington, Kyle R.; Xiao, Weimin; Gunaratne, Preethi; Pickering, Curtis; Frederick, Mitchell; Myers, Jeffrey N.; Shen, Li; Yao, Hui; Su, Xiaoping; Rapini, Ronald P.; Wheeler, David A.; Hawk, Ernest T.; Flores, Elsa R.; Tsai, Kenneth Y.

2016-01-01

Cutaneous squamous cell carcinoma (cuSCC) comprises 15–20% of all skin cancers, accounting for over 700,000 cases in USA annually. Most cuSCC arise in association with a distinct precancerous lesion, the actinic keratosis (AK). To identify potential targets for molecularly targeted chemoprevention, here we perform integrated cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar ultraviolet radiation-driven Hairless mouse model. We identify the major transcriptional drivers of this progression sequence, showing that the key genomic changes in cuSCC development occur in the normal skin to AK transition. Our data validate the use of this ultraviolet radiation-driven mouse cuSCC model for cross-species analysis and demonstrate that cuSCC bears deep molecular similarities to multiple carcinogen-driven SCCs from diverse sites, suggesting that cuSCC may serve as an effective, accessible model for multiple SCC types and that common treatment and prevention strategies may be feasible. PMID:27574101

Complex multi-enhancer contacts captured by Genome Architecture Mapping (GAM)

PubMed Central

Beagrie, Robert A.; Scialdone, Antonio; Schueler, Markus; Kraemer, Dorothee C.A.; Chotalia, Mita; Xie, Sheila Q.; Barbieri, Mariano; de Santiago, Inês; Lavitas, Liron-Mark; Branco, Miguel R.; Fraser, James; Dostie, Josée; Game, Laurence; Dillon, Niall; Edwards, Paul A.W.; Nicodemi, Mario; Pombo, Ana

2017-01-01

Summary The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. We developed a novel genome-wide method, Genome Architecture Mapping (GAM), for measuring chromatin contacts, and other features of three-dimensional chromatin topology, based on sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify an enrichment for specific interactions between active genes and enhancers across very large genomic distances, using a mathematical model ‘SLICE’ (Statistical Inference of Co-segregation). GAM also reveals an abundance of three-way contacts genome-wide, especially between regions that are highly transcribed or contain super-enhancers, highlighting a previously inaccessible complexity in genome architecture and a major role for gene-expression specific contacts in organizing the genome in mammalian nuclei. PMID:28273065

Host subspecific viral strains in European house mice: Murine cytomegalovirus in the Eastern (Mus musculus musculus) and Western house mouse (Mus musculus domesticus).

PubMed

Čížková, Dagmar; Baird, Stuart J E; Těšíková, Jana; Voigt, Sebastian; Ľudovít, Ďureje; Piálek, Jaroslav; Goüy de Bellocq, Joëlle

2018-06-09

Murine cytomegalovirus (MCMV) has been reported from house mice (Mus musculus) worldwide, but only recently from Eastern house mice (M. m. musculus), of particular interest because they form a semi-permeable species barrier in Europe with Western house mice, M. m. domesticus. Here we report genome sequences of EastMCMV (from Eastern mice), and set these in the context of MCMV genomes from genus Mus hosts. We show EastMCMV and WestMCMV are genetically distinct. Phylogeny splitting analyses show a genome wide (94%) pattern consistent with no West-East introgression, the major exception (3.8%) being a genome-terminal region of duplicated genes involved in host immune system evasion. As expected from its function, this is a region of maintenance of ancestral polymorphism: The lack of clear splitting signal cannot be interpreted as evidence of introgression. The EastMCMV genome sequences reported here can therefore serve as a well-described resource for exploration of murid MCMV diversity. Copyright © 2018 Elsevier Inc. All rights reserved.

Genome analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of the etiologic agent of tuberculosis

PubMed Central

Supply, Philip; Marceau, Michael; Mangenot, Sophie; Roche, David; Rouanet, Carine; Khanna, Varun; Majlessi, Laleh; Criscuolo, Alexis; Tap, Julien; Pawlik, Alexandre; Fiette, Laurence; Orgeur, Mickael; Fabre, Michel; Parmentier, Cécile; Frigui, Wafa; Simeone, Roxane; Boritsch, Eva C.; Debrie, Anne-Sophie; Willery, Eve; Walker, Danielle; Quail, Michael A.; Ma, Laurence; Bouchier, Christiane; Salvignol, Grégory; Sayes, Fadel; Cascioferro, Alessandro; Seemann, Torsten; Barbe, Valérie; Locht, Camille; Gutierrez, Maria-Cristina; Leclerc, Claude; Bentley, Stephen; Stinear, Timothy P.; Brisse, Sylvain; Médigue, Claudine; Parkhill, Julian; Cruveiller, Stéphane; Brosch, Roland

2013-01-01

Global spread and genetic monomorphism are hallmarks of Mycobacterium tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii, and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology, are restricted to East-Africa. Here, we sequenced and analyzed the genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5x coverage), 454/Roche (13-18x coverage) and/or Illumina DNA sequencing (45-105x coverage). We show that STB are highly recombinogenic and evolutionary early-branching, with larger genome sizes, 25-fold more SNPs, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse-infection experiments revealed that STB are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral, STB-like pool of mycobacteria by gain of persistence and virulence mechanisms and we provide genome-wide insights into the molecular events involved. PMID:23291586

Transcriptome-wide investigation of genomic imprinting in chicken.

PubMed

Frésard, Laure; Leroux, Sophie; Servin, Bertrand; Gourichon, David; Dehais, Patrice; Cristobal, Magali San; Marsaud, Nathalie; Vignoles, Florence; Bed'hom, Bertrand; Coville, Jean-Luc; Hormozdiari, Farhad; Beaumont, Catherine; Zerjal, Tatiana; Vignal, Alain; Morisson, Mireille; Lagarrigue, Sandrine; Pitel, Frédérique

2014-04-01

Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing ∼200 Gb of sequences. This allowed the detection of 79 potentially imprinted SNPs, through an analysis method that we validated by detecting imprinting from mouse data already published. However, out of 23 candidates tested by pyrosequencing, none could be confirmed. These results come together, without a priori, with previous statements and phylogenetic considerations assessing the absence of genomic imprinting in chicken.

The open reading frames in the 3' long terminal repeats of several mouse mammary tumor virus integrants encode V beta 3-specific superantigens

PubMed Central

1992-01-01

Mice expressing the minor lymphocyte stimulation antigens, Mls-1a, -2a, or -3a, singly on the B10.BR background have been generated. Mls phenotypes correlate with the integration of mouse mammary tumor viruses (MTV) in the mouse genome. The open reading frames within the 3' long terminal repeats of the integrated MTVs 1, 3, 6, and 13 encode V beta 3-specific superantigens. Sequence data for these viral superantigens is presented, indicating that it is the COOH-terminal portion of the viral superantigen that interacts with the T cell receptor V beta element. PMID:1309854

Genome databases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Courteau, J.

1991-10-11

Since the Genome Project began several years ago, a plethora of databases have been developed or are in the works. They range from the massive Genome Data Base at Johns Hopkins University, the central repository of all gene mapping information, to small databases focusing on single chromosomes or organisms. Some are publicly available, others are essentially private electronic lab notebooks. Still others limit access to a consortium of researchers working on, say, a single human chromosome. An increasing number incorporate sophisticated search and analytical software, while others operate as little more than data lists. In consultation with numerous experts inmore » the field, a list has been compiled of some key genome-related databases. The list was not limited to map and sequence databases but also included the tools investigators use to interpret and elucidate genetic data, such as protein sequence and protein structure databases. Because a major goal of the Genome Project is to map and sequence the genomes of several experimental animals, including E. coli, yeast, fruit fly, nematode, and mouse, the available databases for those organisms are listed as well. The author also includes several databases that are still under development - including some ambitious efforts that go beyond data compilation to create what are being called electronic research communities, enabling many users, rather than just one or a few curators, to add or edit the data and tag it as raw or confirmed.« less

Identification and functional analysis of long non-coding RNAs in human and mouse early embryos based on single-cell transcriptome data

PubMed Central

Qiu, Jia-jun; Ren, Zhao-rui; Yan, Jing-bin

2016-01-01

Epigenetics regulations have an important role in fertilization and proper embryonic development, and several human diseases are associated with epigenetic modification disorders, such as Rett syndrome, Beckwith-Wiedemann syndrome and Angelman syndrome. However, the dynamics and functions of long non-coding RNAs (lncRNAs), one type of epigenetic regulators, in human pre-implantation development have not yet been demonstrated. In this study, a comprehensive analysis of human and mouse early-stage embryonic lncRNAs was performed based on public single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs are expressed in a developmental stage–specific manner during human early-stage embryonic development, whereas a more temporal-specific expression pattern was identified in mouse embryos. Weighted gene co-expression network analysis suggested that lncRNAs involved in human early-stage embryonic development are associated with several important functions and processes, such as oocyte maturation, zygotic genome activation and mitochondrial functions. We also found that the network of lncRNAs involved in zygotic genome activation was highly preservative between human and mouse embryos, whereas in other stages no strong correlation between human and mouse embryo was observed. This study provides insight into the molecular mechanism underlying lncRNA involvement in human pre-implantation embryonic development. PMID:27542205

Highly Efficient Targeted Mutagenesis in Mice Using TALENs

PubMed Central

Panda, Sudeepta Kumar; Wefers, Benedikt; Ortiz, Oskar; Floss, Thomas; Schmid, Bettina; Haass, Christian; Wurst, Wolfgang; Kühn, Ralf

2013-01-01

Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms. PMID:23979585

[Parental genome imprinting].

PubMed

Babinet, C

1993-01-01

Genetical as well as experimental embryology methods have permitted, in recent years, to uncover a very important feature of mammalian embryonic development: it has been shown that female and male genomic complements are differentially imprinted in such a way that contribution of both a maternally and a paternally derived genome are absolutely necessary for the embryo to complete its normal development. Differential genomic imprinting seems therefore to impose some new and essential kind of information to the one already contained in the genomic sequences. The differential imprinting should be imposed on the genetic material during gametogenesis and persist throughout somatic development after fertilization. It should then be erased in the germ cell line and be established again in sperm and egg genomes. The recent discovery of several mouse genes which are imprinted should permit to address the question of the molecular mechanisms of imprinting.

Discovery of the porcine NGN3 gene and testing its endocrine function in the pig

USDA-ARS?s Scientific Manuscript database

Neurogenin 3 (NGN3) is a member of the basic helix-loop-helix transcription factor family. NGN3 is both necessary and sufficient to drive endocrine differentiation in the developing pancreas in mouse and humans. Until now, the sequence for NGN3 eluded discovery despite completion of the pig genome a...

Orthology for comparative genomics in the mouse genome database.

PubMed

Dolan, Mary E; Baldarelli, Richard M; Bello, Susan M; Ni, Li; McAndrews, Monica S; Bult, Carol J; Kadin, James A; Richardson, Joel E; Ringwald, Martin; Eppig, Janan T; Blake, Judith A

2015-08-01

The mouse genome database (MGD) is the model organism database component of the mouse genome informatics system at The Jackson Laboratory. MGD is the international data resource for the laboratory mouse and facilitates the use of mice in the study of human health and disease. Since its beginnings, MGD has included comparative genomics data with a particular focus on human-mouse orthology, an essential component of the use of mouse as a model organism. Over the past 25 years, novel algorithms and addition of orthologs from other model organisms have enriched comparative genomics in MGD data, extending the use of orthology data to support the laboratory mouse as a model of human biology. Here, we describe current comparative data in MGD and review the history and refinement of orthology representation in this resource.

Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved.

PubMed

Long, Hannah K; King, Hamish W; Patient, Roger K; Odom, Duncan T; Klose, Robert J

2016-08-19

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice.

PubMed

Schmouth, Jean-François; Castellarin, Mauro; Laprise, Stéphanie; Banks, Kathleen G; Bonaguro, Russell J; McInerny, Simone C; Borretta, Lisa; Amirabbasi, Mahsa; Korecki, Andrea J; Portales-Casamar, Elodie; Wilson, Gary; Dreolini, Lisa; Jones, Steven J M; Wasserman, Wyeth W; Goldowitz, Daniel; Holt, Robert A; Simpson, Elizabeth M

2013-10-14

The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo. In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.

Prediction of Human Disease Genes by Human-Mouse Conserved Coexpression Analysis

PubMed Central

Grassi, Elena; Damasco, Christian; Silengo, Lorenzo; Oti, Martin; Provero, Paolo; Di Cunto, Ferdinando

2008-01-01

Background Even in the post-genomic era, the identification of candidate genes within loci associated with human genetic diseases is a very demanding task, because the critical region may typically contain hundreds of positional candidates. Since genes implicated in similar phenotypes tend to share very similar expression profiles, high throughput gene expression data may represent a very important resource to identify the best candidates for sequencing. However, so far, gene coexpression has not been used very successfully to prioritize positional candidates. Methodology/Principal Findings We show that it is possible to reliably identify disease-relevant relationships among genes from massive microarray datasets by concentrating only on genes sharing similar expression profiles in both human and mouse. Moreover, we show systematically that the integration of human-mouse conserved coexpression with a phenotype similarity map allows the efficient identification of disease genes in large genomic regions. Finally, using this approach on 850 OMIM loci characterized by an unknown molecular basis, we propose high-probability candidates for 81 genetic diseases. Conclusion Our results demonstrate that conserved coexpression, even at the human-mouse phylogenetic distance, represents a very strong criterion to predict disease-relevant relationships among human genes. PMID:18369433

A new polymorphic and multicopy MHC gene family related to nonmammalian class I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leelayuwat, C.; Degli-Esposti, M.A.; Abraham, L.J.

1994-12-31

The authors have used genomic analysis to characterize a region of the central major histocompatibility complex (MHC) spanning {approximately} 300 kilobases (kb) between TNF and HLA-B. This region has been suggested to carry genetic factors relevant to the development of autoimmune diseases such as myasthenia gravis (MG) and insulin dependent diabetes mellitus (IDDM). Genomic sequence was analyzed for coding potential, using two neural network programs, GRAIL and GeneParser. A genomic probe, JAB, containing putative coding sequences (PERB11) located 60 kb centromeric of HLA-B, was used for northern analysis of human tissues. Multiple transcripts were detected. Southern analysis of genomic DNAmore » and overlapping YAC clones, covering the region from BAT1 to HLA-F, indicated that there are at least five copies of PERB11, four of which are located within this region of the MHC. The partial cDNA sequence of PERB11 was obtained from poly-A RNA derived from skeletal muscle. The putative amino acid sequence of PERB11 shares {approximately} 30% identity to MHC class I molecules from various species, including reptiles, chickens, and frogs, as well as to other MHC class I-like molecules, such as the IgG FcR of the mouse and rat and the human Zn-{alpha}2-glycoprotein. From direct comparison of amino acid sequences, it is concluded that PERB11 is a distinct molecule more closely related to nonmammalian than known mammalian MHC class I molecules. Genomic sequence analysis of PERB11 from five MHC ancestral haplotypes (AH) indicated that the gene is polymorphic at both DNA and protein level. The results suggest that the authors have identified a novel polymorphic gene family with multiple copies within the MHC. 48 refs., 10 figs., 2 tabs.« less

eShadow: A tool for comparing closely related sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ovcharenko, Ivan; Boffelli, Dario; Loots, Gabriela G.

2004-01-15

Primate sequence comparisons are difficult to interpret due to the high degree of sequence similarity shared between such closely related species. Recently, a novel method, phylogenetic shadowing, has been pioneered for predicting functional elements in the human genome through the analysis of multiple primate sequence alignments. We have expanded this theoretical approach to create a computational tool, eShadow, for the identification of elements under selective pressure in multiple sequence alignments of closely related genomes, such as in comparisons of human to primate or mouse to rat DNA. This tool integrates two different statistical methods and allows for the dynamic visualizationmore » of the resulting conservation profile. eShadow also includes a versatile optimization module capable of training the underlying Hidden Markov Model to differentially predict functional sequences. This module grants the tool high flexibility in the analysis of multiple sequence alignments and in comparing sequences with different divergence rates. Here, we describe the eShadow comparative tool and its potential uses for analyzing both multiple nucleotide and protein alignments to predict putative functional elements. The eShadow tool is publicly available at http://eshadow.dcode.org/« less

Virulence-Associated Genome Mutations of Murine Rotavirus Identified by Alternating Serial Passages in Mice and Cell Cultures

PubMed Central

Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

2014-01-01

ABSTRACT Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3′ consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. IMPORTANCE Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. PMID:24599996

Virulence-associated genome mutations of murine rotavirus identified by alternating serial passages in mice and cell cultures.

PubMed

Tsugawa, Takeshi; Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

2014-05-01

Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3' consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence.

Murine norovirus infection in Brazilian animal facilities

PubMed Central

Rodrigues, Daniele Masselli; Moreira, Josélia Cristina de Oliveira; Lancellotti, Marcelo; Gilioli, Rovilson; Corat, Marcus Alexandre Finzi

2016-01-01

Murine norovirus (MNV) is a single-stranded positive-sense RNA virus of the Caliciviridae family. MNV has been reported to infect laboratory mice with the ability to cause lethal infections in strains lacking components of the innate immune response. Currently, MNV is considered the most prevalent infectious agent detected in laboratory mouse facilities. In this study, mice in 22 laboratory animal facilities within Brazil were analyzed for MNV infection. Using primers targeting a conserved region of the viral capsid, MNV was detected by RT-PCR in 137 of 359 mice from all 22 facilities. Nucleotide sequencing and phylogenetic analysis of the capsid region from the viral genome showed identity ranging from 87% to 99% when compared to reported MNV sequences. In addition, RAW264.7 cells inoculated with a mouse fecal suspension displayed cytopathic effect after the fifth passage. This study represents the first report of MNV in mouse colonies in Brazilian laboratory animal facilities, emphasizing the relevance of a health surveillance program in such environments. PMID:28049885

Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

2003-12-31

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involvedmore » in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.« less

Comparative genome and evolutionary analysis of naturally occurring Beilong virus in brown and black rats.

PubMed

Woo, Patrick C Y; Wong, Annette Y P; Wong, Beatrice H L; Lam, Carol S F; Fan, Rachel Y Y; Lau, Susanna K P; Yuen, Kwok-Yung

2016-11-01

Recently, we reported the presence of Beilong virus in spleen and kidney samples of brown rats and black rats, suggesting that these rodents could be natural reservoirs of Beilong virus. In this study, four genomes of Beilong virus from brown rats and black rats were sequenced. Similar to the Beilong virus genome sequenced from kidney mesangial cell line culture, those of J-virus from house mouse and Tailam virus from Sikkim rats, these four genomes from naturally occurring Beilong virus also contain the eight genes (3'-N-P/V/C-M-F-SH-TM-G-L-5'). In these four genomes, the attachment glycoprotein encoded by the G gene consists of 1046 amino acids; but for the original Beilong virus genome sequenced from kidney mesangial cell line, the G CDS was predicted to be prematurely terminated at position 2205 (TGG→TAG), resulting in a 734-amino-acid truncated G protein. This phenomenon of a lack of nonsense mutation in naturally occurring Beilong viruses was confirmed by sequencing this region of 15 additional rodent samples. Phylogenetic analyses showed that the cell line and naturally occurring Beilong viruses were closely clustered, without separation into subgroups. In addition, these viruses were further clustered with J-virus and Tailam virus, with high bootstrap supports of >90%, forming a distinct group in Paramyxoviridae. Brown rats and black rats are natural reservoirs of Beilong virus. Our results also supports that the recently proposed genus, Jeilongvirus, should encompass Beilong virus, J-virus and Tailam virus as members. Copyright © 2016 Elsevier B.V. All rights reserved.

Mouse Models for Drug Discovery. Can New Tools and Technology Improve Translational Power?

PubMed

Zuberi, Aamir; Lutz, Cathleen

2016-12-01

The use of mouse models in biomedical research and preclinical drug evaluation is on the rise. The advent of new molecular genome-altering technologies such as CRISPR/Cas9 allows for genetic mutations to be introduced into the germ line of a mouse faster and less expensively than previous methods. In addition, the rapid progress in the development and use of somatic transgenesis using viral vectors, as well as manipulations of gene expression with siRNAs and antisense oligonucleotides, allow for even greater exploration into genomics and systems biology. These technological advances come at a time when cost reductions in genome sequencing have led to the identification of pathogenic mutations in patient populations, providing unprecedented opportunities in the use of mice to model human disease. The ease of genetic engineering in mice also offers a potential paradigm shift in resource sharing and the speed by which models are made available in the public domain. Predictively, the knowledge alone that a model can be quickly remade will provide relief to resources encumbered by licensing and Material Transfer Agreements. For decades, mouse strains have provided an exquisite experimental tool to study the pathophysiology of the disease and assess therapeutic options in a genetically defined system. However, a major limitation of the mouse has been the limited genetic diversity associated with common laboratory mice. This has been overcome with the recent development of the Collaborative Cross and Diversity Outbred mice. These strains provide new tools capable of replicating genetic diversity to that approaching the diversity found in human populations. The Collaborative Cross and Diversity Outbred strains thus provide a means to observe and characterize toxicity or efficacy of new therapeutic drugs for a given population. The combination of traditional and contemporary mouse genome editing tools, along with the addition of genetic diversity in new modeling systems, are synergistic and serve to make the mouse a better model for biomedical research, enhancing the potential for preclinical drug discovery and personalized medicine. © The Author 2016. Published by Oxford University Press.

Identification of a QTL in Mus musculus for Alcohol Preference, Withdrawal, and Ap3m2 Expression Using Integrative Functional Genomics and Precision Genetics

PubMed Central

Bubier, Jason A.; Jay, Jeremy J.; Baker, Christopher L.; Bergeson, Susan E.; Ohno, Hiroshi; Metten, Pamela; Crabbe, John C.; Chesler, Elissa J.

2014-01-01

Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with >60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits. PMID:24923803

Identification of a QTL in Mus musculus for alcohol preference, withdrawal, and Ap3m2 expression using integrative functional genomics and precision genetics.

PubMed

Bubier, Jason A; Jay, Jeremy J; Baker, Christopher L; Bergeson, Susan E; Ohno, Hiroshi; Metten, Pamela; Crabbe, John C; Chesler, Elissa J

2014-08-01

Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with >60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits. Copyright © 2014 by the Genetics Society of America.

Molecular cloning and expression of the CRISP family of proteins in the boar.

PubMed

Vadnais, Melissa L; Foster, Douglas N; Roberts, Kenneth P

2008-12-01

The family of mammalian cysteine-rich secretory proteins (CRISP) have been well characterized in the rat, mouse, and human. Here we report the molecular cloning and expression analysis of CRISP1, CRISP2, and CRISP3 in the boar. A partial sequence published in the National Center for Biotechnology Information (NCBI) database was used to derive the full-length sequences for CRISP1 and CRISP2 using rapid amplification of cDNA ends. RT-PCR confirmed the expression of these mRNAs in the boar reproductive tract, and real time RT-PCR showed CRISP1 to be highly expressed throughout the epididymis, with CRISP2 highly expressed in the testis. A search of the porcine genomic sequence in the NCBI database identified a BAC (CH242-199E6) encoding the CRISP1 gene. This BAC is derived from porcine Chromosome 7 and is syntenic with the regions of the mouse, rat, and human genomes encoding the CRISP gene family. This BAC was found to encode a third CRISP protein with a predicted amino acid sequence of high similarity to human CRISP3. Using RT-PCR we show that CRISP3 expression in the boar reproductive tract is confined to the prostate. Recombinant porcine (rp) CRISP2 protein was produced and purified. When incubated with capacitated boar sperm, rpCRISP2 induced an acrosome reaction, consistent with its demonstrated ability to alter the activity of calcium channels.

Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in the HoxD Locus

PubMed Central

Mason, Mandy K.; VanderMeer, Julia E.; Zhao, Jingjing; Eckalbar, Walter L.; Logan, Malcolm; Illing, Nicola; Pollard, Katherine S.; Ahituv, Nadav

2016-01-01

The molecular events leading to the development of the bat wing remain largely unknown, and are thought to be caused, in part, by changes in gene expression during limb development. These expression changes could be instigated by variations in gene regulatory enhancers. Here, we used a comparative genomics approach to identify regions that evolved rapidly in the bat ancestor, but are highly conserved in other vertebrates. We discovered 166 bat accelerated regions (BARs) that overlap H3K27ac and p300 ChIP-seq peaks in developing mouse limbs. Using a mouse enhancer assay, we show that five Myotis lucifugus BARs drive gene expression in the developing mouse limb, with the majority showing differential enhancer activity compared to the mouse orthologous BAR sequences. These include BAR116, which is located telomeric to the HoxD cluster and had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis of Hoxd10-Hoxd13 in Miniopterus natalensis bats showed a high-forelimb weak-hindlimb expression for Hoxd10-Hoxd11, similar to the expression trend observed for M. lucifugus BAR116 in mice, suggesting that it could be involved in the regulation of the bat HoxD complex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing. PMID:27019019

The PRC2-binding long non-coding RNAs in human and mouse genomes are associated with predictive sequence features

NASA Astrophysics Data System (ADS)

Tu, Shiqi; Yuan, Guo-Cheng; Shao, Zhen

2017-01-01

Recently, long non-coding RNAs (lncRNAs) have emerged as an important class of molecules involved in many cellular processes. One of their primary functions is to shape epigenetic landscape through interactions with chromatin modifying proteins. However, mechanisms contributing to the specificity of such interactions remain poorly understood. Here we took the human and mouse lncRNAs that were experimentally determined to have physical interactions with Polycomb repressive complex 2 (PRC2), and systematically investigated the sequence features of these lncRNAs by developing a new computational pipeline for sequences composition analysis, in which each sequence is considered as a series of transitions between adjacent nucleotides. Through that, PRC2-binding lncRNAs were found to be associated with a set of distinctive and evolutionarily conserved sequence features, which can be utilized to distinguish them from the others with considerable accuracy. We further identified fragments of PRC2-binding lncRNAs that are enriched with these sequence features, and found they show strong PRC2-binding signals and are more highly conserved across species than the other parts, implying their functional importance.

Motif mismatches in microsatellites: insights from genome-wide investigation among 20 insect species.

PubMed

Behura, Susanta K; Severson, David W

2015-02-01

We present a detailed genome-wide comparative study of motif mismatches of microsatellites among 20 insect species representing five taxonomic orders. The results show that varying proportions (∼15-46%) of microsatellites identified in these species are imperfect in motif structure, and that they also vary in chromosomal distribution within genomes. It was observed that the genomic abundance of imperfect repeats is significantly associated with the length and number of motif mismatches of microsatellites. Furthermore, microsatellites with a higher number of mismatches tend to have lower abundance in the genome, suggesting that sequence heterogeneity of repeat motifs is a key determinant of genomic abundance of microsatellites. This relationship seems to be a general feature of microsatellites even in unrelated species such as yeast, roundworm, mouse and human. We provide a mechanistic explanation of the evolutionary link between motif heterogeneity and genomic abundance of microsatellites by examining the patterns of motif mismatches and allele sequences of single-nucleotide polymorphisms identified within microsatellite loci. Using Drosophila Reference Genetic Panel data, we further show that pattern of allelic variation modulates motif heterogeneity of microsatellites, and provide estimates of allele age of specific imperfect microsatellites found within protein-coding genes. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Adaptive Evolution as a Predictor of Species-Specific Innate Immune Response.

PubMed

Webb, Andrew E; Gerek, Z Nevin; Morgan, Claire C; Walsh, Thomas A; Loscher, Christine E; Edwards, Scott V; O'Connell, Mary J

2015-07-01

It has been proposed that positive selection may be associated with protein functional change. For example, human and macaque have different outcomes to HIV infection and it has been shown that residues under positive selection in the macaque TRIM5α receptor locate to the region known to influence species-specific response to HIV. In general, however, the relationship between sequence and function has proven difficult to fully elucidate, and it is the role of large-scale studies to help bridge this gap in our understanding by revealing major patterns in the data that correlate genotype with function or phenotype. In this study, we investigate the level of species-specific positive selection in innate immune genes from human and mouse. In total, we analyzed 456 innate immune genes using codon-based models of evolution, comparing human, mouse, and 19 other vertebrate species to identify putative species-specific positive selection. Then we used population genomic data from the recently completed Neanderthal genome project, the 1000 human genomes project, and the 17 laboratory mouse genomes project to determine whether the residues that were putatively positively selected are fixed or variable in these populations. We find evidence of species-specific positive selection on both the human and the mouse branches and we show that the classes of genes under positive selection cluster by function and by interaction. Data from this study provide us with targets to test the relationship between positive selection and protein function and ultimately to test the relationship between positive selection and discordant phenotypes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Predicting Survival within the Lung Cancer Histopathological Hierarchy Using a Multi-Scale Genomic Model of Development

PubMed Central

Liu, Hongye; Kho, Alvin T; Kohane, Isaac S; Sun, Yao

2006-01-01

Background The histopathologic heterogeneity of lung cancer remains a significant confounding factor in its diagnosis and prognosis—spurring numerous recent efforts to find a molecular classification of the disease that has clinical relevance. Methods and Findings Molecular profiles of tumors from 186 patients representing four different lung cancer subtypes (and 17 normal lung tissue samples) were compared with a mouse lung development model using principal component analysis in both temporal and genomic domains. An algorithm for the classification of lung cancers using a multi-scale developmental framework was developed. Kaplan–Meier survival analysis was conducted for lung adenocarcinoma patient subgroups identified via their developmental association. We found multi-scale genomic similarities between four human lung cancer subtypes and the developing mouse lung that are prognostically meaningful. Significant association was observed between the localization of human lung cancer cases along the principal mouse lung development trajectory and the corresponding patient survival rate at three distinct levels of classical histopathologic resolution: among different lung cancer subtypes, among patients within the adenocarcinoma subtype, and within the stage I adenocarcinoma subclass. The earlier the genomic association between a human tumor profile and the mouse lung development sequence, the poorer the patient's prognosis. Furthermore, decomposing this principal lung development trajectory identified a gene set that was significantly enriched for pyrimidine metabolism and cell-adhesion functions specific to lung development and oncogenesis. Conclusions From a multi-scale disease modeling perspective, the molecular dynamics of murine lung development provide an effective framework that is not only data driven but also informed by the biology of development for elucidating the mechanisms of human lung cancer biology and its clinical outcome. PMID:16800721

In silico identification and comparative analysis of differentially expressed genes in human and mouse tissues

PubMed Central

Pao, Sheng-Ying; Lin, Win-Li; Hwang, Ming-Jing

2006-01-01

Background Screening for differentially expressed genes on the genomic scale and comparative analysis of the expression profiles of orthologous genes between species to study gene function and regulation are becoming increasingly feasible. Expressed sequence tags (ESTs) are an excellent source of data for such studies using bioinformatic approaches because of the rich libraries and tremendous amount of data now available in the public domain. However, any large-scale EST-based bioinformatics analysis must deal with the heterogeneous, and often ambiguous, tissue and organ terms used to describe EST libraries. Results To deal with the issue of tissue source, in this work, we carefully screened and organized more than 8 million human and mouse ESTs into 157 human and 108 mouse tissue/organ categories, to which we applied an established statistic test using different thresholds of the p value to identify genes differentially expressed in different tissues. Further analysis of the tissue distribution and level of expression of human and mouse orthologous genes showed that tissue-specific orthologs tended to have more similar expression patterns than those lacking significant tissue specificity. On the other hand, a number of orthologs were found to have significant disparity in their expression profiles, hinting at novel functions, divergent regulation, or new ortholog relationships. Conclusion Comprehensive statistics on the tissue-specific expression of human and mouse genes were obtained in this very large-scale, EST-based analysis. These statistical results have been organized into a database, freely accessible at our website , for easy searching of human and mouse tissue-specific genes and for investigating gene expression profiles in the context of comparative genomics. Comparative analysis showed that, although highly tissue-specific genes tend to exhibit similar expression profiles in human and mouse, there are significant exceptions, indicating that orthologous genes, while sharing basic genomic properties, could result in distinct phenotypes. PMID:16626500

Targeted disruption of the murine Facc gene: Towards the establishment of a mouse model for Fanconi anemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, M.; Auerbach, W.; Buchwald, M.

1994-09-01

Fanconi anemia (FA) is an autosomal recessive disease characterized by bone marrow failure, congenital malformations and predisposition to malignancies. The gene responsible for the defect in FA group C has been cloned and designated the Fanconi Anemia Complementation Group C gene (FACC). A murine cDNA for this gene (Facc) was also cloned. Here we report our progress in the establishment of a mouse model for FA. The mouse Facc cDNA was used as probe to screen a genomic library of mouse strain 129. More than twenty positive clones were isolated. Three of them were mapped and found to be overlappingmore » clones, encompassing the genomic region from exon 8 to the end of the 3{prime} UTR of the mouse cDNA. A targeting vector was constructed using the most 5{prime} mouse genomic sequence available. The end result of the homologous recombination is that exon 8 is deleted and the neo gene is inserted. The last exon, exon 14, is essential for the complementing function of the FACC gene product; the disruption in the middle of the murine Facc gene should render this locus biologically inactive. This targeting vector was linearized and electroporated into R1 embryonic stem (ES) cells which were derived from the 129 mouse. Of 102 clones screened, 19 positive cell lines were identified. Four targeted cell lines have been used to produce chimeric mice. 129-derived ES cells were aggregated ex vivo into the morulas derived from CD1 mice and then implanted into foster mothers. 22 chimeras have been obtained. Moderately and strongly chimeric mice have been bred to test for germline transmission. Progeny with the expected coat color derived from 2 chimeras are currently being examined to confirm transmission of the targeted allele.« less

In silico Analysis of 2085 Clones from a Normalized Rat Vestibular Periphery 3′ cDNA Library

PubMed Central

Roche, Joseph P.; Cioffi, Joseph A.; Kwitek, Anne E.; Erbe, Christy B.; Popper, Paul

2005-01-01

The inserts from 2400 cDNA clones isolated from a normalized Rattus norvegicus vestibular periphery cDNA library were sequenced and characterized. The Wackym-Soares vestibular 3′ cDNA library was constructed from the saccular and utricular maculae, the ampullae of all three semicircular canals and Scarpa's ganglia containing the somata of the primary afferent neurons, microdissected from 104 male and female rats. The inserts from 2400 randomly selected clones were sequenced from the 5′ end. Each sequence was analyzed using the BLAST algorithm compared to the Genbank nonredundant, rat genome, mouse genome and human genome databases to search for high homology alignments. Of the initial 2400 clones, 315 (13%) were found to be of poor quality and did not yield useful information, and therefore were eliminated from the analysis. Of the remaining 2085 sequences, 918 (44%) were found to represent 758 unique genes having useful annotations that were identified in databases within the public domain or in the published literature; these sequences were designated as known characterized sequences. 1141 sequences (55%) aligned with 1011 unique sequences had no useful annotations and were designated as known but uncharacterized sequences. Of the remaining 26 sequences (1%), 24 aligned with rat genomic sequences, but none matched previously described rat expressed sequence tags or mRNAs. No significant alignment to the rat or human genomic sequences could be found for the remaining 2 sequences. Of the 2085 sequences analyzed, 86% were singletons. The known, characterized sequences were analyzed with the FatiGO online data-mining tool (http://fatigo.bioinfo.cnio.es/) to identify level 5 biological process gene ontology (GO) terms for each alignment and to group alignments with similar or identical GO terms. Numerous genes were identified that have not been previously shown to be expressed in the vestibular system. Further characterization of the novel cDNA sequences may lead to the identification of genes with vestibular-specific functions. Continued analysis of the rat vestibular periphery transcriptome should provide new insights into vestibular function and generate new hypotheses. Physiological studies are necessary to further elucidate the roles of the identified genes and novel sequences in vestibular function. PMID:16103642

Bacterial Artificial Chromosome Libraries for Mouse Sequencing and Functional Analysis

PubMed Central

Osoegawa, Kazutoyo; Tateno, Minako; Woon, Peng Yeong; Frengen, Eirik; Mammoser, Aaron G.; Catanese, Joseph J.; Hayashizaki, Yoshihide; de Jong, Pieter J.

2000-01-01

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9–14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, ∼1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research. [The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AQ797173–AQ797398.] PMID:10645956

The murine Cd48 gene: allelic polymorphism in the IgV-like region.

PubMed

Cabrero, J G; Freeman, G J; Reiser, H

1998-12-01

The murine CD48 molecule is a member of the immunoglobulin superfamily which regulates the activation of T lymphocytes. prior cloning experiments using mRNA from two different mouse strains had yielded discrepant sequences within the IgV-like domain of murine CD48. To resolve this issue, we have directly sequenced genomic DNA of 10 laboratory strains and two inbred strains of wild origin. The results of our analysis reveal an allelic polymorphism within the IgV-like domain of murine CD48.

Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes

PubMed Central

Terrados, Gloria; Finkernagel, Florian; Stielow, Bastian; Sadic, Dennis; Neubert, Juliane; Herdt, Olga; Krause, Michael; Scharfe, Maren; Jarek, Michael; Suske, Guntram

2012-01-01

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes. PMID:22684502

The genome of Eimeria falciformis--reduction and specialization in a single host apicomplexan parasite.

PubMed

Heitlinger, Emanuel; Spork, Simone; Lucius, Richard; Dieterich, Christoph

2014-08-20

The phylum Apicomplexa comprises important unicellular human parasites such as Toxoplasma and Plasmodium. Eimeria is the largest and most diverse genus of apicomplexan parasites and some species of the genus are the causative agent of coccidiosis, a disease economically devastating in poultry. We report a complete genome sequence of the mouse parasite Eimeria falciformis. We assembled and annotated the genome sequence to study host-parasite interactions in this understudied genus in a model organism host. The genome of E. falciformis is 44 Mb in size and contains 5,879 predicted protein coding genes. Comparative analysis of E. falciformis with Toxoplasma gondii shows an emergence and diversification of gene families associated with motility and invasion mainly at the level of the Coccidia. Many rhoptry kinases, among them important virulence factors in T. gondii, are absent from the E. falciformis genome. Surface antigens are divergent between Eimeria species. Comparisons with T. gondii showed differences between genes involved in metabolism, N-glycan and GPI-anchor synthesis. E. falciformis possesses a reduced set of transmembrane transporters and we suggest an altered mode of iron uptake in the genus Eimeria. Reduced diversity of genes required for host-parasite interaction and transmembrane transport allow hypotheses on host adaptation and specialization of a single host parasite. The E. falciformis genome sequence sheds light on the evolution of the Coccidia and helps to identify determinants of host-parasite interaction critical for drug and vaccine development.

Progressive Loss of Function in a Limb Enhancer during Snake Evolution.

PubMed

Kvon, Evgeny Z; Kamneva, Olga K; Melo, Uirá S; Barozzi, Iros; Osterwalder, Marco; Mannion, Brandon J; Tissières, Virginie; Pickle, Catherine S; Plajzer-Frick, Ingrid; Lee, Elizabeth A; Kato, Momoe; Garvin, Tyler H; Akiyama, Jennifer A; Afzal, Veena; Lopez-Rios, Javier; Rubin, Edward M; Dickel, Diane E; Pennacchio, Len A; Visel, Axel

2016-10-20

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP. Copyright © 2016 Elsevier Inc. All rights reserved.

Genomic analysis of a new mammalian distal-less gene: Dlx7.

PubMed

Nakamura, S; Stock, D W; Wydner, K L; Bollekens, J A; Takeshita, K; Nagai, B M; Chiba, S; Kitamura, T; Freeland, T M; Zhao, Z; Minowada, J; Lawrence, J B; Weiss, K M; Ruddle, F H

1996-12-15

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt gene NvHBox-5. The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouse Dlx7 upstream of the homeodomain that may account for some of this divergence. We have mapped human DLX7 distal to the 5' end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.

Transcriptome-wide investigation of genomic imprinting in chicken

PubMed Central

Frésard, Laure; Leroux, Sophie; Servin, Bertrand; Gourichon, David; Dehais, Patrice; Cristobal, Magali San; Marsaud, Nathalie; Vignoles, Florence; Bed'hom, Bertrand; Coville, Jean-Luc; Hormozdiari, Farhad; Beaumont, Catherine; Zerjal, Tatiana; Vignal, Alain; Morisson, Mireille; Lagarrigue, Sandrine; Pitel, Frédérique

2014-01-01

Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing ∼200 Gb of sequences. This allowed the detection of 79 potentially imprinted SNPs, through an analysis method that we validated by detecting imprinting from mouse data already published. However, out of 23 candidates tested by pyrosequencing, none could be confirmed. These results come together, without a priori, with previous statements and phylogenetic considerations assessing the absence of genomic imprinting in chicken. PMID:24452801

Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora.

PubMed

Qi, Ce; Li, Ya; Yu, Ren-Qiang; Zhou, Sheng-Li; Wang, Xing-Guo; Le, Guo-Wei; Jin, Qing-Zhe; Xiao, Hang; Sun, Jin

2017-11-28

To demonstrate that specific bacteria might release bacterial extracellular DNA (eDNA) to exert immunomodulatory functions in the mouse small intestine. Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of eDNA in the mucus layers of the small intestine and colon in healthy Male C57BL/6 mice. Composition difference of eDNA and intracellular DNA (iDNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism (T-RFLP). Stimulation of cytokine production by eDNA was studied in RAW264.7 cells in vitro . TOTO-1 iodide staining confirmed existence of eDNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the eDNA in the small intestinal mucus was significantly different from that of the iDNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the eDNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Gram-positive bacteria. Both eDNA and iDNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The eDNA induced significantly lower tumor necrosis factor-α/interleukin-10 (IL-10) and IL-6/IL-10 ratios than iDNA, suggesting the predominance for maintaining immune homeostasis of the gut. Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.

Reviving the Dead: History and Reactivation of an Extinct L1

PubMed Central

Yang, Lei; Brunsfeld, John; Scott, LuAnn; Wichman, Holly

2014-01-01

Although L1 sequences are present in the genomes of all placental mammals and marsupials examined to date, their activity was lost in the megabat family, Pteropodidae, ∼24 million years ago. To examine the characteristics of L1s prior to their extinction, we analyzed the evolutionary history of L1s in the genome of a megabat, Pteropus vampyrus, and found a pattern of periodic L1 expansion and quiescence. In contrast to the well-characterized L1s in human and mouse, megabat genomes have accommodated two or more simultaneously active L1 families throughout their evolutionary history, and major peaks of L1 deposition into the genome always involved multiple families. We compared the consensus sequences of the two major megabat L1 families at the time of their extinction to consensus L1s of a variety of mammalian species. Megabat L1s are comparable to the other mammalian L1s in terms of adenosine content and conserved amino acids in the open reading frames (ORFs). However, the intergenic region (IGR) of the reconstructed element from the more active family is dramatically longer than the IGR of well-characterized human and mouse L1s. We synthesized the reconstructed element from this L1 family and tested the ability of its components to support retrotransposition in a tissue culture assay. Both ORFs are capable of supporting retrotransposition, while the IGR is inhibitory to retrotransposition, especially when combined with either of the reconstructed ORFs. We dissected the inhibitory effect of the IGR by testing truncated and shuffled versions and found that length is a key factor, but not the only one affecting inhibition of retrotransposition. Although the IGR is inhibitory to retrotransposition, this inhibition does not account for the extinction of L1s in megabats. Overall, the evolution of the L1 sequence or the quiescence of L1 is unlikely the reason of L1 extinction. PMID:24968166

Reviving the dead: history and reactivation of an extinct l1.

PubMed

Yang, Lei; Brunsfeld, John; Scott, LuAnn; Wichman, Holly

2014-06-01

Although L1 sequences are present in the genomes of all placental mammals and marsupials examined to date, their activity was lost in the megabat family, Pteropodidae, ∼24 million years ago. To examine the characteristics of L1s prior to their extinction, we analyzed the evolutionary history of L1s in the genome of a megabat, Pteropus vampyrus, and found a pattern of periodic L1 expansion and quiescence. In contrast to the well-characterized L1s in human and mouse, megabat genomes have accommodated two or more simultaneously active L1 families throughout their evolutionary history, and major peaks of L1 deposition into the genome always involved multiple families. We compared the consensus sequences of the two major megabat L1 families at the time of their extinction to consensus L1s of a variety of mammalian species. Megabat L1s are comparable to the other mammalian L1s in terms of adenosine content and conserved amino acids in the open reading frames (ORFs). However, the intergenic region (IGR) of the reconstructed element from the more active family is dramatically longer than the IGR of well-characterized human and mouse L1s. We synthesized the reconstructed element from this L1 family and tested the ability of its components to support retrotransposition in a tissue culture assay. Both ORFs are capable of supporting retrotransposition, while the IGR is inhibitory to retrotransposition, especially when combined with either of the reconstructed ORFs. We dissected the inhibitory effect of the IGR by testing truncated and shuffled versions and found that length is a key factor, but not the only one affecting inhibition of retrotransposition. Although the IGR is inhibitory to retrotransposition, this inhibition does not account for the extinction of L1s in megabats. Overall, the evolution of the L1 sequence or the quiescence of L1 is unlikely the reason of L1 extinction.

Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an amazingly versatile tool for the study of mammalian biology.

PubMed

Babinet, C; Cohen-Tannoudji, M

2001-09-01

The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene "targeting"). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions, etc.) and chromosomal rearrangements such as large deletions, duplications and translocations. Finally, methods have been devised which permit the creation of conditional mutations, allowing the study of gene function throughout the life of an animal, when gene inactivation entails embryonic lethality. In this paper, we present an overview of the methods and scenarios used for the programmed modification of mouse genome, and we underline their enormous interest for the study of mammalian biology.

Complex multi-enhancer contacts captured by genome architecture mapping.

PubMed

Beagrie, Robert A; Scialdone, Antonio; Schueler, Markus; Kraemer, Dorothee C A; Chotalia, Mita; Xie, Sheila Q; Barbieri, Mariano; de Santiago, Inês; Lavitas, Liron-Mark; Branco, Miguel R; Fraser, James; Dostie, Josée; Game, Laurence; Dillon, Niall; Edwards, Paul A W; Nicodemi, Mario; Pombo, Ana

2017-03-23

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Characterization of Aeromonas hydrophila Wound Pathotypes by Comparative Genomic and Functional Analyses of Virulence Genes

PubMed Central

Grim, Christopher J.; Kozlova, Elena V.; Sha, Jian; Fitts, Eric C.; van Lier, Christina J.; Kirtley, Michelle L.; Joseph, Sandeep J.; Read, Timothy D.; Burd, Eileen M.; Tall, Ben D.; Joseph, Sam W.; Horneman, Amy J.; Chopra, Ashok K.; Shak, Joshua R.

2013-01-01

ABSTRACT Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A. hydrophila (E2). To better understand the differences between pathogenic and environmental strains of A. hydrophila, we conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966T, and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966T. The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966T and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, we characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity. PMID:23611906

Identification of novel non-coding small RNAs from Streptococcus pneumoniae TIGR4 using high-resolution genome tiling arrays

PubMed Central

2010-01-01

Background The identification of non-coding transcripts in human, mouse, and Escherichia coli has revealed their widespread occurrence and functional importance in both eukaryotic and prokaryotic life. In prokaryotes, studies have shown that non-coding transcripts participate in a broad range of cellular functions like gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Streptococcus pneumoniae (pneumococcus), an obligate human respiratory pathogen responsible for significant worldwide morbidity and mortality. Tiling microarrays enable genome wide mRNA profiling as well as identification of novel transcripts at a high-resolution. Results Here, we describe a high-resolution transcription map of the S. pneumoniae clinical isolate TIGR4 using genomic tiling arrays. Our results indicate that approximately 66% of the genome is expressed under our experimental conditions. We identified a total of 50 non-coding small RNAs (sRNAs) from the intergenic regions, of which 36 had no predicted function. Half of the identified sRNA sequences were found to be unique to S. pneumoniae genome. We identified eight overrepresented sequence motifs among sRNA sequences that correspond to sRNAs in different functional categories. Tiling arrays also identified approximately 202 operon structures in the genome. Conclusions In summary, the pneumococcal operon structures and novel sRNAs identified in this study enhance our understanding of the complexity and extent of the pneumococcal 'expressed' genome. Furthermore, the results of this study open up new avenues of research for understanding the complex RNA regulatory network governing S. pneumoniae physiology and virulence. PMID:20525227

Whipworm genome and dual-species transcriptome analyses provide molecular insights into an intimate host-parasite interaction.

PubMed

Foth, Bernardo J; Tsai, Isheng J; Reid, Adam J; Bancroft, Allison J; Nichol, Sarah; Tracey, Alan; Holroyd, Nancy; Cotton, James A; Stanley, Eleanor J; Zarowiecki, Magdalena; Liu, Jimmy Z; Huckvale, Thomas; Cooper, Philip J; Grencis, Richard K; Berriman, Matthew

2014-07-01

Whipworms are common soil-transmitted helminths that cause debilitating chronic infections in man. These nematodes are only distantly related to Caenorhabditis elegans and have evolved to occupy an unusual niche, tunneling through epithelial cells of the large intestine. We report here the whole-genome sequences of the human-infective Trichuris trichiura and the mouse laboratory model Trichuris muris. On the basis of whole-transcriptome analyses, we identify many genes that are expressed in a sex- or life stage-specific manner and characterize the transcriptional landscape of a morphological region with unique biological adaptations, namely, bacillary band and stichosome, found only in whipworms and related parasites. Using RNA sequencing data from whipworm-infected mice, we describe the regulated T helper 1 (TH1)-like immune response of the chronically infected cecum in unprecedented detail. In silico screening identified numerous new potential drug targets against trichuriasis. Together, these genomes and associated functional data elucidate key aspects of the molecular host-parasite interactions that define chronic whipworm infection.

An interactive environment for agile analysis and visualization of ChIP-sequencing data.

PubMed

Lerdrup, Mads; Johansen, Jens Vilstrup; Agrawal-Singh, Shuchi; Hansen, Klaus

2016-04-01

To empower experimentalists with a means for fast and comprehensive chromatin immunoprecipitation sequencing (ChIP-seq) data analyses, we introduce an integrated computational environment, EaSeq. The software combines the exploratory power of genome browsers with an extensive set of interactive and user-friendly tools for genome-wide abstraction and visualization. It enables experimentalists to easily extract information and generate hypotheses from their own data and public genome-wide datasets. For demonstration purposes, we performed meta-analyses of public Polycomb ChIP-seq data and established a new screening approach to analyze more than 900 datasets from mouse embryonic stem cells for factors potentially associated with Polycomb recruitment. EaSeq, which is freely available and works on a standard personal computer, can substantially increase the throughput of many analysis workflows, facilitate transparency and reproducibility by automatically documenting and organizing analyses, and enable a broader group of scientists to gain insights from ChIP-seq data.

Molecular cloning of the mouse gene coding for {alpha}{sub 2}-macroglobulin and targeting of the gene in embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Umans, L.; Serneels, L.; Hilliker, C.

1994-08-01

The authors have cloned the mouse gene coding for {alpha}{sub 2}-macroglobulin in overlapping {lambda} clones and have analyzed its structure. The gene contains 36 exons, coding for the 4.8-kb cDNA that we cloned previously. Including putative control elements in the 5{prime} flanking region, the gene covers about 45 kb. A region of 3.8 kb, stretching from 835 bases upstream of the cDNA start site to exon 4, including all intervening sequences, was sequenced completely. The analysis demonstrated that the putative promoter region of the mouse A2M gene differed considerably from the known promoter sequences of the human A2M gene andmore » of the rat acute-phas A2M gene. Comparison of the exon-intron structure of all known genes of the A2M family confirmed that the rat acute phase A2M gene is more closely related to the human gene than to the mouse A2M gene. To generate mice with the A2M gene inactivated, an insertion type of construct containing 7.5 kb of genomic DNA of the mouse strain 129/J, encompassing exons 16 to 19, was synthesized. A hygromycin marker gene was embedded in intron 17. After electroporation, 198 hygromycin-resistant ES cell lines were isolated and analyzed by Southern blotting. Five ES cell lines were obtained with one allele of the mouse A2M gene targeted by this insertion construct, demonstrating that the position and the characteristics of the vector served the intended goal.« less

Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias

PubMed Central

2012-01-01

Background High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs) in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. Results We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO) probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno) that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. Conclusion The problems of ascertainment bias and missing information due to genotyping errors are widely recognized as limiting factors in genetic studies. We have conducted the first formal analysis of the effect of novel variants on genotyping arrays, and we have shown that these variants account for a large portion of miscalled and uncalled genotypes. Genetic studies will benefit from substantial improvements in the accuracy of their results by incorporating VINOs in their analyses. PMID:22260749

The role of retrotransposons in gene family expansions: insights from the mouse Abp gene family.

PubMed

Janoušek, Václav; Karn, Robert C; Laukaitis, Christina M

2013-05-29

Retrotransposons have been suggested to provide a substrate for non-allelic homologous recombination (NAHR) and thereby promote gene family expansion. Their precise role, however, is controversial. Here we ask whether retrotransposons contributed to the recent expansions of the Androgen-binding protein (Abp) gene families that occurred independently in the mouse and rat genomes. Using dot plot analysis, we found that the most recent duplication in the Abp region of the mouse genome is flanked by L1Md_T elements. Analysis of the sequence of these elements revealed breakpoints that are the relicts of the recombination that caused the duplication, confirming that the duplication arose as a result of NAHR using L1 elements as substrates. L1 and ERVII retrotransposons are considerably denser in the Abp regions than in one Mb flanking regions, while other repeat types are depleted in the Abp regions compared to flanking regions. L1 retrotransposons preferentially accumulated in the Abp gene regions after lineage separation and roughly followed the pattern of Abp gene expansion. By contrast, the proportion of shared vs. lineage-specific ERVII repeats in the Abp region resembles the rest of the genome. We confirmed the role of L1 repeats in Abp gene duplication with the identification of recombinant L1Md_T elements at the edges of the most recent mouse Abp gene duplication. High densities of L1 and ERVII repeats were found in the Abp gene region with abrupt transitions at the region boundaries, suggesting that their higher densities are tightly associated with Abp gene duplication. We observed that the major accumulation of L1 elements occurred after the split of the mouse and rat lineages and that there is a striking overlap between the timing of L1 accumulation and expansion of the Abp gene family in the mouse genome. Establishing a link between the accumulation of L1 elements and the expansion of the Abp gene family and identification of an NAHR-related breakpoint in the most recent duplication are the main contributions of our study.

The role of retrotransposons in gene family expansions: insights from the mouse Abp gene family

PubMed Central

2013-01-01

Background Retrotransposons have been suggested to provide a substrate for non-allelic homologous recombination (NAHR) and thereby promote gene family expansion. Their precise role, however, is controversial. Here we ask whether retrotransposons contributed to the recent expansions of the Androgen-binding protein (Abp) gene families that occurred independently in the mouse and rat genomes. Results Using dot plot analysis, we found that the most recent duplication in the Abp region of the mouse genome is flanked by L1Md_T elements. Analysis of the sequence of these elements revealed breakpoints that are the relicts of the recombination that caused the duplication, confirming that the duplication arose as a result of NAHR using L1 elements as substrates. L1 and ERVII retrotransposons are considerably denser in the Abp regions than in one Mb flanking regions, while other repeat types are depleted in the Abp regions compared to flanking regions. L1 retrotransposons preferentially accumulated in the Abp gene regions after lineage separation and roughly followed the pattern of Abp gene expansion. By contrast, the proportion of shared vs. lineage-specific ERVII repeats in the Abp region resembles the rest of the genome. Conclusions We confirmed the role of L1 repeats in Abp gene duplication with the identification of recombinant L1Md_T elements at the edges of the most recent mouse Abp gene duplication. High densities of L1 and ERVII repeats were found in the Abp gene region with abrupt transitions at the region boundaries, suggesting that their higher densities are tightly associated with Abp gene duplication. We observed that the major accumulation of L1 elements occurred after the split of the mouse and rat lineages and that there is a striking overlap between the timing of L1 accumulation and expansion of the Abp gene family in the mouse genome. Establishing a link between the accumulation of L1 elements and the expansion of the Abp gene family and identification of an NAHR-related breakpoint in the most recent duplication are the main contributions of our study. PMID:23718880

Heat*seq: an interactive web tool for high-throughput sequencing experiment comparison with public data.

PubMed

Devailly, Guillaume; Mantsoki, Anna; Joshi, Anagha

2016-11-01

Better protocols and decreasing costs have made high-throughput sequencing experiments now accessible even to small experimental laboratories. However, comparing one or few experiments generated by an individual lab to the vast amount of relevant data freely available in the public domain might be limited due to lack of bioinformatics expertise. Though several tools, including genome browsers, allow such comparison at a single gene level, they do not provide a genome-wide view. We developed Heat*seq, a web-tool that allows genome scale comparison of high throughput experiments chromatin immuno-precipitation followed by sequencing, RNA-sequencing and Cap Analysis of Gene Expression) provided by a user, to the data in the public domain. Heat*seq currently contains over 12 000 experiments across diverse tissues and cell types in human, mouse and drosophila. Heat*seq displays interactive correlation heatmaps, with an ability to dynamically subset datasets to contextualize user experiments. High quality figures and tables are produced and can be downloaded in multiple formats. Web application: http://www.heatstarseq.roslin.ed.ac.uk/ Source code: https://github.com/gdevailly CONTACT: Guillaume.Devailly@roslin.ed.ac.uk or Anagha.Joshi@roslin.ed.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

microPIR2: a comprehensive database for human–mouse comparative study of microRNA–promoter interactions

PubMed Central

Piriyapongsa, Jittima; Bootchai, Chaiwat; Ngamphiw, Chumpol; Tongsima, Sissades

2014-01-01

microRNA (miRNA)–promoter interaction resource (microPIR) is a public database containing over 15 million predicted miRNA target sites located within human promoter sequences. These predicted targets are presented along with their related genomic and experimental data, making the microPIR database the most comprehensive repository of miRNA promoter target sites. Here, we describe major updates of the microPIR database including new target predictions in the mouse genome and revised human target predictions. The updated database (microPIR2) now provides ∼80 million human and 40 million mouse predicted target sites. In addition to being a reference database, microPIR2 is a tool for comparative analysis of target sites on the promoters of human–mouse orthologous genes. In particular, this new feature was designed to identify potential miRNA–promoter interactions conserved between species that could be stronger candidates for further experimental validation. We also incorporated additional supporting information to microPIR2 such as nuclear and cytoplasmic localization of miRNAs and miRNA–disease association. Extra search features were also implemented to enable various investigations of targets of interest. Database URL: http://www4a.biotec.or.th/micropir2 PMID:25425035

HMG-CoA lyase (HL) gene: Cloning and characterization of the 5{prime} end of the mouse gene, gene targeting in ES cells, and demonstration of large deletions in three HL-deficient patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, S.; Robert, M.F.; Mitchell, G.A.

1994-09-01

3-hydroxy-3-methylglutaryl CoA lyase (HL) is a mitochondrial matrix enzyme which catalyzes the last step of leucine catabolism and of ketogenesis. Autosomal recessive HL deficiency in humans results in episodes of hypoglycemia and coma. We are interested in the pathophysiology of HL deficiency as a model for both amino acid and fatty acid inborn errors. We have cloned the human and mouse HL genes. In order to analyze the 5{prime} nontranslated region of mouse HL gene, we cloned and sequenced a 1.8 kb fragment containing the 5{prime} extremity including exon 1 and about 1.6 kb of 5{prime} nontranslated sequence. The regionmore » surrounding exon 1 is CpG-rich (66.4%). Using the criteria of West, the Observed/Expected ratio for CpG dinucleotides is 0.7 ({ge}0.6 is consistent with a CpG island). We are carrying out primer extension and RNase protection experiments to determine the transcription initiation site. We constructed a gene targeting vector by introducing the neomycin resistance gene into exon 2 of a 7.5 kb genomic subclone of the mouse HL gene. Targeting was performed by electroporating 10 mg linearized vector into 10{sup 7} ES cells and selecting for 12 days with G418. 5/228 colonies (2.2%) had homologous recombination as shown by PCR screening and Southern analysis. We are microinjecting the 5 targeted clones into blastocysts to create an HL-deficient mouse. To date we have obtained two chimeras with contributions of 95% and 55% from 129, by coat color estimates. Three of 27 (11%) of the HL-deficient patients studied were suggested by genomic Southern analysis to be homozygous for large intragenic deletions. We confirmed this and defined the boundaries using exonic PCR.« less

Prevalence of Avian-Pathogenic Escherichia coli Strain O1 Genomic Islands among Extraintestinal and Commensal E. coli Isolates

PubMed Central

Johnson, Timothy J.; Wannemuehler, Yvonne; Kariyawasam, Subhashinie; Johnson, James R.; Logue, Catherine M.

2012-01-01

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include pathogens of humans and animals. Previously, the genome of avian-pathogenic E. coli (APEC) O1:K1:H7 strain O1, from ST95, was sequenced and compared to those of several other E. coli strains, identifying 43 genomic islands. Here, the genomic islands of APEC O1 were compared to those of other sequenced E. coli strains, and the distribution of 81 genes belonging to 12 APEC O1 genomic islands among 828 human and avian ExPEC and commensal E. coli isolates was determined. Multiple islands were highly prevalent among isolates belonging to the O1 and O18 serogroups within phylogenetic group B2, which are implicated in human neonatal meningitis. Because of the extensive genomic similarities between APEC O1 and other human ExPEC strains belonging to the ST95 phylogenetic lineage, its ability to cause disease in a rat model of sepsis and meningitis was assessed. Unlike other ST95 lineage strains, APEC O1 was unable to cause bacteremia or meningitis in the neonatal rat model and was significantly less virulent than uropathogenic E. coli (UPEC) CFT073 in a mouse sepsis model, despite carrying multiple neonatal meningitis E. coli (NMEC) virulence factors and belonging to the ST95 phylogenetic lineage. These results suggest that host adaptation or genome modifications have occurred either in APEC O1 or in highly virulent ExPEC isolates, resulting in differences in pathogenicity. Overall, the genomic islands examined provide targets for further discrimination of the different ExPEC subpathotypes, serogroups, phylogenetic types, and sequence types. PMID:22467781

Prevalence of avian-pathogenic Escherichia coli strain O1 genomic islands among extraintestinal and commensal E. coli isolates.

PubMed

Johnson, Timothy J; Wannemuehler, Yvonne; Kariyawasam, Subhashinie; Johnson, James R; Logue, Catherine M; Nolan, Lisa K

2012-06-01

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include pathogens of humans and animals. Previously, the genome of avian-pathogenic E. coli (APEC) O1:K1:H7 strain O1, from ST95, was sequenced and compared to those of several other E. coli strains, identifying 43 genomic islands. Here, the genomic islands of APEC O1 were compared to those of other sequenced E. coli strains, and the distribution of 81 genes belonging to 12 APEC O1 genomic islands among 828 human and avian ExPEC and commensal E. coli isolates was determined. Multiple islands were highly prevalent among isolates belonging to the O1 and O18 serogroups within phylogenetic group B2, which are implicated in human neonatal meningitis. Because of the extensive genomic similarities between APEC O1 and other human ExPEC strains belonging to the ST95 phylogenetic lineage, its ability to cause disease in a rat model of sepsis and meningitis was assessed. Unlike other ST95 lineage strains, APEC O1 was unable to cause bacteremia or meningitis in the neonatal rat model and was significantly less virulent than uropathogenic E. coli (UPEC) CFT073 in a mouse sepsis model, despite carrying multiple neonatal meningitis E. coli (NMEC) virulence factors and belonging to the ST95 phylogenetic lineage. These results suggest that host adaptation or genome modifications have occurred either in APEC O1 or in highly virulent ExPEC isolates, resulting in differences in pathogenicity. Overall, the genomic islands examined provide targets for further discrimination of the different ExPEC subpathotypes, serogroups, phylogenetic types, and sequence types.

The Mouse Genome Database (MGD): facilitating mouse as a model for human biology and disease.

PubMed

Eppig, Janan T; Blake, Judith A; Bult, Carol J; Kadin, James A; Richardson, Joel E

2015-01-01

The Mouse Genome Database (MGD, http://www.informatics.jax.org) serves the international biomedical research community as the central resource for integrated genomic, genetic and biological data on the laboratory mouse. To facilitate use of mouse as a model in translational studies, MGD maintains a core of high-quality curated data and integrates experimentally and computationally generated data sets. MGD maintains a unified catalog of genes and genome features, including functional RNAs, QTL and phenotypic loci. MGD curates and provides functional and phenotype annotations for mouse genes using the Gene Ontology and Mammalian Phenotype Ontology. MGD integrates phenotype data and associates mouse genotypes to human diseases, providing critical mouse-human relationships and access to repositories holding mouse models. MGD is the authoritative source of nomenclature for genes, genome features, alleles and strains following guidelines of the International Committee on Standardized Genetic Nomenclature for Mice. A new addition to MGD, the Human-Mouse: Disease Connection, allows users to explore gene-phenotype-disease relationships between human and mouse. MGD has also updated search paradigms for phenotypic allele attributes, incorporated incidental mutation data, added a module for display and exploration of genes and microRNA interactions and adopted the JBrowse genome browser. MGD resources are freely available to the scientific community. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Nonparametric Bayesian clustering to detect bipolar methylated genomic loci.

PubMed

Wu, Xiaowei; Sun, Ming-An; Zhu, Hongxiao; Xie, Hehuang

2015-01-16

With recent development in sequencing technology, a large number of genome-wide DNA methylation studies have generated massive amounts of bisulfite sequencing data. The analysis of DNA methylation patterns helps researchers understand epigenetic regulatory mechanisms. Highly variable methylation patterns reflect stochastic fluctuations in DNA methylation, whereas well-structured methylation patterns imply deterministic methylation events. Among these methylation patterns, bipolar patterns are important as they may originate from allele-specific methylation (ASM) or cell-specific methylation (CSM). Utilizing nonparametric Bayesian clustering followed by hypothesis testing, we have developed a novel statistical approach to identify bipolar methylated genomic regions in bisulfite sequencing data. Simulation studies demonstrate that the proposed method achieves good performance in terms of specificity and sensitivity. We used the method to analyze data from mouse brain and human blood methylomes. The bipolar methylated segments detected are found highly consistent with the differentially methylated regions identified by using purified cell subsets. Bipolar DNA methylation often indicates epigenetic heterogeneity caused by ASM or CSM. With allele-specific events filtered out or appropriately taken into account, our proposed approach sheds light on the identification of cell-specific genes/pathways under strong epigenetic control in a heterogeneous cell population.

Short interfering RNA confers intracellular antiviral immunity in human cells.

PubMed

Gitlin, Leonid; Karelsky, Sveta; Andino, Raul

2002-07-25

Gene silencing mediated by double-stranded RNA (dsRNA) is a sequence-specific, highly conserved mechanism in eukaryotes. In plants, it serves as an antiviral defence mechanism. Animal cells also possess this machinery but its specific function is unclear. Here we demonstrate that dsRNA can effectively protect human cells against infection by a rapidly replicating and highly cytolytic RNA virus. Pre-treatment of human and mouse cells with double-stranded, short interfering RNAs (siRNAs) to the poliovirus genome markedly reduces the titre of virus progeny and promotes clearance of the virus from most of the infected cells. The antiviral effect is sequence-specific and is not attributable to either classical antisense mechanisms or to interferon and the interferon response effectors protein kinase R (PKR) and RNaseL. Protection is the result of direct targeting of the viral genome by siRNA, as sequence analysis of escape virus (resistant to siRNAs) reveals one nucleotide substitution in the middle of the targeted sequence. Thus, siRNAs elicit specific intracellular antiviral resistance that may provide a therapeutic strategy against human viruses.

High-throughput engineering of a mammalian genome reveals building principles of methylation states at CG rich regions.

PubMed

Krebs, Arnaud R; Dessus-Babus, Sophie; Burger, Lukas; Schübeler, Dirk

2014-09-26

The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.

Genomic organization, complete sequence, and chromosomal location of the gene for human eotaxin (SCYA11), an eosinophil-specific CC chemokine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Zepeda, E.A.; Sarafi, M.N.; Luster, A.D.

1997-05-01

Eotaxin is a CC chemokine that is a specific chemoattractant for eosinophils and is implicated in the pathogenesis of eosinophilic inflammatory diseases, such as asthma. We describe the genomic organization, complete sequence, including 1354 bp 5{prime} of the RNA initiation site, and chromosomal localization of the human eotaxin gene. Fluorescence in situ hybridization analysis localized eotaxin to human chromosome 17, in the region q21.1-q21.2, and the human gene name SCYA11 was assigned. We also present the 5{prime} flanking sequence of the mouse eotaxin gene and have identified several regulatory elements that are conserved between the murine and the human promoters.more » In particular, the presence of elements such as NF-{Kappa}B, interferon-{gamma} response element, and glucocorticoid response element may explain the observed regulation of the eotaxin gene by cytokines and glucocorticoids. 17 refs., 4 figs., 1 tab.« less

ARTS: a web-based tool for the set-up of high-throughput genome-wide mapping panels for the SNP genotyping of mouse mutants.

PubMed

Klaften, Matthias; Hrabé de Angelis, Martin

2005-07-01

Genome-wide mapping in the identification of novel candidate genes has always been the standard method in genetics and genomics to correlate a clinically interesting phenotypic trait with a genotype. However, the performance of a mapping experiment using classical microsatellite approaches can be very time consuming. The high-throughput analysis of single-nucleotide polymorphisms (SNPs) has the potential of being the successor of microsatellite analysis routinely used for these mapping approaches, where one of the major obstacles is the design of the appropriate SNP marker set itself. Here we report on ARTS, an advanced retrieval tool for SNPs, which allows researchers to comb freely the public mouse dbSNP database for multiple reference and test strains. Several filters can be applied in order to improve the sensitivity and the specificity of the search results. By employing the panel generator function of this program, it is possible to abbreviate the extraction of reliable sequence data for a large marker panel including several different mouse strains from days to minutes. The concept of ARTS is easily adaptable to other species for which SNP databases are available, making it a versatile tool for the use of SNPs as markers for genotyping. The web interface is accessible at http://andromeda.gsf.de/arts.

Adaptive Evolution of the Insulin Two-Gene System in Mouse

PubMed Central

Shiao, Meng-Shin; Liao, Ben-Yang; Long, Manyuan; Yu, Hon-Tsen

2008-01-01

Insulin genes in mouse and rat compose a two-gene system in which Ins1 was retroposed from the partially processed mRNA of Ins2. When Ins1 originated and how it was retained in genomes still remain interesting problems. In this study, we used genomic approaches to detect insulin gene copy number variation in rodent species and investigated evolutionary forces acting on both Ins1 and Ins2. We characterized the phylogenetic distribution of the new insulin gene (Ins1) by Southern analyses and confirmed by sequencing insulin genes in the rodent genomes. The results demonstrate that Ins1 originated right before the mouse–rat split (∼20 MYA), and both Ins1 and Ins2 are under strong functional constraints in these murine species. Interestingly, by examining a range of nucleotide polymorphisms, we detected positive selection acting on both Ins2 and Ins1 gene regions in the Mus musculus domesticus populations. Furthermore, three amino acid sites were also identified as having evolved under positive selection in two insulin peptides: two are in the signal peptide and one is in the C-peptide. Our data suggest an adaptive divergence in the mouse insulin two-gene system, which may result from the response to environmental change caused by the rise of agricultural civilization, as proposed by the thrifty-genotype hypothesis. PMID:18245324

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

A High-Resolution Enhancer Atlas of the Developing Telencephalon

PubMed Central

Visel, Axel; Taher, Leila; Girgis, Hani; May, Dalit; Golonzhka, Olga; Hoch, Renee; McKinsey, Gabriel L.; Pattabiraman, Kartik; Silberberg, Shanni N.; Blow, Matthew J.; Hansen, David V.; Nord, Alex S.; Akiyama, Jennifer A.; Holt, Amy; Hosseini, Roya; Phouanenavong, Sengthavy; Plajzer-Frick, Ingrid; Shoukry, Malak; Afzal, Veena; Kaplan, Tommy; Kriegstein, Arnold R.; Rubin, Edward M.; Ovcharenko, Ivan; Pennacchio, Len A.; Rubenstein, John L. R.

2013-01-01

Summary The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. While many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified over 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising over 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders. PMID:23375746

Ancient genomic architecture for mammalian olfactory receptor clusters

PubMed Central

Aloni, Ronny; Olender, Tsviya; Lancet, Doron

2006-01-01

Background Mammalian olfactory receptor (OR) genes reside in numerous genomic clusters of up to several dozen genes. Whole-genome sequence alignment nets of five mammals allow their comprehensive comparison, aimed at reconstructing the ancestral olfactory subgenome. Results We developed a new and general tool for genome-wide definition of genomic gene clusters conserved in multiple species. Syntenic orthologs, defined as gene pairs showing conservation of both genomic location and coding sequence, were subjected to a graph theory algorithm for discovering CLICs (clusters in conservation). When applied to ORs in five mammals, including the marsupial opossum, more than 90% of the OR genes were found within a framework of 48 multi-species CLICs, invoking a general conservation of gene order and composition. A detailed analysis of individual CLICs revealed multiple differences among species, interpretable through species-specific genomic rearrangements and reflecting complex mammalian evolutionary dynamics. One significant instance involves CLIC #1, which lacks a human member, implying the human-specific deletion of an OR cluster, whose mouse counterpart has been tentatively associated with isovaleric acid odorant detection. Conclusion The identified multi-species CLICs demonstrate that most of the mammalian OR clusters have a common ancestry, preceding the split between marsupials and placental mammals. However, only two of these CLICs were capable of incorporating chicken OR genes, parsimoniously implying that all other CLICs emerged subsequent to the avian-mammalian divergence. PMID:17010214

A novel class of small RNAs bind to MILI protein in mouse testes.

PubMed

Aravin, Alexei; Gaidatzis, Dimos; Pfeffer, Sébastien; Lagos-Quintana, Mariana; Landgraf, Pablo; Iovino, Nicola; Morris, Patricia; Brownstein, Michael J; Kuramochi-Miyagawa, Satomi; Nakano, Toru; Chien, Minchen; Russo, James J; Ju, Jingyue; Sheridan, Robert; Sander, Chris; Zavolan, Mihaela; Tuschl, Thomas

2006-07-13

Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins--known as the 'Piwi family'--is required for germ- and stem-cell development in invertebrates, and two Piwi members--MILI and MIWI--are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5' uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26-31 nucleotides (nt) in length--clearly distinct from the 21-23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)--and we refer to them as 'Piwi-interacting RNAs' or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.

Identification of an Internal Ribosome Entry Segment in the 5′ Region of the Mouse VL30 Retrotransposon and Its Use in the Development of Retroviral Vectors

PubMed Central

López-Lastra, Marcelo; Ulrici, Sandrine; Gabus, Caroline; Darlix, Jean-Luc

1999-01-01

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5′ region of VL30m could replace the 5′ leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5′ region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5′ region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5′ region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors. PMID:10482590

Identification of an internal ribosome entry segment in the 5' region of the mouse VL30 retrotransposon and its use in the development of retroviral vectors.

PubMed

López-Lastra, M; Ulrici, S; Gabus, C; Darlix, J L

1999-10-01

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5' region of VL30m could replace the 5' leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5' region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5' region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5' region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors.

Isolation of a promoter region in mouse cytochrome P450 3A (Cyp3A16) gene and its transcriptional control.

PubMed

Itoh, S; Abe, Y; Kubo, A; Okuda, M; Shimoji, M; Nakayama, K; Kamataki, T

1997-02-07

An 11.5 kb fragment of the mouse Cyp3a16 gene containing the 5' flanking region was isolated from the lambda DASHII mouse genomic library. A part of the 5' flanking region and the first exon of Cyp3a16 gene were sequenced. S1 mapping analysis showed the presence of two transcriptional initiation sites. The first exon was completely identical to Cyp3a16 cDNA. The identity of 5' flanking sequences between Cyp3a16 and Cyp3a11 genes was about 69%. A typical TATA box and a basic transcription element (BTE) were found as seen with other CYP3A genes from various animal species Moreover, some putative transcriptional regulatory elements were also found in addition to the sequence motif seen for the formation of Z-type DNA. To examine the transcriptional activity of Cyp3a11 gene, DNA fragments in the 5'-flanking region of the gene were inserted front of the luciferase structural gene, and the constructs were transfected in primary hepatocytes. The analysis of the luciferase activity indicated that the region between -146 and -56 was necessary for the transcription of CYP3a16 gene.

Extreme variability among mammalian V1R gene families.

PubMed

Young, Janet M; Massa, Hillary F; Hsu, Li; Trask, Barbara J

2010-01-01

We report an evolutionary analysis of the V1R gene family across 37 mammalian genomes. V1Rs comprise one of three chemosensory receptor families expressed in the vomeronasal organ, and contribute to pheromone detection. We first demonstrate that Trace Archive data can be used effectively to determine V1R family sizes and to obtain sequences of most V1R family members. Analyses of V1R sequences from trace data and genome assemblies show that species-specific expansions previously observed in only eight species were prevalent throughout mammalian evolution, resulting in "semi-private" V1R repertoires for most mammals. The largest families are found in mouse and platypus, whose V1R repertoires have been published previously, followed by mouse lemur and rabbit (approximately 215 and approximately 160 intact V1Rs, respectively). In contrast, two bat species and dolphin possess no functional V1Rs, only pseudogenes, and suffered inactivating mutations in the vomeronasal signal transduction gene Trpc2. We show that primate V1R decline happened prior to acquisition of trichromatic vision, earlier during evolution than was previously thought. We also show that it is extremely unlikely that decline of the dog V1R repertoire occurred in response to selective pressures imposed by humans during domestication. Functional repertoire sizes in each species correlate roughly with anatomical observations of vomeronasal organ size and quality; however, no single ecological correlate explains the very diverse fates of this gene family in different mammalian genomes. V1Rs provide one of the most extreme examples observed to date of massive gene duplication in some genomes, with loss of all functional genes in other species.

Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18

PubMed Central

2011-01-01

Background Our previously published reports have described an effective biocontrol agent named Pseudomonas sp. M18 as its 16S rDNA sequence and several regulator genes share homologous sequences with those of P. aeruginosa, but there are several unusual phenotypic features. This study aims to explore its strain specific genomic features and gene expression patterns at different temperatures. Results The complete M18 genome is composed of a single chromosome of 6,327,754 base pairs containing 5684 open reading frames. Seven genomic islands, including two novel prophages and five specific non-phage islands were identified besides the conserved P. aeruginosa core genome. Each prophage contains a putative chitinase coding gene, and the prophage II contains a capB gene encoding a putative cold stress protein. The non-phage genomic islands contain genes responsible for pyoluteorin biosynthesis, environmental substance degradation and type I and III restriction-modification systems. Compared with other P. aeruginosa strains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18 genome may contribute to the relative genome stability. Although the M18 genome is most closely related to that of P. aeruginosa strain LESB58, the strain M18 is more susceptible to several antimicrobial agents and easier to be erased in a mouse acute lung infection model than the strain LESB58. The whole M18 transcriptomic analysis indicated that 10.6% of the expressed genes are temperature-dependent, with 22 genes up-regulated at 28°C in three non-phage genomic islands and one prophage but none at 37°C. Conclusions The P. aeruginosa strain M18 has evolved its specific genomic structures and temperature dependent expression patterns to meet the requirement of its fitness and competitiveness under selective pressures imposed on the strain in rhizosphere niche. PMID:21884571

Evolutionary History of LINE-1 in the Major Clades of Placental Mammals

PubMed Central

Waters, Paul D.; Dobigny, Gauthier; Waddell, Peter J.; Robinson, Terence J.

2007-01-01

Background LINE-1 constitutes an important component of mammalian genomes. It has a dynamic evolutionary history characterized by the rise, fall and replacement of subfamilies. Most data concerning LINE-1 biology and evolution are derived from the human and mouse genomes and are often assumed to hold for all placentals. Methodology To examine LINE-1 relationships, sequences from the 3′ region of the reverse transcriptase from 21 species (representing 13 orders across Afrotheria, Xenarthra, Supraprimates and Laurasiatheria) were obtained from whole genome sequence assemblies, or by PCR with degenerate primers. These sequences were aligned and analysed. Principal Findings Our analysis reflects accepted placental relationships suggesting mostly lineage-specific LINE-1 families. The data provide clear support for several clades including Glires, Supraprimates, Laurasiatheria, Boreoeutheria, Xenarthra and Afrotheria. Within the afrotherian LINE-1 (AfroLINE) clade, our tree supports Paenungulata, Afroinsectivora and Afroinsectiphillia. Xenarthran LINE-1 (XenaLINE) falls sister to AfroLINE, providing some support for the Atlantogenata (Xenarthra+Afrotheria) hypothesis. Significance LINEs and SINEs make up approximately half of all placental genomes, so understanding their dynamics is an essential aspect of comparative genomics. Importantly, a tree of LINE-1 offers a different view of the root, as long edges (branches) such as that to marsupials are shortened and/or broken up. Additionally, a robust phylogeny of diverse LINE-1 is essential in testing that site-specific LINE-1 insertions, often regarded as homoplasy-free phylogenetic markers, are indeed unique and not convergent. PMID:17225861

Global Metabolic Reconstruction and Metabolic Gene Evolution in the Cattle Genome

PubMed Central

Kim, Woonsu; Park, Hyesun; Seo, Seongwon

2016-01-01

The sequence of cattle genome provided a valuable opportunity to systematically link genetic and metabolic traits of cattle. The objectives of this study were 1) to reconstruct genome-scale cattle-specific metabolic pathways based on the most recent and updated cattle genome build and 2) to identify duplicated metabolic genes in the cattle genome for better understanding of metabolic adaptations in cattle. A bioinformatic pipeline of an organism for amalgamating genomic annotations from multiple sources was updated. Using this, an amalgamated cattle genome database based on UMD_3.1, was created. The amalgamated cattle genome database is composed of a total of 33,292 genes: 19,123 consensus genes between NCBI and Ensembl databases, 8,410 and 5,493 genes only found in NCBI or Ensembl, respectively, and 266 genes from NCBI scaffolds. A metabolic reconstruction of the cattle genome and cattle pathway genome database (PGDB) was also developed using Pathway Tools, followed by an intensive manual curation. The manual curation filled or revised 68 pathway holes, deleted 36 metabolic pathways, and added 23 metabolic pathways. Consequently, the curated cattle PGDB contains 304 metabolic pathways, 2,460 reactions including 2,371 enzymatic reactions, and 4,012 enzymes. Furthermore, this study identified eight duplicated genes in 12 metabolic pathways in the cattle genome compared to human and mouse. Some of these duplicated genes are related with specific hormone biosynthesis and detoxifications. The updated genome-scale metabolic reconstruction is a useful tool for understanding biology and metabolic characteristics in cattle. There has been significant improvements in the quality of cattle genome annotations and the MetaCyc database. The duplicated metabolic genes in the cattle genome compared to human and mouse implies evolutionary changes in the cattle genome and provides a useful information for further research on understanding metabolic adaptations of cattle. PMID:26992093

Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS)

PubMed Central

Matsuyama, Tomoki; Kimura, Makoto T.; Koike, Kuniaki; Abe, Tomoko; Nakano, Takeshi; Asami, Tadao; Ebisuzaki, Toshikazu; Held, William A.; Yoshida, Shigeo; Nagase, Hiroki

2003-01-01

Understanding the role of ‘epigenetic’ changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development. PMID:12888509

The uncharacterized gene 1700093K21Rik and flanking regions are correlated with reproductive isolation in the house mouse, Mus musculus.

PubMed

Kass, David H; Janoušek, Václav; Wang, Liuyang; Tucker, Priscilla K

2014-06-01

Reproductive barriers exist between the house mouse subspecies, Mus musculus musculus and M. m. domesticus, members of the Mus musculus species complex, primarily as a result of hybrid male infertility, and a hybrid zone exists where their ranges intersect in Europe. Using single nucleotide polymorphisms (SNPs) diagnostic for the two taxa, the extent of introgression across the genome was previously compared in these hybrid populations. Sixty-nine of 1316 autosomal SNPs exhibited reduced introgression in two hybrid zone transects suggesting maladaptive interactions among certain loci. One of these markers is within a region on chromosome 11 that, in other studies, has been associated with hybrid male sterility of these subspecies. We assessed sequence variation in a 20 Mb region on chromosome 11 flanking this marker, and observed its inclusion within a roughly 150 kb stretch of DNA showing elevated sequence differentiation between the two subspecies. Four genes are associated with this genomic subregion, with two entirely encompassed. One of the two genes, the uncharacterized 1700093K21Rik gene, displays distinguishing features consistent with a potential role in reproductive isolation between these subspecies. Along with its expression specifically within spermatogenic cells, we present various sequence analyses that demonstrate a high rate of molecular evolution of this gene, as well as identify a subspecies amino acid variant resulting in a structural difference. Taken together, the data suggest a role for this gene in reproductive isolation.

Assignment of the human PAX4 gene to chromosome band 7q32 by fluorescence in situ hybridization.

PubMed

Tamura, T; Izumikawa, Y; Kishino, T; Soejima, H; Jinno, Y; Niikawa, N

1994-01-01

Of the nine known members of a human paired box-containing gene family (Pax), only PAX4 has not been precisely localized. We screened a cosmid library of human genomic DNA using polymerase chain reaction products for PAX4 as a probe and isolated three positive cosmid clones. Sequence analysis revealed that at least two of them had exon-like sequences and showed extensive homology to Pax-4 in the mouse. These two cosmid clones were mapped to human chromosome band 7q32 by fluorescence in situ hybridization.

Harnessing Omics Big Data in Nine Vertebrate Species by Genome-Wide Prioritization of Sequence Variants with the Highest Predicted Deleterious Effect on Protein Function.

PubMed

Rozman, Vita; Kunej, Tanja

2018-05-10

Harnessing the genomics big data requires innovation in how we extract and interpret biologically relevant variants. Currently, there is no established catalog of prioritized missense variants associated with deleterious protein function phenotypes. We report in this study, to the best of our knowledge, the first genome-wide prioritization of sequence variants with the most deleterious effect on protein function (potentially deleterious variants [pDelVars]) in nine vertebrate species: human, cattle, horse, sheep, pig, dog, rat, mouse, and zebrafish. The analysis was conducted using the Ensembl/BioMart tool. Genes comprising pDelVars in the highest number of examined species were identified using a Python script. Multiple genomic alignments of the selected genes were built to identify interspecies orthologous potentially deleterious variants, which we defined as the "ortho-pDelVars." Genome-wide prioritization revealed that in humans, 0.12% of the known variants are predicted to be deleterious. In seven out of nine examined vertebrate species, the genes encoding the multiple PDZ domain crumbs cell polarity complex component (MPDZ) and the transforming acidic coiled-coil containing protein 2 (TACC2) comprise pDelVars. Five interspecies ortho-pDelVars were identified in three genes. These findings offer new ways to harness genomics big data by facilitating the identification of functional polymorphisms in humans and animal models and thus provide a future basis for optimization of protocols for whole genome prioritization of pDelVars and screening of orthologous sequence variants. The approach presented here can inform various postgenomic applications such as personalized medicine and multiomics study of health interventions (iatromics).

Comparative genomic analysis of the MHC: the evolution of class I duplication blocks, diversity and complexity from shark to man.

PubMed

Kulski, Jerzy K; Shiina, Takashi; Anzai, Tatsuya; Kohara, Sakae; Inoko, Hidetoshi

2002-12-01

The major histocompatibility complex (MHC) genomic region is composed of a group of linked genes involved functionally with the adaptive and innate immune systems. The class I and class II genes are intrinsic features of the MHC and have been found in all the jawed vertebrates studied so far. The MHC genomic regions of the human and the chicken (B locus) have been fully sequenced and mapped, and the mouse MHC sequence is almost finished. Information on the MHC genomic structures (size, complexity, genic and intergenic composition and organization, gene order and number) of other vertebrates is largely limited or nonexistent. Therefore, we are mapping, sequencing and analyzing the MHC genomic regions of different human haplotypes and at least eight nonhuman species. Here, we review our progress with these sequences and compare the human MHC structure with that of the nonhuman primates (chimpanzee and rhesus macaque), other mammals (pigs, mice and rats) and nonmammalian vertebrates such as birds (chicken and quail), bony fish (medaka, pufferfish and zebrafish) and cartilaginous fish (nurse shark). This comparison reveals a complex MHC structure for mammals and a relatively simpler design for nonmammalian animals with a hypothetical prototypic structure for the shark. In the mammalian MHC, there are two to five different class I duplication blocks embedded within a framework of conserved nonclass I and/or nonclass II genes. With a few exceptions, the class I framework genes are absent from the MHC of birds, bony fish and sharks. Comparative genomics of the MHC reveal a highly plastic region with major structural differences between the mammalian and nonmammalian vertebrates. Additional genomic data are needed on animals of the reptilia, crocodilia and marsupial classes to find the origins of the class I framework genes and examples of structures that may be intermediate between the simple and complex MHC organizations of birds and mammals, respectively.

Genomic structure, promoter identification, and chromosomal mapping of a mouse nuclear orphan receptor expressed in embryos and adult testes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, C.H.; Wei, Li-Na; Copeland, N.G.

We have isolated and characterized overlapping genomic clones containing the complete transcribed region of a newly isolated mouse cDNA encoding an orphan receptor expressed specifically in midgestation embryos and adult testis. This gene spans a distance of more than 50 kb and is organized into 13 exons. The transcription initiation site is located at the 158th nucleotide upstream from the translation initiation codon. All the exon/intron junction sequences follow the GT/AG rule. Based upon Northern blot analysis and the size of the transcribed region of the gene, its transcript was determined to be approximately 2.5 kb. Within approximately 500 hpmore » upstream from the transcription initiation site, several immune response regulatory elements were identified but no TATA box was located. This gene was mapped to the distal region of mouse chromosome 10 and its locus has been designated Tr2-11. Immunohistochemical studies show that the Tr2-11 protein is present mainly in advanced germ cell populations of mature testes and that Tr2-11 gene expression is dramatically decreased in vitamin A-depleted animals. 23 refs., 7 figs.« less

Selective Constraints on Coding Sequences of Nervous System Genes Are a Major Determinant of Duplicate Gene Retention in Vertebrates

PubMed Central

Roux, Julien; Liu, Jialin; Robinson-Rechavi, Marc

2017-01-01

Abstract The evolutionary history of vertebrates is marked by three ancient whole-genome duplications: two successive rounds in the ancestor of vertebrates, and a third one specific to teleost fishes. Biased loss of most duplicates enriched the genome for specific genes, such as slow evolving genes, but this selective retention process is not well understood. To understand what drives the long-term preservation of duplicate genes, we characterized duplicated genes in terms of their expression patterns. We used a new method of expression enrichment analysis, TopAnat, applied to in situ hybridization data from thousands of genes from zebrafish and mouse. We showed that the presence of expression in the nervous system is a good predictor of a higher rate of retention of duplicate genes after whole-genome duplication. Further analyses suggest that purifying selection against the toxic effects of misfolded or misinteracting proteins, which is particularly strong in nonrenewing neural tissues, likely constrains the evolution of coding sequences of nervous system genes, leading indirectly to the preservation of duplicate genes after whole-genome duplication. Whole-genome duplications thus greatly contributed to the expansion of the toolkit of genes available for the evolution of profound novelties of the nervous system at the base of the vertebrate radiation. PMID:28981708

GANESH: software for customized annotation of genome regions.

PubMed

Huntley, Derek; Hummerich, Holger; Smedley, Damian; Kittivoravitkul, Sasivimol; McCarthy, Mark; Little, Peter; Sergot, Marek

2003-09-01

GANESH is a software package designed to support the genetic analysis of regions of human and other genomes. It provides a set of components that may be assembled to construct a self-updating database of DNA sequence, mapping data, and annotations of possible genome features. Once one or more remote sources of data for the target region have been identified, all sequences for that region are downloaded, assimilated, and subjected to a (configurable) set of standard database-searching and genome-analysis packages. The results are stored in compressed form in a relational database, and are updated automatically on a regular schedule so that they are always immediately available in their most up-to-date versions. A Java front-end, executed as a stand alone application or web applet, provides a graphical interface for navigating the database and for viewing the annotations. There are facilities for importing and exporting data in the format of the Distributed Annotation System (DAS), enabling a GANESH database to be used as a component of a DAS configuration. The system has been used to construct databases for about a dozen regions of human chromosomes and for three regions of mouse chromosomes.

Influence of radiation quality on mouse chromosome 2 deletions in radiation-induced acute myeloid leukaemia.

PubMed

Brown, Natalie; Finnon, Rosemary; Manning, Grainne; Bouffler, Simon; Badie, Christophe

2015-11-01

Leukaemia is the prevailing neoplastic disorder of the hematopoietic system. Epidemiological analyses of the survivors of the Japanese atomic bombings show that exposure to ionising radiation (IR) can cause leukaemia. Although a clear association between radiation exposure and leukaemia development is acknowledged, the underlying mechanisms remain incompletely understood. A hemizygous deletion on mouse chromosome 2 (del2) is a common feature in several mouse strains susceptible to radiation-induced acute myeloid leukaemia (rAML). The deletion is an early event detectable 24h after exposure in bone marrow cells. Ultimately, 15-25% of exposed animals develop AML with 80-90% of cases carrying del2. Molecular mapping of leukaemic cell genomes identified a minimal deleted region (MDR) on chromosome 2 (chr2) in which a tumour suppressor gene, Sfpi1 is located, encoding the transcription factor PU.1, essential in haematopoiesis. The remaining copy of Sfpi1 has a point mutation in the coding sequence for the DNA-binding domain of the protein in 70% of rAML, which alters a single CpG sequence in the codon for arginine residue R235. In order to identify chr2 deletions and Sfpi.1/PU.1 loss, we performed array comparative genomic hybridization (aCGH) on a unique panel of 79rAMLs. Using a custom made CGH array specifically designed for mouse chr2, we analysed at unprecedentedly high resolution (1.4M array- 148bp resolution) the size of the MDR in low LET and high-LET induced rAMLs (32 X-ray- and 47 neutron-induced). Sequencing of Sfpi1/PU.1DNA binding domain identified the presence of R235 point mutations, showing no influence of radiation quality on R235 type or frequency. We identified for the first time rAML cases with complex del2 in a subset of neutron-induced AMLs. This study allowed us to re-define the MDR to a much smaller 5.5Mb region (still including Sfpi1/PU.1), identical regardless of radiation quality. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

A novel lens epithelium gene, LEP503, is highly conserved in different vertebrate species and is developmentally regulated in postnatal rat lens.

PubMed

Wen, Y; Sachs, G; Athmann, C

2000-02-01

The development of the lens is dependent on the proliferation of lens epithelial cells and their differentiation into fiber cells near the lens bow/equator. Identification of genes specifically expressed in the lens epithelial cells and their functions may provide insight into molecular events that regulate the processes of lens epithelial cell differentiation. In this study, a novel lens epithelium gene product, LEP503, identified from rat by a subtractive cDNA cloning strategy was investigated in the genome organization, mRNA expression and protein localization. The genomic sequences for LEP503 isolated from rat, mouse and human span 1754 bp, 1694 bp and 1895 bp regions encompassing the 5'-flanking region, two exons, one intron and 3'-flanking region. All exon-intron junction sequences conform to the GT/AG rule. Both mouse and human LEP503 genes show very high identity (93% for mouse and 79% for human) to rat LEP503 gene in the exon 1 that contains an open reading frame coding for a protein of 61 amino acid residues with a leucine-rich domain. The deduced protein sequences also show high identity (91% between mouse and rat and 77% between human and rat). Western blot shows that LEP503 is present as a specific approximately 6.9 kDa band in the water-insoluble-urea-soluble fraction of lens cortex where lens epithelium is included. Immuno-staining shows that LEP503 is localized in the epithelial cells along the entire anterior surface of rat lens. Developmentally, LEP503 is expressed at a low level at newborn, and then the expression level increases by about ten-fold around postnatal day 14 and remains at this high level for about 25 days before it drops back to the low level by postnatal day 84. These data suggest that the LEP503 may be an important lens epithelial cell gene involving the processes of epithelial cell differentiation. Copyright 2000 Academic Press.

Knock-in reporter mice demonstrate that DNA repair by non-homologous end joining declines with age.

PubMed

Vaidya, Amita; Mao, Zhiyong; Tian, Xiao; Spencer, Brianna; Seluanov, Andrei; Gorbunova, Vera

2014-07-01

Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs), we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ) pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ) events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age.

Comparative Genomic Analysis of slc39a12/ZIP12: Insight into a Zinc Transporter Required for Vertebrate Nervous System Development

PubMed Central

Chowanadisai, Winyoo

2014-01-01

The zinc transporter ZIP12, which is encoded by the gene slc39a12, has previously been shown to be important for neuronal differentiation in mouse Neuro-2a neuroblastoma cells and primary mouse neurons and necessary for neurulation during Xenopus tropicalis embryogenesis. However, relatively little is known about the biochemical properties, cellular regulation, or the physiological role of this gene. The hypothesis that ZIP12 is a zinc transporter important for nervous system function and development guided a comparative genetics approach to uncover the presence of ZIP12 in various genomes and identify conserved sequences and expression patterns associated with ZIP12. Ortholog detection of slc39a12 was conducted with reciprocal BLAST hits with the amino acid sequence of human ZIP12 in comparison to the human paralog ZIP4 and conserved local synteny between genomes. ZIP12 is present in the genomes of almost all vertebrates examined, from humans and other mammals to most teleost fish. However, ZIP12 appears to be absent from the zebrafish genome. The discrimination of ZIP12 compared to ZIP4 was unsuccessful or inconclusive in other invertebrate chordates and deuterostomes. Splice variation, due to the inclusion or exclusion of a conserved exon, is present in humans, rats, and cows and likely has biological significance. ZIP12 also possesses many putative di-leucine and tyrosine motifs often associated with intracellular trafficking, which may control cellular zinc uptake activity through the localization of ZIP12 within the cell. These findings highlight multiple aspects of ZIP12 at the biochemical, cellular, and physiological levels with likely biological significance. ZIP12 appears to have conserved function as a zinc uptake transporter in vertebrate nervous system development. Consequently, the role of ZIP12 may be an important link to reported congenital malformations in numerous animal models and humans that are caused by zinc deficiency. PMID:25375179

Comparative Genomic Sequencing and Pathogenic Properties of Equine Herpesvirus 1 KyA and RacL11

PubMed Central

Shakya, Akhalesh K.; O’Callaghan, Dennis J.; Kim, Seong K.

2017-01-01

Equine herpesvirus 1 (EHV-1) is a major pathogen affecting equines worldwide. The virus causes respiratory disease, abortion, and, in some cases, neurological disease. EHV-1 Kentucky A (KyA) is attenuated in the mouse and equine, whereas wild-type pathogenic strain RacL11 induces severe inflammatory infiltration of the lung, causing infected mice to succumb. The complete DNA sequencing of the KyA genome revealed that genes UL17 (ORF17), US6 (ORF73; gI), US7 (ORF74; gE), and US8 (ORF75; 10 K) are deleted as compared to the RacL11 and Ab4 genomes. In-frame deletions in the US1 (ORF68), US4 (ORF71; gp2), and UL63 (ORF63; EICP0) genes and point mutations in 14 different open reading frames (ORFs) were detected in the KyA genome. Interestingly, UL1 (ORF1) and UL2 (ORF2) were deleted in both KyA and RacL11. Our previous studies showed that EHV-1 glycoproteins gI, gE, and full-length gp2 contribute to the pathogenesis of the RacL11 strain. The confirmation of these gene deletions in KyA suggests their contribution to the attenuation of this virus. The growth kinetics results revealed that KyA replicates to high titers in cell culture as compared to RacL11 and Ab4, indicating that the above genomic deletions and mutations in KyA do not have an inhibitory effect on KyA replication in cells of mouse, rabbit, equine, or human origin. Studies of EHV-1 pathogenesis in CBA mice showed that KyA is attenuated whereas mice infected with RacL11 succumbed by 3–6 days post-infection, which is consistent with our previous results. PMID:29312962

The roles of gene duplication, gene conversion and positive selection in rodent Esp and Mup pheromone gene families with comparison to the Abp family.

PubMed

Karn, Robert C; Laukaitis, Christina M

2012-01-01

Three proteinaceous pheromone families, the androgen-binding proteins (ABPs), the exocrine-gland secreting peptides (ESPs) and the major urinary proteins (MUPs) are encoded by large gene families in the genomes of Mus musculus and Rattus norvegicus. We studied the evolutionary histories of the Mup and Esp genes and compared them with what is known about the Abp genes. Apparently gene conversion has played little if any role in the expansion of the mouse Class A and Class B Mup genes and pseudogenes, and the rat Mups. By contrast, we found evidence of extensive gene conversion in many Esp genes although not in all of them. Our studies of selection identified at least two amino acid sites in β-sheets as having evolved under positive selection in the mouse Class A and Class B MUPs and in rat MUPs. We show that selection may have acted on the ESPs by determining K(a)/K(s) for Exon 3 sequences with and without the converted sequence segment. While it appears that purifying selection acted on the ESP signal peptides, the secreted portions of the ESPs probably have undergone much more rapid evolution. When the inner gene converted fragment sequences were removed, eleven Esp paralogs were present in two or more pairs with K(a)/K(s) >1.0 and thus we propose that positive selection is detectable by this means in at least some mouse Esp paralogs. We compare and contrast the evolutionary histories of all three mouse pheromone gene families in light of their proposed functions in mouse communication.

ALEA: a toolbox for allele-specific epigenomics analysis.

PubMed

Younesy, Hamid; Möller, Torsten; Heravi-Moussavi, Alireza; Cheng, Jeffrey B; Costello, Joseph F; Lorincz, Matthew C; Karimi, Mohammad M; Jones, Steven J M

2014-04-15

The assessment of expression and epigenomic status using sequencing based methods provides an unprecedented opportunity to identify and correlate allelic differences with epigenomic status. We present ALEA, a computational toolbox for allele-specific epigenomics analysis, which incorporates allelic variation data within existing resources, allowing for the identification of significant associations between epigenetic modifications and specific allelic variants in human and mouse cells. ALEA provides a customizable pipeline of command line tools for allele-specific analysis of next-generation sequencing data (ChIP-seq, RNA-seq, etc.) that takes the raw sequencing data and produces separate allelic tracks ready to be viewed on genome browsers. The pipeline has been validated using human and hybrid mouse ChIP-seq and RNA-seq data. The package, test data and usage instructions are available online at http://www.bcgsc.ca/platform/bioinfo/software/alea CONTACT: : mkarimi1@interchange.ubc.ca or sjones@bcgsc.ca Supplementary information: Supplementary data are available at Bioinformatics online. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genomic identification and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion.

PubMed Central

Vujcic, Slavoljub; Liang, Ping; Diegelman, Paula; Kramer, Debora L; Porter, Carl W

2003-01-01

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1 -acetyltransferase (SSAT) and then oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine respectively. Although PAO was first purified more than two decades ago, the protein has not yet been linked to genomic sequences. In the present study, we apply a BLAST search strategy to identify novel oxidase sequences located on human chromosome 10 and mouse chromosome 7. Homologous mammalian cDNAs derived from human brain and mouse mammary tumour were deduced to encode proteins of approx. 55 kDa having 82% sequence identity. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by approx. 30%, whereas spermidine increased 2-4-fold. Lysates of human PAO cDNA-transfected HEK-293 cells, but not vector-transfected cells, rapidly oxidized N1-acetylspermine to spermidine. Substrate specificity determinations with the lysate assay revealed a preference ranking of N1-acetylspermine= N1-acetylspermidine> N1,N12-diacetylspermine>>spermine; spermidine was not acted upon. This ranking is identical to that reported for purified PAO and distinctly different from the recently identified spermine oxidase (SMO), which prefers spermine over N1-acetylspermine. Monoethyl- and diethylspermine analogues also served as substrates for PAO, and were internally cleaved adjacent to a secondary amine. We deduce that the present oxidase sequences are those of the FAD-dependent PAO involved in the polyamine back-conversion pathway. In Northern blot analysis, PAO mRNA was much less abundant in HEK-293 cells than SMO or SSAT mRNA, and all three were differentially induced in a similar manner by selected polyamine analogues. The identification of PAO sequences, together with the recently identified SMO sequences, provides new opportunities for understanding the dynamics of polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically-relevant polyamine analogues and inhibitors. PMID:12477380

Molecular cloning of a gene encoding translation initiation factor (TIF) from Candida albicans.

PubMed

Mirbod, F; Nakashima, S; Kitajima, Y; Ghannoum, M A; Cannon, R D; Nozawa, Y

1996-01-01

The differential display technique was applied to compare mRNAs from two clinical isolates of Candida albicans with different virulence; high (potent strain, 16240) and low (weak strain, 18084) extracellular phospholipase activities. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. A complementary DNA fragment seen distinctly in the potent phospholipase producing strain was highly homologous to the yeast translation initiation factor (TIF). The selected DNA fragment was then used as a probe to isolate its corresponding complementary DNA clone from a library of C. albicans genomic DNA. The sequence of isolated gene revealed an open reading frame of 1194 nucleotides with the potential to encode a protein of 397 amino acids with a predicted molecular weight of 43 kDa. Over its entire length, the amino acid sequence showed strong homology (78-89%) to Saccharomyces cerevisiae TIF and (63-80%) to mouse eIF-4A proteins. Therefore, our C. albicans gene was identified to be TIF (Ca TIF). Northern blot analysis in the two strains of C. albicans revealed that Ca TIF expression is 1.5-fold higher in the potent phospholipase producing strain. The restriction endonuclease digestion of genomic DNA from this potent strain revealed at least two hybridized bands in Southern blot analysis, suggesting two or more closely related sequences in the C. albicans genome.

SPAR: small RNA-seq portal for analysis of sequencing experiments.

PubMed

Kuksa, Pavel P; Amlie-Wolf, Alexandre; Katanic, Živadin; Valladares, Otto; Wang, Li-San; Leung, Yuk Yee

2018-05-04

The introduction of new high-throughput small RNA sequencing protocols that generate large-scale genomics datasets along with increasing evidence of the significant regulatory roles of small non-coding RNAs (sncRNAs) have highlighted the urgent need for tools to analyze and interpret large amounts of small RNA sequencing data. However, it remains challenging to systematically and comprehensively discover and characterize sncRNA genes and specifically-processed sncRNA products from these datasets. To fill this gap, we present Small RNA-seq Portal for Analysis of sequencing expeRiments (SPAR), a user-friendly web server for interactive processing, analysis, annotation and visualization of small RNA sequencing data. SPAR supports sequencing data generated from various experimental protocols, including smRNA-seq, short total RNA sequencing, microRNA-seq, and single-cell small RNA-seq. Additionally, SPAR includes publicly available reference sncRNA datasets from our DASHR database and from ENCODE across 185 human tissues and cell types to produce highly informative small RNA annotations across all major small RNA types and other features such as co-localization with various genomic features, precursor transcript cleavage patterns, and conservation. SPAR allows the user to compare the input experiment against reference ENCODE/DASHR datasets. SPAR currently supports analyses of human (hg19, hg38) and mouse (mm10) sequencing data. SPAR is freely available at https://www.lisanwanglab.org/SPAR.

DNA sequence responsible for the amplification of adjacent genes.

PubMed

Pasion, S G; Hartigan, J A; Kumar, V; Biswas, D K

1987-10-01

A 10.3-kb DNA fragment in the 5'-flanking region of the rat prolactin (rPRL) gene was isolated from F1BGH(1)2C1, a strain of rat pituitary tumor cells (GH cells) that produces prolactin in response to 5-bromodeoxyuridine (BrdU). Following transfection and integration into genomic DNA of recipient mouse L cells, this DNA induced amplification of the adjacent thymidine kinase gene from Herpes simplex virus type 1 (HSV1TK). We confirmed the ability of this "Amplicon" sequence to induce amplification of other linked or unlinked genes in DNA-mediated gene transfer studies. When transferred into the mouse L cells with the 10.3-5'rPRL gene sequence of BrdU-responsive cells, both the human growth hormone and the HSV1TK genes are amplified in response to 5-bromodeoxyuridine. This observation is substantiated by BrdU-induced amplification of the cotransferred bacterial Neo gene. Cotransfection studies reveal that the BrdU-induced amplification capability is associated with a 4-kb DNA sequence in the 5'-flanking region of the rPRL gene of BrdU-responsive cells. These results demonstrate that genes of heterologous origin, linked or unlinked, and selected or unselected, can be coamplified when located within the amplification boundary of the Amplicon sequence.

The evolutionary capacitor HSP90 buffers the regulatory effects of mammalian endogenous retroviruses.

PubMed

Hummel, Barbara; Hansen, Erik C; Yoveva, Aneliya; Aprile-Garcia, Fernando; Hussong, Rebecca; Sawarkar, Ritwick

2017-03-01

Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.

BGDB: a database of bivalent genes.

PubMed

Li, Qingyan; Lian, Shuabin; Dai, Zhiming; Xiang, Qian; Dai, Xianhua

2013-01-01

Bivalent gene is a gene marked with both H3K4me3 and H3K27me3 epigenetic modification in the same area, and is proposed to play a pivotal role related to pluripotency in embryonic stem (ES) cells. Identification of these bivalent genes and understanding their functions are important for further research of lineage specification and embryo development. So far, lots of genome-wide histone modification data were generated in mouse and human ES cells. These valuable data make it possible to identify bivalent genes, but no comprehensive data repositories or analysis tools are available for bivalent genes currently. In this work, we develop BGDB, the database of bivalent genes. The database contains 6897 bivalent genes in human and mouse ES cells, which are manually collected from scientific literature. Each entry contains curated information, including genomic context, sequences, gene ontology and other relevant information. The web services of BGDB database were implemented with PHP + MySQL + JavaScript, and provide diverse query functions. Database URL: http://dailab.sysu.edu.cn/bgdb/

Cloning-free CRISPR

PubMed Central

Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

2015-01-01

Summary We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. PMID:26527385

Simultaneous gene finding in multiple genomes.

PubMed

König, Stefanie; Romoth, Lars W; Gerischer, Lizzy; Stanke, Mario

2016-11-15

As the tree of life is populated with sequenced genomes ever more densely, the new challenge is the accurate and consistent annotation of entire clades of genomes. We address this problem with a new approach to comparative gene finding that takes a multiple genome alignment of closely related species and simultaneously predicts the location and structure of protein-coding genes in all input genomes, thereby exploiting negative selection and sequence conservation. The model prefers potential gene structures in the different genomes that are in agreement with each other, or-if not-where the exon gains and losses are plausible given the species tree. We formulate the multi-species gene finding problem as a binary labeling problem on a graph. The resulting optimization problem is NP hard, but can be efficiently approximated using a subgradient-based dual decomposition approach. The proposed method was tested on whole-genome alignments of 12 vertebrate and 12 Drosophila species. The accuracy was evaluated for human, mouse and Drosophila melanogaster and compared to competing methods. Results suggest that our method is well-suited for annotation of (a large number of) genomes of closely related species within a clade, in particular, when RNA-Seq data are available for many of the genomes. The transfer of existing annotations from one genome to another via the genome alignment is more accurate than previous approaches that are based on protein-spliced alignments, when the genomes are at close to medium distances. The method is implemented in C ++ as part of Augustus and available open source at http://bioinf.uni-greifswald.de/augustus/ CONTACT: stefaniekoenig@ymail.com or mario.stanke@uni-greifswald.deSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells

PubMed Central

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-01-01

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics. PMID:25762467

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells.

PubMed

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-03-12

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics.

CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation.

PubMed

Miki, Daisuke; Zhang, Wenxin; Zeng, Wenjie; Feng, Zhengyan; Zhu, Jian-Kang

2018-05-17

Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.

RatMap--rat genome tools and data.

PubMed

Petersen, Greta; Johnson, Per; Andersson, Lars; Klinga-Levan, Karin; Gómez-Fabre, Pedro M; Ståhl, Fredrik

2005-01-01

The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB-Genetics at Goteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided.

RatMap—rat genome tools and data

PubMed Central

Petersen, Greta; Johnson, Per; Andersson, Lars; Klinga-Levan, Karin; Gómez-Fabre, Pedro M.; Ståhl, Fredrik

2005-01-01

The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB–Genetics at Göteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided. PMID:15608244

PGMapper: a web-based tool linking phenotype to genes.

PubMed

Xiong, Qing; Qiu, Yuhui; Gu, Weikuan

2008-04-01

With the availability of whole genome sequence in many species, linkage analysis, positional cloning and microarray are gradually becoming powerful tools for investigating the links between phenotype and genotype or genes. However, in these methods, causative genes underlying a quantitative trait locus, or a disease, are usually located within a large genomic region or a large set of genes. Examining the function of every gene is very time consuming and needs to retrieve and integrate the information from multiple databases or genome resources. PGMapper is a software tool for automatically matching phenotype to genes from a defined genome region or a group of given genes by combining the mapping information from the Ensembl database and gene function information from the OMIM and PubMed databases. PGMapper is currently available for candidate gene search of human, mouse, rat, zebrafish and 12 other species. Available online at http://www.genediscovery.org/pgmapper/index.jsp.

Developmental history and application of CRISPR in human disease.

PubMed

Liang, Puping; Zhang, Xiya; Chen, Yuxi; Huang, Junjiu

2017-06-01

Genome-editing tools are programmable artificial nucleases, mainly including zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeat (CRISPR). By recognizing and cleaving specific DNA sequences, genome-editing tools make it possible to generate site-specific DNA double-strand breaks (DSBs) in the genome. DSBs will then be repaired by either error-prone nonhomologous end joining or high-fidelity homologous recombination mechanisms. Through these two different mechanisms, endogenous genes can be knocked out or precisely repaired/modified. Rapid developments in genome-editing tools, especially CRISPR, have revolutionized human disease models generation, for example, various zebrafish, mouse, rat, pig, monkey and human cell lines have been constructed. Here, we review the developmental history of CRISPR and its application in studies of human diseases. In addition, we also briefly discussed the therapeutic application of CRISPR in the near future. Copyright © 2017 John Wiley & Sons, Ltd.

Chromosomal localization and partial genomic structure of the human peroxisome proliferator activated receptor-gamma (hPPAR gamma) gene.

PubMed

Beamer, B A; Negri, C; Yen, C J; Gavrilova, O; Rumberger, J M; Durcan, M J; Yarnall, D P; Hawkins, A L; Griffin, C A; Burns, D K; Roth, J; Reitman, M; Shuldiner, A R

1997-04-28

We determined the chromosomal localization and partial genomic structure of the coding region of the human PPAR gamma gene (hPPAR gamma), a nuclear receptor important for adipocyte differentiation and function. Sequence analysis and long PCR of human genomic DNA with primers that span putative introns revealed that intron positions and sizes of hPPAR gamma are similar to those previously determined for the mouse PPAR gamma gene[13]. Fluorescent in situ hybridization localized hPPAR gamma to chromosome 3, band 3p25. Radiation hybrid mapping with two independent primer pairs was consistent with hPPAR gamma being within 1.5 Mb of marker D3S1263 on 3p25-p24.2. These sequences of the intron/exon junctions of the 6 coding exons shared by hPPAR gamma 1 and hPPAR gamma 2 will facilitate screening for possible mutations. Furthermore, D3S1263 is a suitable polymorphic marker for linkage analysis to evaluate PPAR gamma's potential contribution to genetic susceptibility to obesity, lipoatrophy, insulin resistance, and diabetes.

CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening.

PubMed

Köferle, Anna; Worf, Karolina; Breunig, Christopher; Baumann, Valentin; Herrero, Javier; Wiesbeck, Maximilian; Hutter, Lukas H; Götz, Magdalena; Fuchs, Christiane; Beck, Stephan; Stricker, Stefan H

2016-11-14

The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs. The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.

The evolution of the search for novel genes in mammalian sex determination: from mice to men.

PubMed

Arboleda, Valerie A; Vilain, Eric

2011-01-01

Disorders of sex determination are a genetically heterogeneous group of rare disorders, presenting with sex-specific phenotypes and variable expressivity. Prior to the advent of the Human Genome Project, the identification of novel mammalian sex determination genes was hindered by the rarity of disorders of sex determination and small family sizes that made traditional linkage approaches difficult, if not impossible. This article reviews the revolutionary role of the Human Genome Project in the history of sex determination research and highlights the important role of inbred mouse models in elucidating the role of identified sex determination genes in mammalian sex determination. Next generation sequencing technologies has made it possible to sequence complete human genomes or exomes for the purpose of providing a genetic diagnosis to more patients with unexplained disorders of sex determination and identifying novel sex determination genes. However, beyond novel gene discovery, these tools have the power to inform us on more intricate and complex regulation-taking place within the heterogeneous cells that make up the testis and ovary. Copyright © 2011 Elsevier Inc. All rights reserved.

Database resources of the National Center for Biotechnology Information

PubMed Central

Wheeler, David L.; Church, Deanna M.; Lash, Alex E.; Leipe, Detlef D.; Madden, Thomas L.; Pontius, Joan U.; Schuler, Gregory D.; Schriml, Lynn M.; Tatusova, Tatiana A.; Wagner, Lukas; Rapp, Barbara A.

2001-01-01

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI’s Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, GeneMap’99, Human–Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP), SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheri­tance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov. PMID:11125038

Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study.

PubMed

Ji, Xinglai; Tang, Jie; Halberg, Richard; Busam, Dana; Ferriera, Steve; Peña, Maria Marjorette O; Venkataramu, Chinnambally; Yeatman, Timothy J; Zhao, Shaying

2010-08-13

We are developing a cross-species comparison strategy to distinguish between cancer driver- and passenger gene alteration candidates, by utilizing the difference in genomic location of orthologous genes between the human and other mammals. As an initial test of this strategy, we conducted a pilot study with human colorectal cancer (CRC) and its mouse model C57BL/6J ApcMin/+, focusing on human 5q22.2 and 18q21.1-q21.2. We first performed bioinformatics analysis on the evolution of 5q22.2 and 18q21.1-q21.2 regions. Then, we performed exon-targeted sequencing, real time quantitative polymerase chain reaction (qPCR), and real time quantitative reverse transcriptase PCR (qRT-PCR) analyses on a number of genes of both regions with both human and mouse colon tumors. These two regions (5q22.2 and 18q21.1-q21.2) are frequently deleted in human CRCs and encode genuine colorectal tumor suppressors APC and SMAD4. They also encode genes such as MCC (mutated in colorectal cancer) with their role in CRC etiology unknown. We have discovered that both regions are evolutionarily unstable, resulting in genes that are clustered in each human region being found scattered at several distinct loci in the genome of many other species. For instance, APC and MCC are within 200 kb apart in human 5q22.2 but are 10 Mb apart in the mouse genome. Importantly, our analyses revealed that, while known CRC driver genes APC and SMAD4 were disrupted in both human colorectal tumors and tumors from ApcMin/+ mice, the questionable MCC gene was disrupted in human tumors but appeared to be intact in mouse tumors. These results indicate that MCC may not actually play any causative role in early colorectal tumorigenesis. We also hypothesize that its disruption in human CRCs is likely a mere result of its close proximity to APC in the human genome. Expanding this pilot study to the entire genome may identify more questionable genes like MCC, facilitating the discovery of new CRC driver gene candidates.

Estimation of pairwise sequence similarity of mammalian enhancers with word neighbourhood counts.

PubMed

Göke, Jonathan; Schulz, Marcel H; Lasserre, Julia; Vingron, Martin

2012-03-01

The identity of cells and tissues is to a large degree governed by transcriptional regulation. A major part is accomplished by the combinatorial binding of transcription factors at regulatory sequences, such as enhancers. Even though binding of transcription factors is sequence-specific, estimating the sequence similarity of two functionally similar enhancers is very difficult. However, a similarity measure for regulatory sequences is crucial to detect and understand functional similarities between two enhancers and will facilitate large-scale analyses like clustering, prediction and classification of genome-wide datasets. We present the standardized alignment-free sequence similarity measure N2, a flexible framework that is defined for word neighbourhoods. We explore the usefulness of adding reverse complement words as well as words including mismatches into the neighbourhood. On simulated enhancer sequences as well as functional enhancers in mouse development, N2 is shown to outperform previous alignment-free measures. N2 is flexible, faster than competing methods and less susceptible to single sequence noise and the occurrence of repetitive sequences. Experiments on the mouse enhancers reveal that enhancers active in different tissues can be separated by pairwise comparison using N2. N2 represents an improvement over previous alignment-free similarity measures without compromising speed, which makes it a good candidate for large-scale sequence comparison of regulatory sequences. The software is part of the open-source C++ library SeqAn (www.seqan.de) and a compiled version can be downloaded at http://www.seqan.de/projects/alf.html. Supplementary data are available at Bioinformatics online.

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

PubMed Central

Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

2013-01-01

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

PubMed

Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin

2017-03-01

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

Global survey of genomic imprinting by transcriptome sequencing.

PubMed

Babak, Tomas; Deveale, Brian; Armour, Christopher; Raymond, Christopher; Cleary, Michele A; van der Kooy, Derek; Johnson, Jason M; Lim, Lee P

2008-11-25

Genomic imprinting restricts gene expression to a paternal or maternal allele. To date, approximately 90 imprinted transcripts have been identified in mouse, of which the majority were detected after intense interrogation of clusters of imprinted genes identified by phenotype-driven assays in mice with uniparental disomies [1]. Here we use selective priming and parallel sequencing to measure allelic bias in whole transcriptomes. By distinguishing parent-of-origin bias from strain-specific bias in embryos derived from a reciprocal cross of mice, we constructed a genome-wide map of imprinted transcription. This map was able to objectively locate over 80% of known imprinted loci and allowed the detection and confirmation of six novel imprinted genes. Even in the intensely studied embryonic day 9.5 developmental stage that we analyzed, more than half of all imprinted single-nucleotide polymorphisms did not overlap previously discovered imprinted transcripts; a large fraction of these represent novel noncoding RNAs within known imprinted loci. For example, a previously unnoticed, maternally expressed antisense transcript was mapped within the Grb10 locus. This study demonstrates the feasibility of using transcriptome sequencing for mapping of imprinted gene expression in physiologically normal animals. Such an approach will allow researchers to study imprinting without restricting themselves to individual loci or specific transcripts.

Extraordinary Sequence Divergence at Tsga8, an X-linked Gene Involved in Mouse Spermiogenesis

PubMed Central

Good, Jeffrey M.; Vanderpool, Dan; Smith, Kimberly L.; Nachman, Michael W.

2011-01-01

The X chromosome plays an important role in both adaptive evolution and speciation. We used a molecular evolutionary screen of X-linked genes potentially involved in reproductive isolation in mice to identify putative targets of recurrent positive selection. We then sequenced five very rapidly evolving genes within and between several closely related species of mice in the genus Mus. All five genes were involved in male reproduction and four of the genes showed evidence of recurrent positive selection. The most remarkable evolutionary patterns were found at Testis-specific gene a8 (Tsga8), a spermatogenesis-specific gene expressed during postmeiotic chromatin condensation and nuclear transformation. Tsga8 was characterized by extremely high levels of insertion–deletion variation of an alanine-rich repetitive motif in natural populations of Mus domesticus and M. musculus, differing in length from the reference mouse genome by up to 89 amino acids (27% of the total protein length). This population-level variation was coupled with striking divergence in protein sequence and length between closely related mouse species. Although no clear orthologs had previously been described for Tsga8 in other mammalian species, we have identified a highly divergent hypothetical gene on the rat X chromosome that shares clear orthology with the 5′ and 3′ ends of Tsga8. Further inspection of this ortholog verified that it is expressed in rat testis and shares remarkable similarity with mouse Tsga8 across several general features of the protein sequence despite no conservation of nucleotide sequence across over 60% of the rat-coding domain. Overall, Tsga8 appears to be one of the most rapidly evolving genes to have been described in rodents. We discuss the potential evolutionary causes and functional implications of this extraordinary divergence and the possible contribution of Tsga8 and the other four genes we examined to reproductive isolation in mice. PMID:21186189

Microarray Analysis of LTR Retrotransposon Silencing Identifies Hdac1 as a Regulator of Retrotransposon Expression in Mouse Embryonic Stem Cells

PubMed Central

Madej, Monika J.; Taggart, Mary; Gautier, Philippe; Garcia-Perez, Jose Luis; Meehan, Richard R.; Adams, Ian R.

2012-01-01

Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells. PMID:22570599

REDIdb: the RNA editing database.

PubMed

Picardi, Ernesto; Regina, Teresa Maria Rosaria; Brennicke, Axel; Quagliariello, Carla

2007-01-01

The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at http://biologia.unical.it/py_script/search.html.

Genetic recombination is directed away from functional genomic elements in mice.

PubMed

Brick, Kevin; Smagulova, Fatima; Khil, Pavel; Camerini-Otero, R Daniel; Petukhova, Galina V

2012-05-13

Genetic recombination occurs during meiosis, the key developmental programme of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals. PRDM9 is thought to determine the preferred recombination sites--recombination hotspots--through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyse the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F(1) hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the exception of the pseudo-autosomal region (PAR)--the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice, and as in wild type, these hotspots are found at H3 lysine 4 (H3K4) trimethylation marks. However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice, indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene-promoter regions and other functional genomic elements.

Human HST1 (HSTF1) gene maps to chromosome band 11q13 and coamplifies with the INT2 gene in human cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Michihiro C.; Wada, Makio; Satoh, Hitoshi

1988-07-01

The human HST1 gene, previously designated the hst gene, and now assigned the name HSTF1 for heparin-binding secretory transforming factor in human gene nomenclature, was originally identified as a transforming gene in DNAs from human stomach cancers by transfection assay with mouse NIH 3T3 cells. The amino acid sequence of the product deduced from DNA sequences of the HST1 cDNA and genomic clones had approximately 40% homology to human basic and acidic fibroblast growth factors and mouse Int-2-encoded protein. The authors have mapped the human HST1 gene to chromosome 11 at band q13.3 by Southern blot hybridization analysis of amore » panel of human and mouse somatic cell hybrids and in situ hybridization with an HST1 cDNA probe. The HST1 gene was found to be amplified in DNAs obtained from a stomach cancer and a vulvar carcinoma cell line, A431. In all of these samples of DNA, the INT2 gene, previously mapped to human chromosome 11q13, was also amplified to the same degree as the HST1 gene.« less

Function and regulation of AUTS2, a gene implicated in autism and human evolution.

PubMed

Oksenberg, Nir; Stevison, Laurie; Wall, Jeffrey D; Ahituv, Nadav

2013-01-01

Nucleotide changes in the AUTS2 locus, some of which affect only noncoding regions, are associated with autism and other neurological disorders, including attention deficit hyperactivity disorder, epilepsy, dyslexia, motor delay, language delay, visual impairment, microcephaly, and alcohol consumption. In addition, AUTS2 contains the most significantly accelerated genomic region differentiating humans from Neanderthals, which is primarily composed of noncoding variants. However, the function and regulation of this gene remain largely unknown. To characterize auts2 function, we knocked it down in zebrafish, leading to a smaller head size, neuronal reduction, and decreased mobility. To characterize AUTS2 regulatory elements, we tested sequences for enhancer activity in zebrafish and mice. We identified 23 functional zebrafish enhancers, 10 of which were active in the brain. Our mouse enhancer assays characterized three mouse brain enhancers that overlap an ASD-associated deletion and four mouse enhancers that reside in regions implicated in human evolution, two of which are active in the brain. Combined, our results show that AUTS2 is important for neurodevelopment and expose candidate enhancer sequences in which nucleotide variation could lead to neurological disease and human-specific traits.

Mouse Genome Informatics (MGI): Resources for Mining Mouse Genetic, Genomic, and Biological Data in Support of Primary and Translational Research.

PubMed

Eppig, Janan T; Smith, Cynthia L; Blake, Judith A; Ringwald, Martin; Kadin, James A; Richardson, Joel E; Bult, Carol J

2017-01-01

The Mouse Genome Informatics (MGI), resource ( www.informatics.jax.org ) has existed for over 25 years, and over this time its data content, informatics infrastructure, and user interfaces and tools have undergone dramatic changes (Eppig et al., Mamm Genome 26:272-284, 2015). Change has been driven by scientific methodological advances, rapid improvements in computational software, growth in computer hardware capacity, and the ongoing collaborative nature of the mouse genomics community in building resources and sharing data. Here we present an overview of the current data content of MGI, describe its general organization, and provide examples using simple and complex searches, and tools for mining and retrieving sets of data.

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Po-E; Lo, Chien -Chi; Anderson, Joseph J.

Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the easemore » of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. As a result, this bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research.« less

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform

PubMed Central

Li, Po-E; Lo, Chien-Chi; Anderson, Joseph J.; Davenport, Karen W.; Bishop-Lilly, Kimberly A.; Xu, Yan; Ahmed, Sanaa; Feng, Shihai; Mokashi, Vishwesh P.; Chain, Patrick S.G.

2017-01-01

Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the ease of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. This bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research. PMID:27899609

Integrated systems analysis reveals a molecular network underlying autism spectrum disorders

PubMed Central

Li, Jingjing; Shi, Minyi; Ma, Zhihai; Zhao, Shuchun; Euskirchen, Ghia; Ziskin, Jennifer; Urban, Alexander; Hallmayer, Joachim; Snyder, Michael

2014-01-01

Autism is a complex disease whose etiology remains elusive. We integrated previously and newly generated data and developed a systems framework involving the interactome, gene expression and genome sequencing to identify a protein interaction module with members strongly enriched for autism candidate genes. Sequencing of 25 patients confirmed the involvement of this module in autism, which was subsequently validated using an independent cohort of over 500 patients. Expression of this module was dichotomized with a ubiquitously expressed subcomponent and another subcomponent preferentially expressed in the corpus callosum, which was significantly affected by our identified mutations in the network center. RNA-sequencing of the corpus callosum from patients with autism exhibited extensive gene mis-expression in this module, and our immunochemical analysis showed that the human corpus callosum is predominantly populated by oligodendrocyte cells. Analysis of functional genomic data further revealed a significant involvement of this module in the development of oligodendrocyte cells in mouse brain. Our analysis delineates a natural network involved in autism, helps uncover novel candidate genes for this disease and improves our understanding of its molecular pathology. PMID:25549968

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform

DOE PAGES

Li, Po-E; Lo, Chien -Chi; Anderson, Joseph J.; ...

2016-11-24

Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the easemore » of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. As a result, this bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research.« less

Identification of estrogen-responsive genes using a genome-wide analysis of promoter elements for transcription factor binding sites.

PubMed

Kamalakaran, Sitharthan; Radhakrishnan, Senthil K; Beck, William T

2005-06-03

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA.

PubMed

Mustafa, Farah; Vivet-Boudou, Valérie; Jabeen, Ayesha; Ali, Lizna M; Kalloush, Rawan M; Marquet, Roland; Rizvi, Tahir A

2018-06-21

Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

Identification of both copy number variation-type and constant-type core elements in a large segmental duplication region of the mouse genome

PubMed Central

2013-01-01

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 large-scale-homology search algorithm SHEAP. For eight chromosomes, we identified various types of large SD as tartan-checked patterns within the self-comparative plots. A complex arrangement of diagonal split lines in the self-comparative-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. PMID:23834397

Comparative analysis and visualization of multiple collinear genomes

PubMed Central

2012-01-01

Background Genome browsers are a common tool used by biologists to visualize genomic features including genes, polymorphisms, and many others. However, existing genome browsers and visualization tools are not well-suited to perform meaningful comparative analysis among a large number of genomes. With the increasing quantity and availability of genomic data, there is an increased burden to provide useful visualization and analysis tools for comparison of multiple collinear genomes such as the large panels of model organisms which are the basis for much of the current genetic research. Results We have developed a novel web-based tool for visualizing and analyzing multiple collinear genomes. Our tool illustrates genome-sequence similarity through a mosaic of intervals representing local phylogeny, subspecific origin, and haplotype identity. Comparative analysis is facilitated through reordering and clustering of tracks, which can vary throughout the genome. In addition, we provide local phylogenetic trees as an alternate visualization to assess local variations. Conclusions Unlike previous genome browsers and viewers, ours allows for simultaneous and comparative analysis. Our browser provides intuitive selection and interactive navigation about features of interest. Dynamic visualizations adjust to scale and data content making analysis at variable resolutions and of multiple data sets more informative. We demonstrate our genome browser for an extensive set of genomic data sets composed of almost 200 distinct mouse laboratory strains. PMID:22536897

Genomic Organization of the Murine Miller–Dieker/Lissencephaly Region: Conservation of Linkage with the Human Region

PubMed Central

Hirotsune, Shinji; Pack, Svetlana D.; Chong, Samuel S.; Robbins, Christiane M.; Pavan, William J.; Ledbetter, David H.; Wynshaw-Boris, Anthony

1997-01-01

Several human syndromes are associated with haploinsufficiency of chromosomal regions secondary to microdeletions. Isolated lissencephaly sequence (ILS), a human developmental disease characterized by a smooth cerebral surface (classical lissencephaly) and microscopic evidence of incomplete neuronal migration, is often associated with small deletions or translocations at chromosome 17p13.3. Miller–Dieker syndrome (MDS) is associated with larger deletions of 17p13.3 and consists of classical lissencephaly with additional phenotypes including facial abnormalities. We have isolated the murine homologs of three genes located inside and outside the MDS region: Lis1, Mnt/Rox, and 14-3-3ε. These genes are all located on mouse chromosome 11B2, as determined by metaphase FISH, and the relative order and approximate gene distance was determined by interphase FISH analysis. The transcriptional orientation and intergenic distance of Lis1 and Mnt/Rox were ascertained by fragmentation analysis of a mouse yeast artificial chromosome containing both genes. To determine the distance and orientation of 14-3-3ε with respect to Lis1 and Mnt/Rox, we introduced a super-rare cutter site (VDE) that is unique in the mouse genome into 14-3-3ε by gene targeting. Using the introduced VDE site, the orientation of this gene was determined by pulsed field gel electrophoresis and Southern blot analysis. Our results demonstrate that the MDS region is conserved between human and mouse. This conservation of linkage suggests that the mouse can be used to model microdeletions that occur in ILS and MDS. PMID:9199935

Carcinogens induce reversion of the mouse pink-eyed unstable mutation

PubMed Central

Schiestl, Robert H.; Aubrecht, Jiri; Khogali, Fathia; Carls, Nicholas

1997-01-01

Deletions and other genome rearrangements are associated with carcinogenesis and inheritable diseases. The pink-eyed unstable (pun) mutation in the mouse is caused by duplication of a 70-kb internal fragment of the p gene. Spontaneous reversion events in homozygous pun/pun mice occur through deletion of a duplicated sequence. Reversion events in premelanocytes in the mouse embryo detected as black spots on the gray fur of the offspring were inducible by the carcinogen x-rays, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, benzo[a]pyrene, trichloroethylene, benzene, and sodium arsenate. The latter three carcinogens are not detectable with several in vitro or in vivo mutagenesis assays. We studied the molecular mechanism of the carcinogen-induced reversion events by cDNA analysis using reverse transcriptase–PCR method and identified the induced reversion events as deletions. DNA deletion assays may be sensitive indicators for carcinogen exposure. PMID:9114032

C57BL/6N mutation in Cytoplasmic FMR interacting protein 2 regulates cocaine response

PubMed Central

Kumar, Vivek; Kim, Kyungin; Joseph, Chryshanthi; Kourrich, Saïd; Yoo, Seung Hee; Huang, Hung Chung; Vitaterna, Martha H.; de Villena, Fernando Pardo-Manuel; Churchill, Gary; Bonci, Antonello; Takahashi, Joseph S.

2015-01-01

The inbred mouse C57BL/6J is the reference strain for genome sequence and for most behavioral and physiological phenotypes. However the International Knockout Mouse Consortium uses an embryonic stem cell line derived from a related C57BL/6N substrain. We found that C57BL/6N has lower acute and sensitized response to cocaine and methamphetamine. We mapped a single causative locus and identified a non-synonymous mutation of serine to phenylalanine (S968F) in Cytoplasmic FMR interacting protein 2 (Cyfip2) as the causative variant. The S968F mutation destabilizes CYFIP2 and deletion of the C57BL/6N mutant allele leads to acute and sensitized cocaine response phenotypes. We propose CYFIP2 is a key regulator of cocaine response in mammals and present a framework to utilize mouse substrains to discover novel genes and alleles regulating behavior. PMID:24357318

Identification of a novel box C/D snoRNA from mouse nucleolar cDNA library.

PubMed

Zhou, Hui; Zhao, Jin; Yu, Chuan-He; Luo, Qing-Jun; Chen, Yue-Qin; Xiao, Yu; Qu, Liang-Hu

2004-02-18

By construction and screen of mouse nucleolar cDNA library, a novel mammalian small nucleolar RNAs (snoRNA) was identified. The novel snoRNA, 70 nt in length, displays structural features typical of C/D box snoRNA family. The snoRNA possesses an 11-nt-long rRNA antisense element and is predicted to guide the 2'-O-methylation of mouse 28S rRNA at G4043, a site unknown so far to be modified in vertebrates. The comparison of functional element of snoRNA guides among eukaryotes reveals that the novel snoRNA is a mammalian counterpart of yeast snR38 despite highly divergent sequence between them. Mouse and human snR38 and other cognates in distant vertebrates were positively detected with slight length variability. As expected, the rRNA ribose-methylation site predicted by mouse snR38 was precisely mapped by specific-primer extension assay. Furthermore, our analyses show that mouse and human snR38 gene have multiple variants and are nested in the introns of different host genes with unknown function. Thus, snR38 is a phylogenetically conserved methylation guide but exhibits different genomic organization in eukaryotes.

A high-resolution enhancer atlas of the developing telencephalon.

PubMed

Visel, Axel; Taher, Leila; Girgis, Hani; May, Dalit; Golonzhka, Olga; Hoch, Renee V; McKinsey, Gabriel L; Pattabiraman, Kartik; Silberberg, Shanni N; Blow, Matthew J; Hansen, David V; Nord, Alex S; Akiyama, Jennifer A; Holt, Amy; Hosseini, Roya; Phouanenavong, Sengthavy; Plajzer-Frick, Ingrid; Shoukry, Malak; Afzal, Veena; Kaplan, Tommy; Kriegstein, Arnold R; Rubin, Edward M; Ovcharenko, Ivan; Pennacchio, Len A; Rubenstein, John L R

2013-02-14

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

Role of H1 Linker Histones in Mammalian Development and Stem Cell Differentiation

PubMed Central

Pan, Chenyi; Fan, Yuhong

2016-01-01

H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. PMID:26689747

Mammalian cDNA Library from the NIH Mammalian Gene Collection (MGC) | Office of Cancer Genomics

Cancer.gov

The MGC provides the research community full-length clones for most of the defined (as of 2006) human and mouse genes, along with selected clones of cow and rat genes. Clones were designed to allow easy transfer of the ORF sequences into nearly any type of expression vector. MGC provides protein ‘expression-ready’ clones for each of the included human genes. MGC is part of the ORFeome Collaboration (OC).

Comparative sequence analysis of the X-inactivation center region in mouse, human, and bovine.

PubMed

Chureau, Corinne; Prissette, Marine; Bourdet, Agnès; Barbe, Valérie; Cattolico, Laurence; Jones, Louis; Eggen, André; Avner, Philip; Duret, Laurent

2002-06-01

We have sequenced to high levels of accuracy 714-kb and 233-kb regions of the mouse and bovine X-inactivation centers (Xic), respectively, centered on the Xist gene. This has provided the basis for a fully annotated comparative analysis of the mouse Xic with the 2.3-Mb orthologous region in human and has allowed a three-way species comparison of the core central region, including the Xist gene. These comparisons have revealed conserved genes, both coding and noncoding, conserved CpG islands and, more surprisingly, conserved pseudogenes. The distribution of repeated elements, especially LINE repeats, in the mouse Xic region when compared to the rest of the genome does not support the hypothesis of a role for these repeat elements in the spreading of X inactivation. Interestingly, an asymmetric distribution of LINE elements on the two DNA strands was observed in the three species, not only within introns but also in intergenic regions. This feature is suggestive of important transcriptional activity within these intergenic regions. In silico prediction followed by experimental analysis has allowed four new genes, Cnbp2, Ftx, Jpx, and Ppnx, to be identified and novel, widespread, complex, and apparently noncoding transcriptional activity to be characterized in a region 5' of Xist that was recently shown to attract histone modification early after the onset of X inactivation.

A novel humanized mouse model of Huntington disease for preclinical development of therapeutics targeting mutant huntingtin alleles.

PubMed

Southwell, Amber L; Skotte, Niels H; Villanueva, Erika B; Østergaard, Michael E; Gu, Xiaofeng; Kordasiewicz, Holly B; Kay, Chris; Cheung, Daphne; Xie, Yuanyun; Waltl, Sabine; Dal Cengio, Louisa; Findlay-Black, Hailey; Doty, Crystal N; Petoukhov, Eugenia; Iworima, Diepiriye; Slama, Ramy; Ooi, Jolene; Pouladi, Mahmoud A; Yang, X William; Swayze, Eric E; Seth, Punit P; Hayden, Michael R

2017-03-15

Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global benefit. Thus there is a need for preclinical models of HD recapitulating human HTT genetics. We previously generated a humanized mouse model of HD, Hu97/18, by intercrossing BACHD and YAC18 mice with knockout of the endogenous mouse HD homolog (Hdh). Hu97/18 mice recapitulate the genetics of HD, having two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of Caucasian descent. We have now generated a companion model, Hu128/21, by intercrossing YAC128 and BAC21 mice on the Hdh-/- background. Hu128/21 mice have two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1, and for preclinical screening of personalized HTT lowering therapies in HD patients of East Asian descent. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Selective Constraints on Coding Sequences of Nervous System Genes Are a Major Determinant of Duplicate Gene Retention in Vertebrates.

PubMed

Roux, Julien; Liu, Jialin; Robinson-Rechavi, Marc

2017-11-01

The evolutionary history of vertebrates is marked by three ancient whole-genome duplications: two successive rounds in the ancestor of vertebrates, and a third one specific to teleost fishes. Biased loss of most duplicates enriched the genome for specific genes, such as slow evolving genes, but this selective retention process is not well understood. To understand what drives the long-term preservation of duplicate genes, we characterized duplicated genes in terms of their expression patterns. We used a new method of expression enrichment analysis, TopAnat, applied to in situ hybridization data from thousands of genes from zebrafish and mouse. We showed that the presence of expression in the nervous system is a good predictor of a higher rate of retention of duplicate genes after whole-genome duplication. Further analyses suggest that purifying selection against the toxic effects of misfolded or misinteracting proteins, which is particularly strong in nonrenewing neural tissues, likely constrains the evolution of coding sequences of nervous system genes, leading indirectly to the preservation of duplicate genes after whole-genome duplication. Whole-genome duplications thus greatly contributed to the expansion of the toolkit of genes available for the evolution of profound novelties of the nervous system at the base of the vertebrate radiation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis.

PubMed

Sabrautzki, Sibylle; Rubio-Aliaga, Isabel; Hans, Wolfgang; Fuchs, Helmut; Rathkolb, Birgit; Calzada-Wack, Julia; Cohrs, Christian M; Klaften, Matthias; Seedorf, Hartwig; Eck, Sebastian; Benet-Pagès, Ana; Favor, Jack; Esposito, Irene; Strom, Tim M; Wolf, Eckhard; Lorenz-Depiereux, Bettina; Hrabě de Angelis, Martin

2012-08-01

Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.

Genomic Organization of the Drosophila Telomere RetrotransposableElements

DOE Office of Scientific and Technical Information (OSTI.GOV)

George, J.A.; DeBaryshe, P.G.; Traverse, K.L.

2006-10-16

The emerging sequence of the heterochromatic portion of the Drosophila melanogaster genome, with the most recent update of euchromatic sequence, gives the first genome-wide view of the chromosomal distribution of the telomeric retrotransposons, HeT-A, TART, and Tahre. As expected, these elements are entirely excluded from euchromatin, although sequence fragments of HeT-A and TART 3 untranslated regions are found in nontelomeric heterochromatin on the Y chromosome. The proximal ends of HeT-A/TART arrays appear to be a transition zone because only here do other transposable elements mix in the array. The sharp distinction between the distribution of telomeric elements and that ofmore » other transposable elements suggests that chromatin structure is important in telomere element localization. Measurements reported here show (1) D. melanogaster telomeres are very long, in the size range reported for inbred mouse strains (averaging 46 kb per chromosome end in Drosophila stock 2057). As in organisms with telomerase, their length varies depending on genotype. There is also slight under-replication in polytene nuclei. (2) Surprisingly, the relationship between the number of HeT-A and TART elements is not stochastic but is strongly correlated across stocks, supporting the idea that the two elements are interdependent. Although currently assembled portions of the HeT-A/TART arrays are from the most-proximal part of long arrays, {approx}61% of the total HeT-A sequence in these regions consists of intact, potentially active elements with little evidence of sequence decay, making it likely that the content of the telomere arrays turns over more extensively than has been thought.« less

RIKEN Integrated Sequence Analysis (RISA) System—384-Format Sequencing Pipeline with 384 Multicapillary Sequencer

PubMed Central

Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Nagaoka, Sumiharu; Sasaki, Nobuya; Carninci, Piero; Konno, Hideaki; Akiyama, Junichi; Nishi, Katsuo; Kitsunai, Tokuji; Tashiro, Hideo; Itoh, Mari; Sumi, Noriko; Ishii, Yoshiyuki; Nakamura, Shin; Hazama, Makoto; Nishine, Tsutomu; Harada, Akira; Yamamoto, Rintaro; Matsumoto, Hiroyuki; Sakaguchi, Sumito; Ikegami, Takashi; Kashiwagi, Katsuya; Fujiwake, Syuji; Inoue, Kouji; Togawa, Yoshiyuki; Izawa, Masaki; Ohara, Eiji; Watahiki, Masanori; Yoneda, Yuko; Ishikawa, Tomokazu; Ozawa, Kaori; Tanaka, Takumi; Matsuura, Shuji; Kawai, Jun; Okazaki, Yasushi; Muramatsu, Masami; Inoue, Yorinao; Kira, Akira; Hayashizaki, Yoshihide

2000-01-01

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3′ end and 5′ end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month. PMID:11076861

A Simple and Efficient Method for Assembling TALE Protein Based on Plasmid Library

PubMed Central

Xu, Huarong; Xin, Ying; Zhang, Tingting; Ma, Lixia; Wang, Xin; Chen, Zhilong; Zhang, Zhiying

2013-01-01

DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate. PMID:23840477

A simple and efficient method for assembling TALE protein based on plasmid library.

PubMed

Zhang, Zhiqiang; Li, Duo; Xu, Huarong; Xin, Ying; Zhang, Tingting; Ma, Lixia; Wang, Xin; Chen, Zhilong; Zhang, Zhiying

2013-01-01

DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.

GWIPS-viz: development of a ribo-seq genome browser

PubMed Central

Michel, Audrey M.; Fox, Gearoid; M. Kiran, Anmol; De Bo, Christof; O’Connor, Patrick B. F.; Heaphy, Stephen M.; Mullan, James P. A.; Donohue, Claire A.; Higgins, Desmond G.; Baranov, Pavel V.

2014-01-01

We describe the development of GWIPS-viz (http://gwips.ucc.ie), an online genome browser for viewing ribosome profiling data. Ribosome profiling (ribo-seq) is a recently developed technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome-protected messenger RNA (mRNA) fragments, which allows the ribosome density along all mRNA transcripts present in the cell to be quantified. Since its inception, ribo-seq has been carried out in a number of eukaryotic and prokaryotic organisms. Owing to the increasing interest in ribo-seq, there is a pertinent demand for a dedicated ribo-seq genome browser. GWIPS-viz is based on The University of California Santa Cruz (UCSC) Genome Browser. Ribo-seq tracks, coupled with mRNA-seq tracks, are currently available for several genomes: human, mouse, zebrafish, nematode, yeast, bacteria (Escherichia coli K12, Bacillus subtilis), human cytomegalovirus and bacteriophage lambda. Our objective is to continue incorporating published ribo-seq data sets so that the wider community can readily view ribosome profiling information from multiple studies without the need to carry out computational processing. PMID:24185699

Syntenic conservation of HSP70 genes in cattle and humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grosz, M.D.; Womack, J.E.; Skow, L.C.

1992-12-01

A phage library of bovine genomic DNA was screened for hybridization with a human HSP70 cDNA probe, and 21 positive plaques were identified and isolated. Restriction mapping and blot hybridization analysis of DNA from the recombinant plaques demonstrated that the cloned DNAs were derived from three different regions of the bovine genome. Ore region contains two tandemly arrayed HSP70 sequences, designated HSP70-1 and HSP70-2, separated by approximately 8 kb of DNA. Single HSP70 sequences, designated HSP70-3 and HSP70-4, were found in two other genomic regions. Locus-specific probes of unique flanking sequences from representative HSP70 clones were hybridized to restriction endonuclease-digestedmore » DNA from bovine-hamster and bovine-mouse somatic cell hybrid panels to determine the chromosomal location of the HSP70 sequences. The probe for the tandemly arrayed HSP70-1 and HSP70-2 sequences mapped to bovine chromosome 23, syntenic with glyoxalase 1, 21 steroid hydroxylase, and major histocompatibility class I loci. HSP70-3 sequences mapped to bovine chromosome 10, syntenic with nucleoside phosphorylase and murine osteosarcoma viral oncogene (v-fos), and HSP70-4 mapped to bovine syntenic group U6, syntenic with amylase 1 and phosphoglucomutase 1. On the basis of these data, the authors propose that bovine HSP70-1,2 are homologous to human HSPA1 and HSPA1L on chromosome 6p21.3, bovine HSP70-3 is the homolog of an unnamed human HSP70 gene on chromosome 14q22-q24, and bovine HSP70-4 is homologous to one of the human HSPA-6,-7 genes on chromosome 1. 34 refs., 2 figs., 1 tab.« less

Virulence factors encoded by Legionella longbeachae identified on the basis of the genome sequence analysis of clinical isolate D-4968.

PubMed

Kozak, Natalia A; Buss, Meghan; Lucas, Claressa E; Frace, Michael; Govil, Dhwani; Travis, Tatiana; Olsen-Rasmussen, Melissa; Benson, Robert F; Fields, Barry S

2010-02-01

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

Cross-species comparison of aCGH data from mouse and human BRCA1- and BRCA2-mutated breast cancers

PubMed Central

2010-01-01

Background Genomic gains and losses are a result of genomic instability in many types of cancers. BRCA1- and BRCA2-mutated breast cancers are associated with increased amounts of chromosomal aberrations, presumably due their functions in genome repair. Some of these genomic aberrations may harbor genes whose absence or overexpression may give rise to cellular growth advantage. So far, it has not been easy to identify the driver genes underlying gains and losses. A powerful approach to identify these driver genes could be a cross-species comparison of array comparative genomic hybridization (aCGH) data from cognate mouse and human tumors. Orthologous regions of mouse and human tumors that are commonly gained or lost might represent essential genomic regions selected for gain or loss during tumor development. Methods To identify genomic regions that are associated with BRCA1- and BRCA2-mutated breast cancers we compared aCGH data from 130 mouse Brca1Δ/Δ;p53Δ/Δ, Brca2Δ/Δ;p53Δ/Δ and p53Δ/Δ mammary tumor groups with 103 human BRCA1-mutated, BRCA2-mutated and non-hereditary breast cancers. Results Our genome-wide cross-species analysis yielded a complete collection of loci and genes that are commonly gained or lost in mouse and human breast cancer. Principal common CNAs were the well known MYC-associated gain and RB1/INTS6-associated loss that occurred in all mouse and human tumor groups, and the AURKA-associated gain occurred in BRCA2-related tumors from both species. However, there were also important differences between tumor profiles of both species, such as the prominent gain on chromosome 10 in mouse Brca2Δ/Δ;p53Δ/Δ tumors and the PIK3CA associated 3q gain in human BRCA1-mutated tumors, which occurred in tumors from one species but not in tumors from the other species. This disparity in recurrent aberrations in mouse and human tumors might be due to differences in tumor cell type or genomic organization between both species. Conclusions The selection of the oncogenome during mouse and human breast tumor development is markedly different, apart from the MYC gain and RB1-associated loss. These differences should be kept in mind when using mouse models for preclinical studies. PMID:20735817

Obesity genetics in mouse and human: back and forth, and back again

PubMed Central

Yazdi, Fereshteh T.; Clee, Susanne M.

2015-01-01

Obesity is a major public health concern. This condition results from a constant and complex interplay between predisposing genes and environmental stimuli. Current attempts to manage obesity have been moderately effective and a better understanding of the etiology of obesity is required for the development of more successful and personalized prevention and treatment options. To that effect, mouse models have been an essential tool in expanding our understanding of obesity, due to the availability of their complete genome sequence, genetically identified and defined strains, various tools for genetic manipulation and the accessibility of target tissues for obesity that are not easily attainable from humans. Our knowledge of monogenic obesity in humans greatly benefited from the mouse obesity genetics field. Genes underlying highly penetrant forms of monogenic obesity are part of the leptin-melanocortin pathway in the hypothalamus. Recently, hypothesis-generating genome-wide association studies for polygenic obesity traits in humans have led to the identification of 119 common gene variants with modest effect, most of them having an unknown function. These discoveries have led to novel animal models and have illuminated new biologic pathways. Integrated mouse-human genetic approaches have firmly established new obesity candidate genes. Innovative strategies recently developed by scientists are described in this review to accelerate the identification of causal genes and deepen our understanding of obesity etiology. An exhaustive dissection of the molecular roots of obesity may ultimately help to tackle the growing obesity epidemic worldwide. PMID:25825681

Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus

PubMed Central

Wang, Yupeng; Khan, Iram F.; Boissel, Sandrine; Jarjour, Jordan; Pangallo, Joseph; Thyme, Summer; Baker, David; Scharenberg, Andrew M.; Rawlings, David J.

2014-01-01

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20–22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)—a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering. PMID:24682825

Genetic Diversity and Reassortment of Hantaan Virus Tripartite RNA Genomes in Nature, the Republic of Korea

PubMed Central

Kim, Jeong-Ah; Kim, Won-keun; No, Jin Sun; Lee, Seung-Ho; Lee, Sook-Young; Kim, Ji Hye; Kho, Jeong Hoon; Lee, Daesang; Song, Dong Hyun; Gu, Se Hun; Jeong, Seong Tae; Park, Man-Seong; Kim, Heung-Chul; Klein, Terry A.; Song, Jin-Won

2016-01-01

Background Hantaan virus (HTNV), a negative sense tripartite RNA virus of the Family Bunyaviridae, is the most prevalent hantavirus in the Republic of Korea (ROK). It is the causative agent of Hemorrhagic Fever with Renal Syndrome (HFRS) in humans and maintained in the striped field mouse, Apodemus agrarius, the primary zoonotic host. Clinical HFRS cases have been reported commonly in HFRS-endemic areas of Gyeonggi province. Recently, the death of a member of the ROK military from Gangwon province due to HFRS prompted an investigation of the epidemiology and distribution of hantaviruses in Gangwon and Gyeonggi provinces that border the demilitarized zone separating North and South Korea. Methodology and Principal Findings To elucidate the geographic distribution and molecular diversity of HTNV, whole genome sequences of HTNV Large (L), Medium (M), and Small (S) segments were acquired from lung tissues of A. agrarius captured from 2003–2014. Consistent with the clinical incidence of HFRS established by the Korea Centers for Disease Control & Prevention (KCDC), the prevalence of HTNV in naturally infected mice in Gangwon province was lower than for Gyeonggi province. Whole genomic sequences of 34 HTNV strains were identified and a phylogenetic analysis showed geographic diversity of the virus in the limited areas. Reassortment analysis first suggested an occurrence of genetic exchange of HTNV genomes in nature, ROK. Conclusion/Significance This study is the first report to demonstrate the molecular prevalence of HTNV in Gangwon province. Whole genome sequencing of HTNV showed well-supported geographic lineages and the molecular diversity in the northern region of ROK due to a natural reassortment of HTNV genomes. These observations contribute to a better understanding of the genetic diversity and molecular evolution of hantaviruses. Also, the full-length of HTNV tripartite genomes will provide a database for phylogeographic analysis of spatial and temporal outbreaks of hantavirus infection. PMID:27315053

Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit whey acidic protein genes.

PubMed Central

Millot, Benjamin; Montoliu, Lluís; Fontaine, Marie-Louise; Mata, Teresa; Devinoy, Eve

2003-01-01

The upstream regulatory regions of the mouse and rabbit whey acidic protein (WAP) genes have been used extensively to target the efficient expression of foreign genes into the mammary gland of transgenic animals. Therefore both regions have been studied to elucidate fully the mechanisms controlling WAP gene expression. Three DNase I-hypersensitive sites (HSS0, HSS1 and HSS2) have been described upstream of the rabbit WAP gene in the lactating mammary gland and correspond to important regulatory regions. These sites are surrounded by variable chromatin structures during mammary-gland development. In the present study, we describe the upstream sequence of the mouse WAP gene. Analysis of genomic sequences shows that the mouse WAP gene is situated between two widely expressed genes (Cpr2 and Ramp3). We show that the hypersensitive sites found upstream of the rabbit WAP gene are also detected in the mouse WAP gene. Further, they encompass functional signal transducer and activator of transcription 5-binding sites, as has been observed in the rabbit. A new hypersensitive site (HSS3), not specific to the mammary gland, was mapped 8 kb upstream of the rabbit WAP gene. Unlike the three HSSs described above, HSS3 is also detected in the liver, but similar to HSS1, it does not depend on lactogenic hormone treatments during cell culture. The region surrounding HSS3 encompasses a potential matrix attachment region, which is also conserved upstream of the mouse WAP gene and contains a functional transcription factor Ets-1 (E26 transformation-specific-1)-binding site. Finally, we demonstrate for the first time that variations in the chromatin structure are dependent on prolactin alone. PMID:12580766

BGDB: a database of bivalent genes

PubMed Central

Li, Qingyan; Lian, Shuabin; Dai, Zhiming; Xiang, Qian; Dai, Xianhua

2013-01-01

Bivalent gene is a gene marked with both H3K4me3 and H3K27me3 epigenetic modification in the same area, and is proposed to play a pivotal role related to pluripotency in embryonic stem (ES) cells. Identification of these bivalent genes and understanding their functions are important for further research of lineage specification and embryo development. So far, lots of genome-wide histone modification data were generated in mouse and human ES cells. These valuable data make it possible to identify bivalent genes, but no comprehensive data repositories or analysis tools are available for bivalent genes currently. In this work, we develop BGDB, the database of bivalent genes. The database contains 6897 bivalent genes in human and mouse ES cells, which are manually collected from scientific literature. Each entry contains curated information, including genomic context, sequences, gene ontology and other relevant information. The web services of BGDB database were implemented with PHP + MySQL + JavaScript, and provide diverse query functions. Database URL: http://dailab.sysu.edu.cn/bgdb/ PMID:23894186

The DNA methylation profile of oocytes in mice with hyperinsulinaemia and hyperandrogenism as detected by single-cell level whole genome bisulphite sequencing (SC-WGBS) technology.

PubMed

Li, Qian-Nan; Guo, Lei; Hou, Yi; Ou, Xiang-Hong; Liu, Zhonghua; Sun, Qing-Yuan

2018-06-22

Polycystic ovary syndrome (PCOS), a familial aggregation disease that causes anovulation in women, has well-recognised characteristics, two of which are hyperinsulinaemia and hyperandrogenaemia. To determine whether the DNA methylation status is altered in oocytes by high insulin and androgen levels, we generated a mouse model with hyperinsulinaemia and hyperandrogenaemia by injection of insulin and human chorionic gonadotrophin and investigated DNA methylation changes through single-cell level whole genome bisulphite sequencing. Our results showed that hyperinsulinaemia and hyperandrogenaemia had no significant effects on the global DNA methylation profile and different functional regions of genes, but did alter methylation status of some genes, which were significantly enriched in 17 gene ontology (GO) terms (P<0.05) by GO analysis. Among differently methylated genes, some were related to the occurrence of PCOS. Based on our results, we suggest that hyperinsulinaemia and hyperandrogenaemia may cause changes in some DNA methylation loci in oocytes.

Basic Helix-Loop-Helix Transcription Factor Gene Family Phylogenetics and Nomenclature

PubMed Central

Skinner, Michael K.; Rawls, Alan; Wilson-Rawls, Jeanne; Roalson, Eric H.

2010-01-01

A phylogenetic analysis of the basic helix-loop-helix (bHLH) gene superfamily was performed using seven different species (human, mouse, rat, worm, fly, yeast, and plant Arabidopsis) and involving over 600 bHLH genes [1]. All bHLH genes were identified in the genomes of the various species, including expressed sequence tags, and the entire coding sequence was used in the analysis. Nearly 15% of the gene family has been updated or added since the original publication. A super-tree involving six clades and all structural relationships was established and is now presented for four of the species. The wealth of functional data available for members of the bHLH gene superfamily provides us with the opportunity to use this exhaustive phylogenetic tree to predict potential functions of uncharacterized members of the family. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique elements of the evolution and functional relationships of the different genes in the bHLH gene family. PMID:20219281

High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing.

PubMed

Lagarde, Julien; Uszczynska-Ratajczak, Barbara; Carbonell, Silvia; Pérez-Lluch, Sílvia; Abad, Amaya; Davis, Carrie; Gingeras, Thomas R; Frankish, Adam; Harrow, Jennifer; Guigo, Roderic; Johnson, Rory

2017-12-01

Accurate annotation of genes and their transcripts is a foundation of genomics, but currently no annotation technique combines throughput and accuracy. As a result, reference gene collections remain incomplete-many gene models are fragmentary, and thousands more remain uncataloged, particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), which combines targeted RNA capture with third-generation long-read sequencing. Here we present an experimental reannotation of the GENCODE intergenic lncRNA populations in matched human and mouse tissues that resulted in novel transcript models for 3,574 and 561 gene loci, respectively. CLS approximately doubled the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enabled us to definitively characterize the genomic features of lncRNAs, including promoter and gene structure, and protein-coding potential. Thus, CLS removes a long-standing bottleneck in transcriptome annotation and generates manual-quality full-length transcript models at high-throughput scales.

GenExp: an interactive web-based genomic DAS client with client-side data rendering.

PubMed

Gel Moreno, Bernat; Messeguer Peypoch, Xavier

2011-01-01

The Distributed Annotation System (DAS) offers a standard protocol for sharing and integrating annotations on biological sequences. There are more than 1000 DAS sources available and the number is steadily increasing. Clients are an essential part of the DAS system and integrate data from several independent sources in order to create a useful representation to the user. While web-based DAS clients exist, most of them do not have direct interaction capabilities such as dragging and zooming with the mouse. Here we present GenExp, a web based and fully interactive visual DAS client. GenExp is a genome oriented DAS client capable of creating informative representations of genomic data zooming out from base level to complete chromosomes. It proposes a novel approach to genomic data rendering and uses the latest HTML5 web technologies to create the data representation inside the client browser. Thanks to client-side rendering most position changes do not need a network request to the server and so responses to zooming and panning are almost immediate. In GenExp it is possible to explore the genome intuitively moving it with the mouse just like geographical map applications. Additionally, in GenExp it is possible to have more than one data viewer at the same time and to save the current state of the application to revisit it later on. GenExp is a new interactive web-based client for DAS and addresses some of the short-comings of the existing clients. It uses client-side data rendering techniques resulting in easier genome browsing and exploration. GenExp is open source under the GPL license and it is freely available at http://gralggen.lsi.upc.edu/recerca/genexp.

Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation* ♦

PubMed Central

Chakraborty, Anirban; Wakamiya, Maki; Venkova-Canova, Tatiana; Pandita, Raj K.; Aguilera-Aguirre, Leopoldo; Sarker, Altaf H.; Singh, Dharmendra Kumar; Hosoki, Koa; Wood, Thomas G.; Sharma, Gulshan; Cardenas, Victor; Sarkar, Partha S.; Sur, Sanjiv; Pandita, Tej K.; Boldogh, Istvan; Hazra, Tapas K.

2015-01-01

Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans. PMID:26245904

GenExp: An Interactive Web-Based Genomic DAS Client with Client-Side Data Rendering

PubMed Central

Gel Moreno, Bernat; Messeguer Peypoch, Xavier

2011-01-01

Background The Distributed Annotation System (DAS) offers a standard protocol for sharing and integrating annotations on biological sequences. There are more than 1000 DAS sources available and the number is steadily increasing. Clients are an essential part of the DAS system and integrate data from several independent sources in order to create a useful representation to the user. While web-based DAS clients exist, most of them do not have direct interaction capabilities such as dragging and zooming with the mouse. Results Here we present GenExp, a web based and fully interactive visual DAS client. GenExp is a genome oriented DAS client capable of creating informative representations of genomic data zooming out from base level to complete chromosomes. It proposes a novel approach to genomic data rendering and uses the latest HTML5 web technologies to create the data representation inside the client browser. Thanks to client-side rendering most position changes do not need a network request to the server and so responses to zooming and panning are almost immediate. In GenExp it is possible to explore the genome intuitively moving it with the mouse just like geographical map applications. Additionally, in GenExp it is possible to have more than one data viewer at the same time and to save the current state of the application to revisit it later on. Conclusions GenExp is a new interactive web-based client for DAS and addresses some of the short-comings of the existing clients. It uses client-side data rendering techniques resulting in easier genome browsing and exploration. GenExp is open source under the GPL license and it is freely available at http://gralggen.lsi.upc.edu/recerca/genexp. PMID:21750706

Characterization of mouse and human GTP cyclohydrolase I genes. Mutations in patients with GTP cyclohydrolase I deficiency.

PubMed

Ichinose, H; Ohye, T; Matsuda, Y; Hori, T; Blau, N; Burlina, A; Rouse, B; Matalon, R; Fujita, K; Nagatsu, T

1995-04-28

GTP cyclohydrolase I is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in mammals. Previously, we reported three species of human GTP cyclohydrolase I cDNA in a human liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita, K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187, 359-365). Furthermore, very recently, we found that the GTP cyclohydrolase I gene is causative for hereditary progressive dystonia with marked diurnal fluctuation, also known as DOPA-responsive dystonia (Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu, T. (1994) Nature Genetics 8, 236-242). To clarify the mechanisms that regulate transcription of the GTP cyclohydrolase I gene and to generate multiple species of mRNA, we isolated genomic DNA clones for the human and mouse GTP cyclohydrolase I genes. Structural analysis of the isolated clones revealed that the GTP cyclohydrolase I gene is encoded by a single copy gene and is composed of six exons spanning approximately 30 kilobases. We sequenced all exon/intron boundaries of the human and mouse genes. Structural analysis also demonstrated that the heterogeneity of GTP cyclohydrolase I mRNA is caused by an alternative usage of the splicing acceptor site at the sixth exon. The transcription start site of the mouse GTP cyclohydrolase I gene and the 5'-flanking sequences of the mouse and human genes were determined. We performed regional mapping of the mouse gene by fluorescence in situ hybridization, and the mouse GTP cyclohydrolase I gene was assigned to region C2-3 of mouse chromosome 14. We identified missense mutations in patients with GTP cyclohydrolase I deficiency and expressed mutated enzymes in Escherichia coli to confirm alterations in the enzyme activity.

Retroviruses Hijack Chromatin Loops to Drive Oncogene Expression and Highlight the Chromatin Architecture around Proto-Oncogenic Loci

PubMed Central

Pattison, Jillian M.; Wright, Jason B.; Cole, Michael D.

2015-01-01

The majority of the genome consists of intergenic and non-coding DNA sequences shown to play a major role in different gene regulatory networks. However, the specific potency of these distal elements as well as how these regions exert function across large genomic distances remains unclear. To address these unresolved issues, we closely examined the chromatin architecture around proto-oncogenic loci in the mouse and human genomes to demonstrate a functional role for chromatin looping in distal gene regulation. Using cell culture models, we show that tumorigenic retroviral integration sites within the mouse genome occur near existing large chromatin loops and that this chromatin architecture is maintained within the human genome as well. Significantly, as mutagenesis screens are not feasible in humans, we demonstrate a way to leverage existing screens in mice to identify disease relevant human enhancers and expose novel disease mechanisms. For instance, we characterize the epigenetic landscape upstream of the human Cyclin D1 locus to find multiple distal interactions that contribute to the complex cis-regulation of this cell cycle gene. Furthermore, we characterize a novel distal interaction upstream of the Cyclin D1 gene which provides mechanistic evidence for the abundant overexpression of Cyclin D1 occurring in multiple myeloma cells harboring a pathogenic translocation event. Through use of mapped retroviral integrations and translocation breakpoints, our studies highlight the importance of chromatin looping in oncogene expression, elucidate the epigenetic mechanisms crucial for distal cis-regulation, and in one particular instance, explain how a translocation event drives tumorigenesis through upregulation of a proto-oncogene. PMID:25799187

A vast genomic deletion in the C56BL/6 genome affects different genes within the Ifi200 cluster on chromosome 1 and mediates obesity and insulin resistance.

PubMed

Vogel, Heike; Jähnert, Markus; Stadion, Mandy; Matzke, Daniela; Scherneck, Stephan; Schürmann, Annette

2017-02-15

Obesity, the excessive accumulation of body fat, is a highly heritable and genetically heterogeneous disorder. The complex, polygenic basis for the disease consisting of a network of different gene variants is still not completely known. In the current study we generated a BAC library of the obese-prone NZO strain to clarify the genomic alteration within the gene cluster Ifi200 on chr.1 including Ifi202b, an obesity gene that is in contrast to NZO not expressed in the lean B6 mouse. With the PacBio sequencing data of NZO BAC clones we identified a deletion spanning approximately 261.8 kb in the B6 reference genome. The deletion affects different members of the Ifi200 gene family which also includes the original first exon and 5'-regulatory parts of the Ifi202b gene and suggests to be the relevant cause of its expression deficiency in B6. In addition, the generation and characterization of congenic mice carrying the critical fragment on the B6 background demonstrate its crucial role for obesity and insulin resistance. Our data reveal the reconstruction of a complex genomic region on mouse chr.1 resulting from deletions and duplications of Ifi200 genes and suggest to be relevant for the development of obesity. The results further demonstrate the complexity of the disease and highlight the importance for studying rare genetic variants as they can be causal for large effects.

Repurposing CRISPR/Cas9 for in situ functional assays.

PubMed

Malina, Abba; Mills, John R; Cencic, Regina; Yan, Yifei; Fraser, James; Schippers, Laura M; Paquet, Marilène; Dostie, Josée; Pelletier, Jerry

2013-12-01

RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.

Repurposing CRISPR/Cas9 for in situ functional assays

PubMed Central

Malina, Abba; Mills, John R.; Cencic, Regina; Yan, Yifei; Fraser, James; Schippers, Laura M.; Paquet, Marilène; Dostie, Josée; Pelletier, Jerry

2013-01-01

RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel “all-in-one” lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an “all-in-one” system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens. PMID:24298059

Transcriptomes of Mouse Olfactory Epithelium Reveal Sexual Differences in Odorant Detection

PubMed Central

Shiao, Meng-Shin; Chang, Andrew Ying-Fei; Liao, Ben-Yang; Ching, Yung-Hao; Lu, Mei-Yeh Jade; Chen, Stella Maris; Li, Wen-Hsiung

2012-01-01

To sense numerous odorants and chemicals, animals have evolved a large number of olfactory receptor genes (Olfrs) in their genome. In particular, the house mouse has ∼1,100 genes in the Olfr gene family. This makes the mouse a good model organism to study Olfr genes and olfaction-related genes. To date, whether male and female mice possess the same ability in detecting environmental odorants is still unknown. Using the next generation sequencing technology (paired-end mRNA-seq), we detected 1,088 expressed Olfr genes in both male and female olfactory epithelium. We found that not only Olfr genes but also odorant-binding protein (Obp) genes have evolved rapidly in the mouse lineage. Interestingly, Olfr genes tend to express at a higher level in males than in females, whereas the Obp genes clustered on the X chromosome show the opposite trend. These observations may imply a more efficient odorant-transporting system in females, whereas a more active Olfr gene expressing system in males. In addition, we detected the expression of two genes encoding major urinary proteins, which have been proposed to bind and transport pheromones or act as pheromones in mouse urine. This observation suggests a role of main olfactory system (MOS) in pheromone detection, contrary to the view that only accessory olfactory system (AOS) is involved in pheromone detection. This study suggests the sexual differences in detecting environmental odorants in MOS and demonstrates that mRNA-seq provides a powerful tool for detecting genes with low expression levels and with high sequence similarities. PMID:22511034

Structural characterization and chromosomal location of the mouse macrophage migration inhibitory factor gene and pseudogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bozza, M.; Gerard, C.; Kolakowski, L.F. Jr.

1995-06-10

Macrophage migration inhibitory factor, MIF, is a cytokine released by T-lymphocytes, macrophages, and the pituitary gland that serves to integrate peripheral and central inflammatory responses. Ubiquitous expression and developmental regulation suggest that MIF may have additional roles outside of the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements inmore » both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5{prime} untranslated region of the cDNA, a 3{prime} poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition. 46 refs., 5 figs., 1 tab.« less

ChIP-seq and RNA-seq methods to study circadian control of transcription in mammals

PubMed Central

Takahashi, Joseph S.; Kumar, Vivek; Nakashe, Prachi; Koike, Nobuya; Huang, Hung-Chung; Green, Carla B.; Kim, Tae-Kyung

2015-01-01

Genome-wide analyses have revolutionized our ability to study the transcriptional regulation of circadian rhythms. The advent of next-generation sequencing methods has facilitated the use of two such technologies, ChIP-seq and RNA-seq. In this chapter, we describe detailed methods and protocols for these two techniques, with emphasis on their usage in circadian rhythm experiments in the mouse liver, a major target organ of the circadian clock system. Critical factors for these methods are highlighted and issues arising with time series samples for ChIP-seq and RNA-seq are discussed. Finally detailed protocols for library preparation suitable for Illumina sequencing platforms are presented. PMID:25662462

Reptile genomes open the frontier for comparative analysis of amniote development and regeneration.

PubMed

Tollis, Marc; Hutchins, Elizabeth D; Kusumi, Kenro

2014-01-01

Developmental genetic studies of vertebrates have focused primarily on zebrafish, frog and mouse models, which have clear application to medicine and well-developed genomic resources. In contrast, reptiles represent the most diverse amniote group, but have only recently begun to gather the attention of genome sequencing efforts. Extant reptilian groups last shared a common ancestor ?280 million years ago and include lepidosaurs, turtles and crocodilians. This phylogenetic diversity is reflected in great morphological and behavioral diversity capturing the attention of biologists interested in mechanisms regulating developmental processes such as somitogenesis and spinal patterning, regeneration, the evolution of "snake-like" morphology, the formation of the unique turtle shell, and the convergent evolution of the four-chambered heart shared by mammals and archosaurs. The complete genome of the first non-avian reptile, the green anole lizard, was published in 2011 and has provided insights into the origin and evolution of amniotes. Since then, the genomes of multiple snakes, turtles, and crocodilians have also been completed. Here we will review the current diversity of available reptile genomes, with an emphasis on their evolutionary relationships, and will highlight how these genomes have and will continue to facilitate research in developmental and regenerative biology.

COSMOS: accurate detection of somatic structural variations through asymmetric comparison between tumor and normal samples

PubMed Central

Yamagata, Koichi; Yamanishi, Ayako; Kokubu, Chikara; Takeda, Junji; Sese, Jun

2016-01-01

An important challenge in cancer genomics is precise detection of structural variations (SVs) by high-throughput short-read sequencing, which is hampered by the high false discovery rates of existing analysis tools. Here, we propose an accurate SV detection method named COSMOS, which compares the statistics of the mapped read pairs in tumor samples with isogenic normal control samples in a distinct asymmetric manner. COSMOS also prioritizes the candidate SVs using strand-specific read-depth information. Performance tests on modeled tumor genomes revealed that COSMOS outperformed existing methods in terms of F-measure. We also applied COSMOS to an experimental mouse cell-based model, in which SVs were induced by genome engineering and gamma-ray irradiation, followed by polymerase chain reaction-based confirmation. The precision of COSMOS was 84.5%, while the next best existing method was 70.4%. Moreover, the sensitivity of COSMOS was the highest, indicating that COSMOS has great potential for cancer genome analysis. PMID:26833260

The immunoglobulin heavy chain locus of the duck. Genomic organization and expression of D, J, and C region genes.

PubMed

Lundqvist, M L; Middleton, D L; Hazard, S; Warr, G W

2001-12-14

The region of the duck IgH locus extending from upstream of the proximal diversity (D) segment to downstream of the constant gene cluster has been cloned and mapped. A sequence contig of 48,796 base pairs established that the organization of the genes is D-J(H)-mu-alpha-upsilon. No evidence for a functional homologue (or remnant) of a delta gene was found. The alpha gene is in inverted transcriptional orientation; class switch to IgA expression thus requires inversion of the approximately 27-kilobase pair region that includes both mu and alpha genes. The secreted forms of duck alpha and mu are each encoded by 4 constant region exons, and the hydrophobic C-terminal regions of the membrane receptor forms of alpha and mu are encoded by one and two transmembrane exons, respectively. Putative switch (S) regions were identified for duck mu and upsilon by comparison with chicken Smu and Supsilon sequences and for duck alpha by comparison with mouse Salpha. The duck IgH locus is rich in complex variable number tandem repeats, which occupy approximately 60% of the sequenced region, and occur at a much higher frequency in the IgH locus than in other sequenced regions of the duck genome.

Dobrava Virus Carried by the Yellow-Necked Field Mouse Apodemus flavicollis, Causing Hemorrhagic Fever with Renal Syndrome in Romania

PubMed Central

Panculescu-Gatej, Raluca Ioana; Sirbu, Anca; Dinu, Sorin; Waldstrom, Maria; Heyman, Paul; Murariu, Dimitru; Petrescu, Angela; Szmal, Camelia; Oprisan, Gabriela; Lundkvist, Åke

2014-01-01

Abstract Hemorrhagic fever with renal syndrome (HFRS) has been confirmed by serological methods during recent years in Romania. In the present study, focus-reduction neutralization tests (FRNT) confirmed Dobrava hantavirus (DOBV) as the causative agent in some HFRS cases, but could not distinguish between DOBV and Saaremaa virus (SAAV) infections in other cases. DOBV was detected by a DOBV-specific TaqMan assay in sera of nine patients out of 22 tested. Partial sequences of the M genomic segment of DOBV were obtained from sera of three patients and revealed the circulation of two DOBV lineages in Romania. Investigation of rodents trapped in Romania found three DOBV-positive Apodemus flavicollis out of 83 rodents tested. Two different DOBV lineages were also detected in A. flavicollis as determined from partial sequences of the M and S genomic segments. Sequences of DOBV in A. flavicollis were either identical or closely related to the sequences obtained from the HFRS patients. The DOBV strains circulating in Romania clustered in two monophyletic groups, together with strains from Slovenia and the north of Greece. This is the first evidence for the circulation of DOBV in wild rodents and for a DOBV etiology of HFRS in Romania. PMID:24746107

Dobrava virus carried by the yellow-necked field mouse Apodemus flavicollis, causing hemorrhagic fever with renal syndrome in Romania.

PubMed

Panculescu-Gatej, Raluca Ioana; Sirbu, Anca; Dinu, Sorin; Waldstrom, Maria; Heyman, Paul; Murariu, Dimitru; Petrescu, Angela; Szmal, Camelia; Oprisan, Gabriela; Lundkvist, Ake; Ceianu, Cornelia S

2014-05-01

Hemorrhagic fever with renal syndrome (HFRS) has been confirmed by serological methods during recent years in Romania. In the present study, focus-reduction neutralization tests (FRNT) confirmed Dobrava hantavirus (DOBV) as the causative agent in some HFRS cases, but could not distinguish between DOBV and Saaremaa virus (SAAV) infections in other cases. DOBV was detected by a DOBV-specific TaqMan assay in sera of nine patients out of 22 tested. Partial sequences of the M genomic segment of DOBV were obtained from sera of three patients and revealed the circulation of two DOBV lineages in Romania. Investigation of rodents trapped in Romania found three DOBV-positive Apodemus flavicollis out of 83 rodents tested. Two different DOBV lineages were also detected in A. flavicollis as determined from partial sequences of the M and S genomic segments. Sequences of DOBV in A. flavicollis were either identical or closely related to the sequences obtained from the HFRS patients. The DOBV strains circulating in Romania clustered in two monophyletic groups, together with strains from Slovenia and the north of Greece. This is the first evidence for the circulation of DOBV in wild rodents and for a DOBV etiology of HFRS in Romania.

The complete sequence and promoter activity of the human A-raf-1 gene (ARAF1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.E.; Beck, T.W.; Brennscheidt, U.

1994-03-01

The raf proto-oncogenes encode cytoplasmic protein serine/threonine kinases, which play a critical role in cell growth and development. One of these, A-raf-1 (human gene symbol, ARAF1), which is predominantly expressed in mouse urogenital tissues, has been mapped to an evolutionarily conserved linkage group composed of ARAF1, SYN1, TIMP, and properdin located at human chromosome Xp11.2. The authors have isolated human genomic DNA clones containing the expressed gene (ARAF1) on the X chromosome and a pseudogene (ARAF2) on chromosome 7p12-q11.21. Analysis of the nucleotide sequence from the ARAF1 genomic clones demonstrated that it consists of 16 exons encoded by minimally 10,776more » nucleotides. The major transcriptional start site (+1) was determined by RNase protection and primer extension assays. Promoter activity was confirmed by functional assays using DNA fragments fused to a CAT reporter gene. The ARAF1 minimal promoter, located between nucleotides -59 and +93, has a low G + C content and lacks consensus TATA and Inr sequences but shows sequence similarity at position -1 to the E box that is known to interact with USF and TFII-I transcription factors. 65 refs., 7 figs., 1 tab.« less

Transcript map of the Ovum mutant (Om) locus: isolation by exon trapping of new candidate genes for the DDK syndrome.

PubMed

Le Bras, Stéphanie; Cohen-Tannoudji, Michel; Guyot, Valérie; Vandormael-Pournin, Sandrine; Coumailleau, Franck; Babinet, Charles; Baldacci, Patricia

2002-08-21

The DDK syndrome is defined as the embryonic lethality of F1 mouse embryos from crosses between DDK females and males from other strains (named hereafter as non-DDK strains). Genetically controlled by the Ovum mutant (Om) locus, it is due to a deleterious interaction between a maternal factor present in DDK oocytes and the non-DDK paternal pronucleus. Therefore, the DDK syndrome constitutes a unique genetic tool to study the crucial interactions that take place between the parental genomes and the egg cytoplasm during mammalian development. In this paper, we present an extensive analysis performed by exon trapping on the Om region. Twenty-seven trapped sequences were from genes in the databases: beta-adaptin, CCT zeta2, DNA LigaseIII, Notchless, Rad51l3 and Scya1. Twenty-eight other sequences presented similarities with expressed sequence tags and genomic sequences whereas 57 did not. The pattern of expression of 37 of these markers was established. Importantly, five of them are expressed in DDK oocytes and are candidate genes for the maternal factor, and 20 are candidate genes for the paternal factor since they are expressed in testis. This data is an important step towards identifying the genes responsible for the DDK syndrome.

cDNA cloning and characterization of the human THRAP2 gene which maps to chromosome 12q24, and its mouse ortholog Thrap2.

PubMed

Musante, Luciana; Bartsch, Oliver; Ropers, Hans-Hilger; Kalscheuer, Vera M

2004-05-12

Characterization of a balanced t(2;12)(q37;q24) translocation in a patient with suspicion of Noonan syndrome revealed that the chromosome 12 breakpoint lies in the vicinity of a novel human gene, thyroid hormone receptor-associated protein 2 (THRAP2). We therefore characterized this gene and its mouse counterpart in more detail. Human and mouse THRAP2/Thrap2 span a genomic region of about 310 and >170 kilobases (kb), and both contain 31 exons. Corresponding transcripts are approximately 9.5 kb long. Their open reading frames code for proteins of 2210 and 2203 amino acids, which are 93% identical. By northern blot analysis, human and mouse THRAP2/Thrap2 genes showed ubiquitous expression. Transcripts were most abundant in human skeletal muscle and in mouse heart. THRAP2 protein is 56% identical to human TRAP240, which belongs to the thyroid hormone receptor associated protein (TRAP) complex and is evolutionary conserved up to yeast. This complex is involved in transcriptional regulation and is believed to serve as adapting interface between regulatory proteins bound to specific DNA sequences and RNA polymerase II.

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

PubMed

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

NASA Astrophysics Data System (ADS)

Licatalosi, Donny D.; Mele, Aldo; Fak, John J.; Ule, Jernej; Kayikci, Melis; Chi, Sung Wook; Clark, Tyson A.; Schweitzer, Anthony C.; Blume, John E.; Wang, Xuning; Darnell, Jennifer C.; Darnell, Robert B.

2008-11-01

Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.

Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.

PubMed

Song, Chun-Xiao; Szulwach, Keith E; Fu, Ye; Dai, Qing; Yi, Chengqi; Li, Xuekun; Li, Yujing; Chen, Chih-Hsin; Zhang, Wen; Jian, Xing; Wang, Jing; Zhang, Li; Looney, Timothy J; Zhang, Baichen; Godley, Lucy A; Hicks, Leslie M; Lahn, Bruce T; Jin, Peng; He, Chuan

2011-01-01

In contrast to 5-methylcytosine (5-mC), which has been studied extensively, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC-containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.

Divergent clonal selection dominates medulloblastoma at recurrence

PubMed Central

Morrissy, A. Sorana; Garzia, Livia; Shih, David J. H.; Zuyderduyn, Scott; Huang, Xi; Skowron, Patryk; Remke, Marc; Cavalli, Florence M. G.; Ramaswamy, Vijay; Lindsay, Patricia E.; Jelveh, Salomeh; Donovan, Laura K.; Wang, Xin; Luu, Betty; Zayne, Kory; Li, Yisu; Mayoh, Chelsea; Thiessen, Nina; Mercier, Eloi; Mungall, Karen L.; Ma, Yusanne; Tse, Kane; Zeng, Thomas; Shumansky, Karey; Roth, Andrew J. L.; Shah, Sohrab; Farooq, Hamza; Kijima, Noriyuki; Holgado, Borja L.; Lee, John J. Y.; Matan-Lithwick, Stuart; Liu, Jessica; Mack, Stephen C.; Manno, Alex; Michealraj, K. A.; Nor, Carolina; Peacock, John; Qin, Lei; Reimand, Juri; Rolider, Adi; Thompson, Yuan Y.; Wu, Xiaochong; Pugh, Trevor; Ally, Adrian; Bilenky, Mikhail; Butterfield, Yaron S. N.; Carlsen, Rebecca; Cheng, Young; Chuah, Eric; Corbett, Richard D.; Dhalla, Noreen; He, An; Lee, Darlene; Li, Haiyan I.; Long, William; Mayo, Michael; Plettner, Patrick; Qian, Jenny Q.; Schein, Jacqueline E.; Tam, Angela; Wong, Tina; Birol, Inanc; Zhao, Yongjun; Faria, Claudia C.; Pimentel, José; Nunes, Sofia; Shalaby, Tarek; Grotzer, Michael; Pollack, Ian F.; Hamilton, Ronald L.; Li, Xiao-Nan; Bendel, Anne E.; Fults, Daniel W.; Walter, Andrew W.; Kumabe, Toshihiro; Tominaga, Teiji; Collins, V. Peter; Cho, Yoon-Jae; Hoffman, Caitlin; Lyden, David; Wisoff, Jeffrey H.; Garvin, James H.; Stearns, Duncan S.; Massimi, Luca; Schüller, Ulrich; Sterba, Jaroslav; Zitterbart, Karel; Puget, Stephanie; Ayrault, Olivier; Dunn, Sandra E.; Tirapelli, Daniela P. C.; Carlotti, Carlos G.; Wheeler, Helen; Hallahan, Andrew R.; Ingram, Wendy; MacDonald, Tobey J.; Olson, Jeffrey J.; Van Meir, Erwin G.; Lee, Ji-Yeoun; Wang, Kyu-Chang; Kim, Seung-Ki; Cho, Byung-Kyu; Pietsch, Torsten; Fleischhack, Gudrun; Tippelt, Stephan; Ra, Young Shin; Bailey, Simon; Lindsey, Janet C.; Clifford, Steven C.; Eberhart, Charles G.; Cooper, Michael K.; Packer, Roger J.; Massimino, Maura; Garre, Maria Luisa; Bartels, Ute; Tabori, Uri; Hawkins, Cynthia E.; Dirks, Peter; Bouffet, Eric; Rutka, James T.; Wechsler-Reya, Robert J.; Weiss, William A.; Collier, Lara S.; Dupuy, Adam J.; Korshunov, Andrey; Jones, David T. W.; Kool, Marcel; Northcott, Paul A.; Pfister, Stefan M.; Largaespada, David A.; Mungall, Andrew J.; Moore, Richard A.; Jabado, Nada; Bader, Gary D.; Jones, Steven J. M.; Malkin, David; Marra, Marco A.; Taylor, Michael D.

2016-01-01

The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon–driven, functional genomic mouse model of medulloblastoma with ‘humanized’ in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (<5%). Whole-genome sequencing of 33 pairs of human diagnostic and post-therapy medulloblastomas demonstrated substantial genetic divergence of the dominant clone after therapy (<12% diagnostic events were retained at recurrence). In both mice and humans, the dominant clone at recurrence arose through clonal selection of a pre-existing minor clone present at diagnosis. Targeted therapy is unlikely to be effective in the absence of the target, therefore our results offer a simple, proximal, and remediable explanation for the failure of prior clinical trials of targeted therapy. PMID:26760213

Identification and characterization of a novel serine-threonine kinase gene from the Xp22 region.

PubMed

Montini, E; Andolfi, G; Caruso, A; Buchner, G; Walpole, S M; Mariani, M; Consalez, G; Trump, D; Ballabio, A; Franco, B

1998-08-01

Eukaryotic protein kinases are part of a large and expanding family of proteins. Through our transcriptional mapping effort in the Xp22 region, we have isolated and sequenced the full-length transcript of STK9, a novel cDNA highly homologous to serine-threonine kinases. A number of human genetic disorders have been mapped to the region where STK9 has been localized including Nance-Horan (NH) syndrome, oral-facial-digital syndrome type 1 (OFD1), and a novel locus for nonsyndromic sensorineural deafness (DFN6). To evaluate the possible involvement of STK9 in any of the above-mentioned disorders, a 2416-bp full-length cDNA was assembled. The entire genomic structure of the gene, which is composed of 20 coding exons, was determined. Northern analysis revealed a transcript larger than 9.5 kb in several tissues including brain, lung, and kidney. The mouse homologue (Stk9) was identified and mapped in the mouse in the region syntenic to human Xp. This location is compatible with the location of the Xcat mutant, which shows congenital cataracts very similar to those observed in NH patients. Sequence homologies, expression pattern, and mapping information in both human and mouse make STK9 a candidate gene for the above-mentioned disorders. Copyright 1998 Academic Press.

Mapping of the X-linked cataract (Xcat) mutation, the gene implicated in the Nance Horan syndrome, on the mouse X chromosome.

PubMed

Stambolian, D; Favor, J; Silvers, W; Avner, P; Chapman, V; Zhou, E

1994-07-15

The Xcat mutation in the mouse, an X-linked inherited disorder, is characterized by the congenital onset of cataracts. The cataracts have morphologies similar to those of cataracts found in the human Nance Horan (X-linked cataract dental) syndrome, suggesting that Xcat is an animal model for Nance Horan. The Xcat mutation provides an opportunity to investigate, at the molecular level, the pathogenesis of cataract. As a first step to cloning the Xcat gene, we report the localization of the Xcat mutation with respect to known molecular markers on the mouse X chromosome. Back-cross progeny carrying the Xcat mutation were obtained from an interspecific cross. Genomic DNA from each mouse was subjected to Southern and PCR analysis to identify restriction fragment length polymorphisms and simple sequence length polymorphisms, respectively. Our results refine the location of Xcat to a 2-cM region, eliminate several genes from consideration as the Xcat mutation, identify molecular probes tightly linked with Xcat, and suggest candidate genes responsible for the Xcat phenotype.

Structural analysis of key gap junction domains--Lessons from genome data and disease-linked mutants.

PubMed

Bai, Donglin

2016-02-01

A gap junction (GJ) channel is formed by docking of two GJ hemichannels and each of these hemichannels is a hexamer of connexins. All connexin genes have been identified in human, mouse, and rat genomes and their homologous genes in many other vertebrates are available in public databases. The protein sequences of these connexins align well with high sequence identity in the same connexin across different species. Domains in closely related connexins and several residues in all known connexins are also well-conserved. These conserved residues form signatures (also known as sequence logos) in these domains and are likely to play important biological functions. In this review, the sequence logos of individual connexins, groups of connexins with common ancestors, and all connexins are analyzed to visualize natural evolutionary variations and the hot spots for human disease-linked mutations. Several gap junction domains are homologous, likely forming similar structures essential for their function. The availability of a high resolution Cx26 GJ structure and the subsequently-derived homology structure models for other connexin GJ channels elevated our understanding of sequence logos at the three-dimensional GJ structure level, thus facilitating the understanding of how disease-linked connexin mutants might impair GJ structure and function. This knowledge will enable the design of complementary variants to rescue disease-linked mutants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Next generation sequencing and analysis of a conserved transcriptome of New Zealand's kiwi.

PubMed

Subramanian, Sankar; Huynen, Leon; Millar, Craig D; Lambert, David M

2010-12-15

Kiwi is a highly distinctive, flightless and endangered ratite bird endemic to New Zealand. To understand the patterns of molecular evolution of the nuclear protein-coding genes in brown kiwi (Apteryx australis mantelli) and to determine the timescale of avian history we sequenced a transcriptome obtained from a kiwi embryo using next generation sequencing methods. We then assembled the conserved protein-coding regions using the chicken proteome as a scaffold. Using 1,543 conserved protein coding genes we estimated the neutral evolutionary divergence between the kiwi and chicken to be ~45%, which is approximately equal to the divergence computed for the human-mouse pair using the same set of genes. A large fraction of genes was found to be under high selective constraint, as most of the expressed genes appeared to be involved in developmental gene regulation. Our study suggests a significant relationship between gene expression levels and protein evolution. Using sequences from over 700 nuclear genes we estimated the divergence between the two basal avian groups, Palaeognathae and Neognathae to be 132 million years, which is consistent with previous studies using mitochondrial genes. The results of this investigation revealed patterns of mutation and purifying selection in conserved protein coding regions in birds. Furthermore this study suggests a relatively cost-effective way of obtaining a glimpse into the fundamental molecular evolutionary attributes of a genome, particularly when no closely related genomic sequence is available.

FULL-GENOME ANALYSIS OF ALTERNATIVE SPLICING IN MOUSE LIVER AFTER HEPATOTOXICANT EXPOSURE

EPA Science Inventory

Alternative splicing plays a role in determining gene function and protein diversity. We have employed whole genome exon profiling using Affymetrix Mouse Exon 1.0 ST arrays to understand the significance of alternative splicing on a genome-wide scale in response to multiple toxic...

Identification and Sequencing of a Novel Rodent Gammaherpesvirus That Establishes Acute and Latent Infection in Laboratory Mice ▿

PubMed Central

Loh, Joy; Zhao, Guoyan; Nelson, Christopher A.; Coder, Penny; Droit, Lindsay; Handley, Scott A.; Johnson, L. Steven; Vachharajani, Punit; Guzman, Hilda; Tesh, Robert B.; Wang, David; Fremont, Daved H.; Virgin, Herbert W.

2011-01-01

Gammaherpesviruses encode numerous immunomodulatory molecules that contribute to their ability to evade the host immune response and establish persistent, lifelong infections. As the human gammaherpesviruses are strictly species specific, small animal models of gammaherpesvirus infection, such as murine gammaherpesvirus 68 (γHV68) infection, are important for studying the roles of gammaherpesvirus immune evasion genes in in vivo infection and pathogenesis. We report here the genome sequence and characterization of a novel rodent gammaherpesvirus, designated rodent herpesvirus Peru (RHVP), that shares conserved genes and genome organization with γHV68 and the primate gammaherpesviruses but is phylogenetically distinct from γHV68. RHVP establishes acute and latent infection in laboratory mice. Additionally, RHVP contains multiple open reading frames (ORFs) not present in γHV68 that have sequence similarity to primate gammaherpesvirus immunomodulatory genes or cellular genes. These include ORFs with similarity to major histocompatibility complex class I (MHC-I), C-type lectins, and the mouse mammary tumor virus and herpesvirus saimiri superantigens. As these ORFs may function as immunomodulatory or virulence factors, RHVP presents new opportunities for the study of mechanisms of immune evasion by gammaherpesviruses. PMID:21209105

Bioinformatics Pipelines for Targeted Resequencing and Whole-Exome Sequencing of Human and Mouse Genomes: A Virtual Appliance Approach for Instant Deployment

PubMed Central

Saeed, Isaam; Wong, Stephen Q.; Mar, Victoria; Goode, David L.; Caramia, Franco; Doig, Ken; Ryland, Georgina L.; Thompson, Ella R.; Hunter, Sally M.; Halgamuge, Saman K.; Ellul, Jason; Dobrovic, Alexander; Campbell, Ian G.; Papenfuss, Anthony T.; McArthur, Grant A.; Tothill, Richard W.

2014-01-01

Targeted resequencing by massively parallel sequencing has become an effective and affordable way to survey small to large portions of the genome for genetic variation. Despite the rapid development in open source software for analysis of such data, the practical implementation of these tools through construction of sequencing analysis pipelines still remains a challenging and laborious activity, and a major hurdle for many small research and clinical laboratories. We developed TREVA (Targeted REsequencing Virtual Appliance), making pre-built pipelines immediately available as a virtual appliance. Based on virtual machine technologies, TREVA is a solution for rapid and efficient deployment of complex bioinformatics pipelines to laboratories of all sizes, enabling reproducible results. The analyses that are supported in TREVA include: somatic and germline single-nucleotide and insertion/deletion variant calling, copy number analysis, and cohort-based analyses such as pathway and significantly mutated genes analyses. TREVA is flexible and easy to use, and can be customised by Linux-based extensions if required. TREVA can also be deployed on the cloud (cloud computing), enabling instant access without investment overheads for additional hardware. TREVA is available at http://bioinformatics.petermac.org/treva/. PMID:24752294

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform.

PubMed

Li, Po-E; Lo, Chien-Chi; Anderson, Joseph J; Davenport, Karen W; Bishop-Lilly, Kimberly A; Xu, Yan; Ahmed, Sanaa; Feng, Shihai; Mokashi, Vishwesh P; Chain, Patrick S G

2017-01-09

Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the ease of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. This bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

PubMed

Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

2017-05-01

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

GraphTeams: a method for discovering spatial gene clusters in Hi-C sequencing data.

PubMed

Schulz, Tizian; Stoye, Jens; Doerr, Daniel

2018-05-08

Hi-C sequencing offers novel, cost-effective means to study the spatial conformation of chromosomes. We use data obtained from Hi-C experiments to provide new evidence for the existence of spatial gene clusters. These are sets of genes with associated functionality that exhibit close proximity to each other in the spatial conformation of chromosomes across several related species. We present the first gene cluster model capable of handling spatial data. Our model generalizes a popular computational model for gene cluster prediction, called δ-teams, from sequences to graphs. Following previous lines of research, we subsequently extend our model to allow for several vertices being associated with the same label. The model, called δ-teams with families, is particular suitable for our application as it enables handling of gene duplicates. We develop algorithmic solutions for both models. We implemented the algorithm for discovering δ-teams with families and integrated it into a fully automated workflow for discovering gene clusters in Hi-C data, called GraphTeams. We applied it to human and mouse data to find intra- and interchromosomal gene cluster candidates. The results include intrachromosomal clusters that seem to exhibit a closer proximity in space than on their chromosomal DNA sequence. We further discovered interchromosomal gene clusters that contain genes from different chromosomes within the human genome, but are located on a single chromosome in mouse. By identifying δ-teams with families, we provide a flexible model to discover gene cluster candidates in Hi-C data. Our analysis of Hi-C data from human and mouse reveals several known gene clusters (thus validating our approach), but also few sparsely studied or possibly unknown gene cluster candidates that could be the source of further experimental investigations.

Identification and Differential Abundance of Mitochondrial Genome Encoding Small RNAs (mitosRNA) in Breast Muscles of Modern Broilers and Unselected Chicken Breed

PubMed Central

Bottje, Walter G.; Khatri, Bhuwan; Shouse, Stephanie A.; Seo, Dongwon; Mallmann, Barbara; Orlowski, Sara K.; Pan, Jeonghoon; Kong, Seongbae; Owens, Casey M.; Anthony, Nicholas B.; Kim, Jae K.; Kong, Byungwhi C.

2017-01-01

Background: Although small non-coding RNAs are mostly encoded by the nuclear genome, thousands of small non-coding RNAs encoded by the mitochondrial genome, termed as mitosRNAs were recently reported in human, mouse and trout. In this study, we first identified chicken mitosRNAs in breast muscle using small RNA sequencing method and the differential abundance was analyzed between modern pedigree male (PeM) broilers (characterized by rapid growth and large muscle mass) and the foundational Barred Plymouth Rock (BPR) chickens (characterized by slow growth and small muscle mass). Methods: Small RNA sequencing was performed with total RNAs extracted from breast muscles of PeM and BPR (n = 6 per group) using the 1 × 50 bp single end read method of Illumina sequencing. Raw reads were processed by quality assessment, adapter trimming, and alignment to the chicken mitochondrial genome (GenBank Accession: X52392.1) using the NGen program. Further statistical analyses were performed using the JMP Genomics 8. Differentially expressed (DE) mitosRNAs between PeM and BPR were confirmed by quantitative PCR. Results: Totals of 183,416 unique small RNA sequences were identified as potential chicken mitosRNAs. After stringent filtering processes, 117 mitosRNAs showing >100 raw read counts were abundantly produced from all 37 mitochondrial genes (except D-loop region) and the length of mitosRNAs ranged from 22 to 46 nucleotides. Of those, abundance of 44 mitosRNAs were significantly altered in breast muscles of PeM compared to those of BPR: all mitosRNAs were higher in PeM breast except those produced from 16S-rRNA gene. Possibly, the higher mitosRNAs abundance in PeM breast may be due to a higher mitochondrial content compared to BPR. Our data demonstrate that in addition to 37 known mitochondrial genes, the mitochondrial genome also encodes abundant mitosRNAs, that may play an important regulatory role in muscle growth via mitochondrial gene expression control. PMID:29104541

Detecting differential DNA methylation from sequencing of bisulfite converted DNA of diverse species.

PubMed

Huh, Iksoo; Wu, Xin; Park, Taesung; Yi, Soojin V

2017-07-21

DNA methylation is one of the most extensively studied epigenetic modifications of genomic DNA. In recent years, sequencing of bisulfite-converted DNA, particularly via next-generation sequencing technologies, has become a widely popular method to study DNA methylation. This method can be readily applied to a variety of species, dramatically expanding the scope of DNA methylation studies beyond the traditionally studied human and mouse systems. In parallel to the increasing wealth of genomic methylation profiles, many statistical tools have been developed to detect differentially methylated loci (DMLs) or differentially methylated regions (DMRs) between biological conditions. We discuss and summarize several key properties of currently available tools to detect DMLs and DMRs from sequencing of bisulfite-converted DNA. However, the majority of the statistical tools developed for DML/DMR analyses have been validated using only mammalian data sets, and less priority has been placed on the analyses of invertebrate or plant DNA methylation data. We demonstrate that genomic methylation profiles of non-mammalian species are often highly distinct from those of mammalian species using examples of honey bees and humans. We then discuss how such differences in data properties may affect statistical analyses. Based on these differences, we provide three specific recommendations to improve the power and accuracy of DML and DMR analyses of invertebrate data when using currently available statistical tools. These considerations should facilitate systematic and robust analyses of DNA methylation from diverse species, thus advancing our understanding of DNA methylation. © The Author 2017. Published by Oxford University Press.

Cloning of human cDNAs for Apg-1 and Apg-2, members of the Hsp110 family, and chromosomal assignment of their genes.

PubMed

Nonoguchi, K; Itoh, K; Xue, J H; Tokuchi, H; Nishiyama, H; Kaneko, Y; Tatsumi, K; Okuno, H; Tomiwa, K; Fujita, J

1999-09-03

In mice, the Hsp110/SSE family is composed of the heat shock protein (Hsp)110/105, Apg-1 and Apg-2. In humans, however, only the Hsp110/105 homolog has been identified as a member, and two cDNAs, Hsp70RY and HS24/p52, potentially encoding proteins structurally similar to, but smaller than, mouse Apg-2 have been reported. To clarify the membership of Hsp110 family in humans, we isolated Apg-1 and Apg-2 cDNAs from a human testis cDNA library. The human Apg-1 was 100% and 91.8% identical in length and amino acid (aa) sequence, respectively, to mouse Apg-1. Human Apg-2 was one aa shorter than and 95.5% identical in sequence to mouse Apg-2. In ECV304, human endothelial cells Apg-1 but not Apg-2 transcripts were induced in 2 h by a temperature shift from 32 degrees C to 39 degrees C. As found in mice, the response was stronger than that to a 37-42 degrees C shift. The human Apg-1 and Apg-2 genes were mapped to the chromosomal loci 4q28 and 5q23.3-q31.1, respectively, by fluorescence in-situ hybridization. We isolated cDNA and genomic clones encompassing the region critical for the difference between Apg-2 and HS24/p52. Although the primer sets used were derived from the sequences common to both cDNAs, all cDNA and genomic clones corresponded to Apg-2. Using a similar approach, the relationship between Apg-2 and Hsp70RY was assessed, and no clone corresponding to Hsp70RY was obtained. These results demonstrated that the Hsp110 family consists of at least three members, Apg-1, Apg-2 and Hsp110 in humans as well as in mice. The significance of HS24/p52 and Hsp70RY cDNAs previously reported remains to be determined.

The structure of the coding and 5'-flanking region of the type 1 iodothyronine deiodinase (dio1) gene is normal in a patient with suspected congenital dio1 deficiency.

PubMed

Toyoda, N; Kleinhaus, N; Larsen, P R

1996-06-01

We analyzed the exon-intron structure of the human type 1 deiodinase gene (dio1) and compared it with that of a patient with suspected congenital type 1 deiodinase (D1) deficiency. The hdio1 gene is identical in exon-intron arrangement to the mouse gene, with coding sequences and a selenocysteine insertion sequence (SECIS) element contained in four exons. There were no mutations in the sequences of exons 1-4 of the patient's genomic DNA. Functional studies by transient expression techniques showed no difference in basal promoter activity or T3 responsiveness between the patient's and the normal dio1 gene. A structural abnormality in the dio1 gene is not a likely explanation for this patient's D1-deficient phenotype.

Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

PubMed Central

Holguín, Angela V.; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C.; Barrios, Andrés Fernando González; Vives, Martha J.

2015-01-01

Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. PMID:26274971

Brain Connectivity as a DNA Sequencing Problem

NASA Astrophysics Data System (ADS)

Zador, Anthony

The mammalian cortex consists of millions or billions of neurons, each connected to thousands of other neurons. Traditional methods for determining the brain connectivity rely on microscopy to visualize neuronal connections, but such methods are slow, labor-intensive and often lack single neuron resolution. We have recently developed a new method, MAPseq, to recast the determination of brain wiring into a form that can exploit the tremendous recent advances in high-throughput DNA sequencing. DNA sequencing technology has outpaced even Moore's law, so that the cost of sequencing the human genome has dropped from a billion dollars in 2001 to below a thousand dollars today. MAPseq works by introducing random sequences of DNA-``barcodes''-to tag neurons uniquely. With MAPseq, we can determine the connectivity of over 50K single neurons in a single mouse cortex in about a week, an unprecedented throughput, ushering in the era of ``big data'' for brain wiring. We are now developing analytical tools and algorithms to make sense of these novel data sets.

A murine host cell factor required for nicking of the dimer bridge of MVM recognizes two CG nucleotides displaced by 10 basepairs.

PubMed

Liu, Q; Astell, C R

1996-10-01

During replication of the minute virus of mice (MVM) genome, a dimer replicative form (RF) intermediate is resolved into two monomer RF molecules in such a way as to retain a unique sequence within the left hand hairpin terminus of the viral genome. Although the proposed mechanism for resolution of the dimer RF remains uncertain, it likely involves site-specific nicking of the dimer bridge. The RF contains two double-stranded copies of the viral genome joined by the extended 3' hairpin. Minor sequence asymmetries within the 3' hairpin allow the two halves of the dimer bridge to be distinguished. The A half contains the sequence [sequence: see text], whereas the B half contains the sequence [sequence: see text]. Using an in vitro assay, we show that only the B half of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular extract. When highly purified NS-1, the major nonstructural protein of MVM, is used in this nicking reaction, there is an absolute requirement for the LA9 cellular extract, suggesting a cellular factor (or factors) is (are) required. A series of mutations were created in the putative host factor binding region (HFBR) on the B half of the MVM dimer bridge adjacent to the NS-1 binding site. Nicking assays of these B half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrated that a host factor(s) can bind to the HFBR of the B half of the dimer bridge and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wild-type HFBR sequence is able to specifically inhibit nicking of the B half, indicating that the host factor(s) bound to the HFBR is(are) essential for site-specific nicking to occur.

E-TALEN: a web tool to design TALENs for genome engineering.

PubMed

Heigwer, Florian; Kerr, Grainne; Walther, Nike; Glaeser, Kathrin; Pelz, Oliver; Breinig, Marco; Boutros, Michael

2013-11-01

Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN (http://www.e-talen.org), a web-based tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. E-TALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis, which can be extended in the future.

Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence.

PubMed

Meuleman, Wouter; Peric-Hupkes, Daan; Kind, Jop; Beaudry, Jean-Bernard; Pagie, Ludo; Kellis, Manolis; Reinders, Marcel; Wessels, Lodewyk; van Steensel, Bas

2013-02-01

In metazoans, the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes, by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific, while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps, we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover, cLADs are depleted of synteny breakpoints, pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically, the overall sequence conservation is low for cLADs. Instead, cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this "A/T rule" in embryonic stem cells, but not in differentiated cells. This suggests that the A/T rule represents a default positioning mechanism that is locally overruled during lineage commitment. Analysis of paralogs suggests that during evolution changes in A/T content have driven the relocation of genes to and from the nuclear lamina, in tight association with changes in expression level. Taken together, these results reveal that the spatial organization of mammalian genomes is highly conserved and tightly linked to local nucleotide composition.

Ancient Exaptation of a CORE-SINE Retroposon into a Highly Conserved Mammalian Neuronal Enhancer of the Proopiomelanocortin Gene

PubMed Central

Bumaschny, Viviana F; Low, Malcolm J; Rubinstein, Marcelo

2007-01-01

The proopiomelanocortin gene (POMC) is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5′ distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE) retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORE-SINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution. PMID:17922573

Predicting Functions of Proteins in Mouse Based on Weighted Protein-Protein Interaction Network and Protein Hybrid Properties

PubMed Central

Shi, Xiaohe; Lu, Wen-Cong; Cai, Yu-Dong; Chou, Kuo-Chen

2011-01-01

Background With the huge amount of uncharacterized protein sequences generated in the post-genomic age, it is highly desirable to develop effective computational methods for quickly and accurately predicting their functions. The information thus obtained would be very useful for both basic research and drug development in a timely manner. Methodology/Principal Findings Although many efforts have been made in this regard, most of them were based on either sequence similarity or protein-protein interaction (PPI) information. However, the former often fails to work if a query protein has no or very little sequence similarity to any function-known proteins, while the latter had similar problem if the relevant PPI information is not available. In view of this, a new approach is proposed by hybridizing the PPI information and the biochemical/physicochemical features of protein sequences. The overall first-order success rates by the new predictor for the functions of mouse proteins on training set and test set were 69.1% and 70.2%, respectively, and the success rate covered by the results of the top-4 order from a total of 24 orders was 65.2%. Conclusions/Significance The results indicate that the new approach is quite promising that may open a new avenue or direction for addressing the difficult and complicated problem. PMID:21283518

Biology and Genomics of an Historic Therapeutic Escherichia coli Bacteriophage Collection.

PubMed

Baig, Abiyad; Colom, Joan; Barrow, Paul; Schouler, Catherine; Moodley, Arshnee; Lavigne, Rob; Atterbury, Robert

2017-01-01

We have performed microbiological and genomic characterization of an historic collection of nine bacteriophages, specifically infecting a K1 E. coli O18:K1:H7 ColV + strain. These phages were isolated from sewage and tested for their efficacy in vivo for the treatment of systemic E. coli infection in a mouse infection model by Smith and Huggins (1982). The aim of the study was to identify common microbiological and genomic characteristics, which co-relate to the performance of these phages in in vivo study. These features will allow an informed selection of phages for use as therapeutic agents. Transmission electron microscopy showed that six of the nine phages were Podoviridae and the remaining three were Siphoviridae . The four best performing phages in vivo belonged to the Podoviridae family. In vitro , these phages exhibited very short latent and rise periods in our study. In agreement with their microbiological profiles, characterization by genome sequencing showed that all six podoviruses belong to the Autographivirinae subfamily. Of these, four were isolates of the same species (99% identity), whereas two had divergent genomes compared to other podoviruses. The Siphoviridae phages, which were moderate to poor performers in vivo , exhibited longer latent and rise periods in vitro . Two of the three siphoviruses were closely related to each other (99% identity), but all can be associated with the Guernseyvirinae subfamily. Genome sequence comparison of both types of phages showed that a gene encoding for DNA-dependent RNA polymerase was only present in phages with faster replication cycle, which may account for their better performance in vivo . These data define a combination of microbiological, genomic and in vivo characteristics which allow a more rational evaluation of the original in vivo data and pave the way for the selection of phages for future phage therapy trails.

Human gastric cancer modelling using organoids.

PubMed

Seidlitz, Therese; Merker, Sebastian R; Rothe, Alexander; Zakrzewski, Falk; von Neubeck, Cläre; Grützmann, Konrad; Sommer, Ulrich; Schweitzer, Christine; Schölch, Sebastian; Uhlemann, Heike; Gaebler, Anne-Marlene; Werner, Kristin; Krause, Mechthild; Baretton, Gustavo B; Welsch, Thilo; Koo, Bon-Kyoung; Aust, Daniela E; Klink, Barbara; Weitz, Jürgen; Stange, Daniel E

2018-04-27

Gastric cancer is the second leading cause of cancer-related deaths and the fifth most common malignancy worldwide. In this study, human and mouse gastric cancer organoids were generated to model the disease and perform drug testing to delineate treatment strategies. Human gastric cancer organoid cultures were established, samples classified according to their molecular profile and their response to conventional chemotherapeutics tested. Targeted treatment was performed according to specific druggable mutations. Mouse gastric cancer organoid cultures were generated carrying molecular subtype-specific alterations. Twenty human gastric cancer organoid cultures were established and four selected for a comprehensive in-depth analysis. Organoids demonstrated divergent growth characteristics and morphologies. Immunohistochemistry showed similar characteristics to the corresponding primary tissue. A divergent response to 5-fluoruracil, oxaliplatin, irinotecan, epirubicin and docetaxel treatment was observed. Whole genome sequencing revealed a mutational spectrum that corresponded to the previously identified microsatellite instable, genomic stable and chromosomal instable subtypes of gastric cancer. The mutational landscape allowed targeted therapy with trastuzumab for ERBB2 alterations and palbociclib for CDKN2A loss. Mouse cancer organoids carrying Kras and Tp53 or Apc and Cdh1 mutations were characterised and serve as model system to study the signalling of induced pathways. We generated human and mouse gastric cancer organoids modelling typical characteristics and altered pathways of human gastric cancer. Successful interference with activated pathways demonstrates their potential usefulness as living biomarkers for therapy response testing. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Pisrt1, a gene implicated in XX sex reversal, is expressed in gonads of both sexes during mouse development.

PubMed

Loffler, Kelly A; Combes, Alexander N; Wilhelm, Dagmar; Beverdam, Annemiek; Bowles, Jo; Koopman, Peter

2005-01-01

XX sex reversal syndromes not involving Sry provide an opportunity to identify and study genes important for sexual development. The polled intersex syndrome (PIS) in goats, which shares some features with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) in humans, exemplifies such syndromes. BPES is caused by defects in the forkhead transcription factor gene FOXL2, while PIS is caused by a large deletion of goat chromosome 1q43 that affects transcription of the genes Pisrt1 and Foxl2. Pisrt1 is a non-translated gene that has a sexually dimorphic expression pattern in goats. Here, we describe the structure and expression of the mouse Pisrt1 locus, to investigate its likely role in ovarian development more broadly in mammals. This gene showed some sequence similarity, and was found in a similar genomic context, to its goat and human orthologues. Expression analyses indicated that Pisrt1 is transcribed, and its mRNA polyadenylated and exported to the cytoplasm, but no significant open reading frames were found in a 1.5kb mouse genomic region corresponding to goat Pisrt1. Pisrt1 transcripts were expressed very broadly among tissues of the developing mouse embryo, and at similar levels in male and female gonads at each stage examined, as determined by in situ hybridisation and RT-PCR. This profile of expression suggests that Pisrt1 is unlikely to contribute to sex-specific events during gonadal development in mice and that divergent pathways of ovarian development operate among different mammalian species.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xusheng; Pandey, Ashutosh K.; Mulligan, Megan K.

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ~5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ~4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets-by far the largest coherent phenome for any experimental cohort (www.genenetwork.org).more » Here, we tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.« less

Joint mouse–human phenome-wide association to test gene function and disease risk

DOE PAGES

Wang, Xusheng; Pandey, Ashutosh K.; Mulligan, Megan K.; ...

2016-02-02

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ~5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ~4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets-by far the largest coherent phenome for any experimental cohort (www.genenetwork.org).more » Here, we tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.« less

GREAM: A Web Server to Short-List Potentially Important Genomic Repeat Elements Based on Over-/Under-Representation in Specific Chromosomal Locations, Such as the Gene Neighborhoods, within or across 17 Mammalian Species

PubMed Central

Chandrashekar, Darshan Shimoga; Dey, Poulami; Acharya, Kshitish K.

2015-01-01

Background Genome-wide repeat sequences, such as LINEs, SINEs and LTRs share a considerable part of the mammalian nuclear genomes. These repeat elements seem to be important for multiple functions including the regulation of transcription initiation, alternative splicing and DNA methylation. But it is not possible to study all repeats and, hence, it would help to short-list before exploring their potential functional significance via experimental studies and/or detailed in silico analyses. Result We developed the ‘Genomic Repeat Element Analyzer for Mammals’ (GREAM) for analysis, screening and selection of potentially important mammalian genomic repeats. This web-server offers many novel utilities. For example, this is the only tool that can reveal a categorized list of specific types of transposons, retro-transposons and other genome-wide repetitive elements that are statistically over-/under-represented in regions around a set of genes, such as those expressed differentially in a disease condition. The output displays the position and frequency of identified elements within the specified regions. In addition, GREAM offers two other types of analyses of genomic repeat sequences: a) enrichment within chromosomal region(s) of interest, and b) comparative distribution across the neighborhood of orthologous genes. GREAM successfully short-listed a repeat element (MER20) known to contain functional motifs. In other case studies, we could use GREAM to short-list repetitive elements in the azoospermia factor a (AZFa) region of the human Y chromosome and those around the genes associated with rat liver injury. GREAM could also identify five over-represented repeats around some of the human and mouse transcription factor coding genes that had conserved expression patterns across the two species. Conclusion GREAM has been developed to provide an impetus to research on the role of repetitive sequences in mammalian genomes by offering easy selection of more interesting repeats in various contexts/regions. GREAM is freely available at http://resource.ibab.ac.in/GREAM/. PMID:26208093

Differential DNA Methylation Analysis without a Reference Genome.

PubMed

Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

2015-12-22

Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.

PubMed

Merienne, Nicolas; Vachey, Gabriel; de Longprez, Lucie; Meunier, Cécile; Zimmer, Virginie; Perriard, Guillaume; Canales, Mathieu; Mathias, Amandine; Herrgott, Lucas; Beltraminelli, Tim; Maulet, Axelle; Dequesne, Thomas; Pythoud, Catherine; Rey, Maria; Pellerin, Luc; Brouillet, Emmanuel; Perrier, Anselme L; du Pasquier, Renaud; Déglon, Nicole

2017-09-19

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Structural Variation Shapes the Landscape of Recombination in Mouse

PubMed Central

Morgan, Andrew P.; Gatti, Daniel M.; Najarian, Maya L.; Keane, Thomas M.; Galante, Raymond J.; Pack, Allan I.; Mott, Richard; Churchill, Gary A.; de Villena, Fernando Pardo-Manuel

2017-01-01

Meiotic recombination is an essential feature of sexual reproduction that ensures faithful segregation of chromosomes and redistributes genetic variants in populations. Multiparent populations such as the Diversity Outbred (DO) mouse stock accumulate large numbers of crossover (CO) events between founder haplotypes, and thus present a unique opportunity to study the role of genetic variation in shaping the recombination landscape. We obtained high-density genotype data from 6886 DO mice, and localized 2.2 million CO events to intervals with a median size of 28 kb. The resulting sex-averaged genetic map of the DO population is highly concordant with large-scale (order 10 Mb) features of previously reported genetic maps for mouse. To examine fine-scale (order 10 kb) patterns of recombination in the DO, we overlaid putative recombination hotspots onto our CO intervals. We found that CO intervals are enriched in hotspots compared to the genomic background. However, as many as 26% of CO intervals do not overlap any putative hotspots, suggesting that our understanding of hotspots is incomplete. We also identified coldspots encompassing 329 Mb, or 12% of observable genome, in which there is little or no recombination. In contrast to hotspots, which are a few kilobases in size, and widely scattered throughout the genome, coldspots have a median size of 2.1 Mb and are spatially clustered. Coldspots are strongly associated with copy-number variant (CNV) regions, especially multi-allelic clusters, identified from whole-genome sequencing of 228 DO mice. Genes in these regions have reduced expression, and epigenetic features of closed chromatin in male germ cells, which suggests that CNVs may repress recombination by altering chromatin structure in meiosis. Our findings demonstrate how multiparent populations, by bridging the gap between large-scale and fine-scale genetic mapping, can reveal new features of the recombination landscape. PMID:28592499

Structural Variation Shapes the Landscape of Recombination in Mouse.

PubMed

Morgan, Andrew P; Gatti, Daniel M; Najarian, Maya L; Keane, Thomas M; Galante, Raymond J; Pack, Allan I; Mott, Richard; Churchill, Gary A; de Villena, Fernando Pardo-Manuel

2017-06-01

Meiotic recombination is an essential feature of sexual reproduction that ensures faithful segregation of chromosomes and redistributes genetic variants in populations. Multiparent populations such as the Diversity Outbred (DO) mouse stock accumulate large numbers of crossover (CO) events between founder haplotypes, and thus present a unique opportunity to study the role of genetic variation in shaping the recombination landscape. We obtained high-density genotype data from [Formula: see text] DO mice, and localized 2.2 million CO events to intervals with a median size of 28 kb. The resulting sex-averaged genetic map of the DO population is highly concordant with large-scale (order 10 Mb) features of previously reported genetic maps for mouse. To examine fine-scale (order 10 kb) patterns of recombination in the DO, we overlaid putative recombination hotspots onto our CO intervals. We found that CO intervals are enriched in hotspots compared to the genomic background. However, as many as [Formula: see text] of CO intervals do not overlap any putative hotspots, suggesting that our understanding of hotspots is incomplete. We also identified coldspots encompassing 329 Mb, or [Formula: see text] of observable genome, in which there is little or no recombination. In contrast to hotspots, which are a few kilobases in size, and widely scattered throughout the genome, coldspots have a median size of 2.1 Mb and are spatially clustered. Coldspots are strongly associated with copy-number variant (CNV) regions, especially multi-allelic clusters, identified from whole-genome sequencing of 228 DO mice. Genes in these regions have reduced expression, and epigenetic features of closed chromatin in male germ cells, which suggests that CNVs may repress recombination by altering chromatin structure in meiosis. Our findings demonstrate how multiparent populations, by bridging the gap between large-scale and fine-scale genetic mapping, can reveal new features of the recombination landscape. Copyright © 2017 by the Genetics Society of America.

Genome-wide Analysis of RARβ Transcriptional Targets in Mouse Striatum Links Retinoic Acid Signaling with Huntington's Disease and Other Neurodegenerative Disorders.

PubMed

Niewiadomska-Cimicka, Anna; Krzyżosiak, Agnieszka; Ye, Tao; Podleśny-Drabiniok, Anna; Dembélé, Doulaye; Dollé, Pascal; Krężel, Wojciech

2017-07-01

Retinoic acid (RA) signaling through retinoic acid receptors (RARs), known for its multiple developmental functions, emerged more recently as an important regulator of adult brain physiology. How RAR-mediated regulation is achieved is poorly known, partly due to the paucity of information on critical target genes in the brain. Also, it is not clear how reduced RA signaling may contribute to pathophysiology of diverse neuropsychiatric disorders. We report the first genome-wide analysis of RAR transcriptional targets in the brain. Using chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis of RARβ-null mutant mice, we identified genomic targets of RARβ in the striatum. Characterization of RARβ transcriptional targets in the mouse striatum points to mechanisms through which RAR may control brain functions and display neuroprotective activity. Namely, our data indicate with statistical significance (FDR 0.1) a strong contribution of RARβ in controlling neurotransmission, energy metabolism, and transcription, with a particular involvement of G-protein coupled receptor (p = 5.0e -5 ), cAMP (p = 4.5e -4 ), and calcium signaling (p = 3.4e -3 ). Many identified RARβ target genes related to these pathways have been implicated in Alzheimer's, Parkinson's, and Huntington's disease (HD), raising the possibility that compromised RA signaling in the striatum may be a mechanistic link explaining the similar affective and cognitive symptoms in these diseases. The RARβ transcriptional targets were particularly enriched for transcripts affected in HD. Using the R6/2 transgenic mouse model of HD, we show that partial sequestration of RARβ in huntingtin protein aggregates may account for reduced RA signaling reported in HD.

The genome of Chelonid herpesvirus 5 harbors atypical genes

USGS Publications Warehouse

Ackermann, Mathias; Koriabine, Maxim; Hartmann-Fritsch, Fabienne; de Jong, Pieter J.; Lewis, Teresa D.; Schetle, Nelli; Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Leong, Jo-Ann C.

2012-01-01

The Chelonid fibropapilloma-associated herpesvirus (CFPHV; ChHV5) is believed to be the causative agent of fibropapillomatosis (FP), a neoplastic disease of marine turtles. While clinical signs and pathology of FP are well known, research on ChHV5 has been impeded because no cell culture system for its propagation exists. We have cloned a BAC containing ChHV5 in pTARBAC2.1 and determined its nucleotide sequence. Accordingly, ChHV5 has a type D genome and its predominant gene order is typical for the varicellovirus genus within thealphaherpesvirinae. However, at least four genes that are atypical for an alphaherpesvirus genome were also detected, i.e. two members of the C-type lectin-like domain superfamily (F-lec1, F-lec2), an orthologue to the mouse cytomegalovirus M04 (F-M04) and a viral sialyltransferase (F-sial). Four lines of evidence suggest that these atypical genes are truly part of the ChHV5 genome: (1) the pTARBAC insertion interrupted the UL52 ORF, leaving parts of the gene to either side of the insertion and suggesting that an intact molecule had been cloned. (2) Using FP-associated UL52 (F-UL52) as an anchor and the BAC-derived sequences as a means to generate primers, overlapping PCR was performed with tumor-derived DNA as template, which confirmed the presence of the same stretch of “atypical” DNA in independent FP cases. (3) Pyrosequencing of DNA from independent tumors did not reveal previously undetected viral sequences, suggesting that no apparent loss of viral sequence had happened due to the cloning strategy. (4) The simultaneous presence of previously known ChHV5 sequences and F-sial as well as F-M04 sequences was also confirmed in geographically distinct Australian cases of FP. Finally, transcripts of F-sial and F-M04 but not transcripts of lytic viral genes were detected in tumors from Hawaiian FP-cases. Therefore, we suggest that F-sial and F-M04 may play a role in FP pathogenesis

GeNemo: a search engine for web-based functional genomic data.

PubMed

Zhang, Yongqing; Cao, Xiaoyi; Zhong, Sheng

2016-07-08

A set of new data types emerged from functional genomic assays, including ChIP-seq, DNase-seq, FAIRE-seq and others. The results are typically stored as genome-wide intensities (WIG/bigWig files) or functional genomic regions (peak/BED files). These data types present new challenges to big data science. Here, we present GeNemo, a web-based search engine for functional genomic data. GeNemo searches user-input data against online functional genomic datasets, including the entire collection of ENCODE and mouse ENCODE datasets. Unlike text-based search engines, GeNemo's searches are based on pattern matching of functional genomic regions. This distinguishes GeNemo from text or DNA sequence searches. The user can input any complete or partial functional genomic dataset, for example, a binding intensity file (bigWig) or a peak file. GeNemo reports any genomic regions, ranging from hundred bases to hundred thousand bases, from any of the online ENCODE datasets that share similar functional (binding, modification, accessibility) patterns. This is enabled by a Markov Chain Monte Carlo-based maximization process, executed on up to 24 parallel computing threads. By clicking on a search result, the user can visually compare her/his data with the found datasets and navigate the identified genomic regions. GeNemo is available at www.genemo.org. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Functional Genomic Characterization of Virulence Factors from Necrotizing Fasciitis-Causing Strains of Aeromonas hydrophila

PubMed Central

Grim, Christopher J.; Kozlova, Elena V.; Ponnusamy, Duraisamy; Fitts, Eric C.; Sha, Jian; Kirtley, Michelle L.; van Lier, Christina J.; Tiner, Bethany L.; Erova, Tatiana E.; Joseph, Sandeep J.; Read, Timothy D.; Shak, Joshua R.; Joseph, Sam W.; Singletary, Ed; Felland, Tracy; Baze, Wallace B.; Horneman, Amy J.

2014-01-01

The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF. PMID:24795370

SC1 Promotes MiR124-3p Expression to Maintain the Self-Renewal of Mouse Embryonic Stem Cells by Inhibiting the MEK/ERK Pathway.

PubMed

Wei, Qing; Liu, Hongliang; Ai, Zhiying; Wu, Yongyan; Liu, Yingxiang; Shi, Zhaopeng; Ren, Xuexue; Guo, Zekun

2017-01-01

Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1. © 2017 The Author(s). Published by S. Karger AG, Basel.

Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

PubMed

Han, Zongchao; Zhong, Li; Maina, Njeri; Hu, Zhongbo; Li, Xiaomiao; Chouthai, Nitin S; Bischof, Daniela; Weigel-Van Aken, Kirsten A; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

2008-03-01

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

Maternal DCAF2 is crucial for maintenance of genome stability during the first cell cycle in mice.

PubMed

Xu, Yi-Wen; Cao, Lan-Rui; Wang, Min; Xu, Ying; Wu, Xin; Liu, Junping; Tong, Chao; Fan, Heng-Yu

2017-10-01

Precise regulation of DNA replication and genome integrity is crucial for gametogenesis and early embryogenesis. Cullin ring-finger ubiquitin ligase 4 (CRL4) has multiple functions in the maintenance of germ cell survival, oocyte meiotic maturation, and maternal-zygotic transition in mammals. DDB1-cullin-4-associated factor-2 (DCAF2, also known as DTL or CDT2) is an evolutionarily conserved substrate receptor of CRL4. To determine whether DCAF2 is a key CRL4 substrate adaptor in mammalian oocytes, we generated a novel mouse strain that carries a Dcaf2 allele flanked by l oxP sequences, and specifically deleted Dcaf2 in oocytes. Dcaf2 knockout in mouse oocytes leads to female infertility. Although Dcaf2 -null oocytes were able to develop and mature normally, the embryos derived from them were arrested at one- to two-cell stage, owing to prolonged DNA replication and accumulation of massive DNA damage. These results indicate that DCAF2 is a previously unrecognized maternal factor that safeguards zygotic genome stability. Maternal DCAF2 protein is crucial for prevention of DNA re-replication in the first and unique mitotic cell cycle of the zygote.This article has an associated First Person interview with the first author of the paper. © 2017. Published by The Company of Biologists Ltd.

Manipulation of the mouse genome: a multiple impact resource for drug discovery and development.

PubMed

Prosser, Haydn; Rastan, Sohaila

2003-05-01

Few would deny that the pharmaceutical industry's investment in genomics throughout the 1990s has yet to deliver in terms of drugs on the market. The reasons are complex and beyond the scope of this review. The unique ability to manipulate the mouse genome, however, has already had a positive impact on all stages of the drug discovery process and, increasingly, on the drug development process too. We give an overview of some recent applications of so-called 'transgenic' mouse technology in pharmaceutical research and development. We show how genetic manipulation in the mouse can be employed at multiple points in the drug discovery and development process, providing new solutions to old problems.

Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

PubMed

Shenoy, Archana; Blelloch, Robert

2009-09-11

The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript. To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8. We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

Chip Based Magnetic Imager for Molecular Profiling of Ovarian Cancer Cells

DTIC Science & Technology

2016-12-01

2015) Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160:1246-1260. PMC4380877, PMID:25748654. Acknowledgement of...Weissleder R, Lee H, Zhang F, Sharp PA (2015) Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160:1246-1260. 5. Im H, Shao H...Lett 32(10):1229–1231. 6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1501815112 Im et al. Resource Genome-wide CRISPR Screen in a Mouse Model of Tumor

18th International Mouse Genome Conference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lossie, Amy C.; Meehan, Thomas P.; Castillo, Andrew

2005-07-01

The 18th International Mouse Genome Conference was held in Seattle, WA, US on October 18-22,2004. The meeting was partially supported by the Department of Energy, Grant No. DE-FG02-04ER63851. Abstracts can be seen at imgs.org and the summary of the meeting was published in Mammalian Genome, Vol 16, Number 7, Pages 471-475.

A universal genomic coordinate translator for comparative genomics

PubMed Central

2014-01-01

Background Genomic duplications constitute major events in the evolution of species, allowing paralogous copies of genes to take on fine-tuned biological roles. Unambiguously identifying the orthology relationship between copies across multiple genomes can be resolved by synteny, i.e. the conserved order of genomic sequences. However, a comprehensive analysis of duplication events and their contributions to evolution would require all-to-all genome alignments, which increases at N2 with the number of available genomes, N. Results Here, we introduce Kraken, software that omits the all-to-all requirement by recursively traversing a graph of pairwise alignments and dynamically re-computing orthology. Kraken scales linearly with the number of targeted genomes, N, which allows for including large numbers of genomes in analyses. We first evaluated the method on the set of 12 Drosophila genomes, finding that orthologous correspondence computed indirectly through a graph of multiple synteny maps comes at minimal cost in terms of sensitivity, but reduces overall computational runtime by an order of magnitude. We then used the method on three well-annotated mammalian genomes, human, mouse, and rat, and show that up to 93% of protein coding transcripts have unambiguous pairwise orthologous relationships across the genomes. On a nucleotide level, 70 to 83% of exons match exactly at both splice junctions, and up to 97% on at least one junction. We last applied Kraken to an RNA-sequencing dataset from multiple vertebrates and diverse tissues, where we confirmed that brain-specific gene family members, i.e. one-to-many or many-to-many homologs, are more highly correlated across species than single-copy (i.e. one-to-one homologous) genes. Not limited to protein coding genes, Kraken also identifies thousands of newly identified transcribed loci, likely non-coding RNAs that are consistently transcribed in human, chimpanzee and gorilla, and maintain significant correlation of expression levels across species. Conclusions Kraken is a computational genome coordinate translator that facilitates cross-species comparisons, distinguishes orthologs from paralogs, and does not require costly all-to-all whole genome mappings. Kraken is freely available under LPGL from http://github.com/nedaz/kraken. PMID:24976580

A universal genomic coordinate translator for comparative genomics.

PubMed

Zamani, Neda; Sundström, Görel; Meadows, Jennifer R S; Höppner, Marc P; Dainat, Jacques; Lantz, Henrik; Haas, Brian J; Grabherr, Manfred G

2014-06-30

Genomic duplications constitute major events in the evolution of species, allowing paralogous copies of genes to take on fine-tuned biological roles. Unambiguously identifying the orthology relationship between copies across multiple genomes can be resolved by synteny, i.e. the conserved order of genomic sequences. However, a comprehensive analysis of duplication events and their contributions to evolution would require all-to-all genome alignments, which increases at N2 with the number of available genomes, N. Here, we introduce Kraken, software that omits the all-to-all requirement by recursively traversing a graph of pairwise alignments and dynamically re-computing orthology. Kraken scales linearly with the number of targeted genomes, N, which allows for including large numbers of genomes in analyses. We first evaluated the method on the set of 12 Drosophila genomes, finding that orthologous correspondence computed indirectly through a graph of multiple synteny maps comes at minimal cost in terms of sensitivity, but reduces overall computational runtime by an order of magnitude. We then used the method on three well-annotated mammalian genomes, human, mouse, and rat, and show that up to 93% of protein coding transcripts have unambiguous pairwise orthologous relationships across the genomes. On a nucleotide level, 70 to 83% of exons match exactly at both splice junctions, and up to 97% on at least one junction. We last applied Kraken to an RNA-sequencing dataset from multiple vertebrates and diverse tissues, where we confirmed that brain-specific gene family members, i.e. one-to-many or many-to-many homologs, are more highly correlated across species than single-copy (i.e. one-to-one homologous) genes. Not limited to protein coding genes, Kraken also identifies thousands of newly identified transcribed loci, likely non-coding RNAs that are consistently transcribed in human, chimpanzee and gorilla, and maintain significant correlation of expression levels across species. Kraken is a computational genome coordinate translator that facilitates cross-species comparisons, distinguishes orthologs from paralogs, and does not require costly all-to-all whole genome mappings. Kraken is freely available under LPGL from http://github.com/nedaz/kraken.

Structure and genomic organization of the human B1 receptor gene for kinins (BDKRB1).

PubMed

Bachvarov, D R; Hess, J F; Menke, J G; Larrivée, J F; Marceau, F

1996-05-01

Two subtypes of mammalian bradykinin receptors, B1 and B2 (BDKRB1 and BDKRB2), have been defined based on their pharmacological properties. The B1 type kinin receptors have weak affinity for intact BK or Lys-BK but strong affinity for kinin metabolites without the C-terminal arginine (e.g., des-Arg9-BK and Lys-des-Arg9-BK, also called des-Arg10-kallidin), which are generated by kininase I. The B1 receptor expression is up-regulated following tissue injury and inflammation (hyperemia, exudation, hyperalgesia, etc.). In the present study, we have cloned and sequenced the gene encoding human B1 receptor from a human genomic library. The human B1 receptor gene contains three exons separated by two introns. The first and the second exon are noncoding, while the coding region and the 3'-flanking region are located entirely on the third exon. The exon-intron arrangement of the human B1 receptor gene shows significant similarity with the genes encoding the B2 receptor subtype in human, mouse, and rat. Sequence analysis of the 5'-flanking region revealed the presence of a consensus TATA box and of numerous candidate transcription factor binding sequences. Primer extension experiments have shown the existence of multiple transcription initiation sites situated downstream and upstream from the consensus TATA box. Genomic Southern blot analysis indicated that the human B1 receptor is encoded by a single-copy gene.

APADB: a database for alternative polyadenylation and microRNA regulation events

PubMed Central

Müller, Sören; Rycak, Lukas; Afonso-Grunz, Fabian; Winter, Peter; Zawada, Adam M.; Damrath, Ewa; Scheider, Jessica; Schmäh, Juliane; Koch, Ina; Kahl, Günter; Rotter, Björn

2014-01-01

Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3′ untranslated region (3′UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3′UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3′ end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3′ end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3′UTR lengths, especially for chicken, and APADB provides necessary information to close this gap. Database URL: http://tools.genxpro.net/apadb/ PMID:25052703

Vitamin K epoxide reductase complex subunit 1 (Vkorc1) haplotype diversity in mouse priority strains

PubMed Central

Song, Ying; Vera, Nicole; Kohn, Michael H

2008-01-01

Background Polymorphisms in the vitamin K-epoxide reductase complex subunit 1 gene, Vkorc1, could affect blood coagulation and other vitamin K-dependent proteins, such as osteocalcin (bone Gla protein, BGP). Here we sequenced the Vkorc1 gene in 40 mouse priority strains. We analyzed Vkorc1 haplotypes with respect to prothrombin time (PT) and bone mineral density and composition (BMD and BMC); phenotypes expected to be vitamin K-dependent and represented by data in the Mouse Phenome Database (MPD). Findings In the commonly used laboratory strains of Mus musculus domesticus we identified only four haplotypes differing in the intron or 5' region sequence of the Vkorc1. Six haplotypes differing by coding and non-coding polymorphisms were identified in the other subspecies of Mus. We detected no significant association of Vkorc1 haplotypes with PT, BMD and BMC within each subspecies of Mus. Vkorc1 haplotype sequences divergence between subspecies was associated with PT, BMD and BMC. Conclusion Phenotypic variation in PT, BMD and BMC within subspecies of Mus, while substantial, appears to be dominated by genetic variation in genes other than the Vkorc1. This was particularly evident for M. m. domesticus, where a single haplotype was observed in conjunction with virtually the entire range of PT, BMD and BMC values of all 5 subspecies of Mus included in this study. Differences in these phenotypes between subspecies also should not be attributed to Vkorc1 variants, but should be viewed as a result of genome wide genetic divergence. PMID:19046458

Comparative analysis of genome maintenance genes in naked mole rat, mouse, and human.

PubMed

MacRae, Sheila L; Zhang, Quanwei; Lemetre, Christophe; Seim, Inge; Calder, Robert B; Hoeijmakers, Jan; Suh, Yousin; Gladyshev, Vadim N; Seluanov, Andrei; Gorbunova, Vera; Vijg, Jan; Zhang, Zhengdong D

2015-04-01

Genome maintenance (GM) is an essential defense system against aging and cancer, as both are characterized by increased genome instability. Here, we compared the copy number variation and mutation rate of 518 GM-associated genes in the naked mole rat (NMR), mouse, and human genomes. GM genes appeared to be strongly conserved, with copy number variation in only four genes. Interestingly, we found NMR to have a higher copy number of CEBPG, a regulator of DNA repair, and TINF2, a protector of telomere integrity. NMR, as well as human, was also found to have a lower rate of germline nucleotide substitution than the mouse. Together, the data suggest that the long-lived NMR, as well as human, has more robust GM than mouse and identifies new targets for the analysis of the exceptional longevity of the NMR. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

CRISPR-Cas9 Targeting of PCSK9 in Human Hepatocytes In Vivo-Brief Report.

PubMed

Wang, Xiao; Raghavan, Avanthi; Chen, Tao; Qiao, Lyon; Zhang, Yongxian; Ding, Qiurong; Musunuru, Kiran

2016-05-01

Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology-permanent alteration of genomic DNA sequences-mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. We used a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing. © 2016 American Heart Association, Inc.

Cloning and sequence analysis of a cDNA clone coding for the mouse GM2 activator protein.

PubMed Central

Bellachioma, G; Stirling, J L; Orlacchio, A; Beccari, T

1993-01-01

A cDNA (1.1 kb) containing the complete coding sequence for the mouse GM2 activator protein was isolated from a mouse macrophage library using a cDNA for the human protein as a probe. There was a single ATG located 12 bp from the 5' end of the cDNA clone followed by an open reading frame of 579 bp. Northern blot analysis of mouse macrophage RNA showed that there was a single band with a mobility corresponding to a size of 2.3 kb. We deduce from this that the mouse mRNA, in common with the mRNA for the human GM2 activator protein, has a long 3' untranslated sequence of approx. 1.7 kb. Alignment of the mouse and human deduced amino acid sequences showed 68% identity overall and 75% identity for the sequence on the C-terminal side of the first 31 residues, which in the human GM2 activator protein contains the signal peptide. Hydropathicity plots showed great similarity between the mouse and human sequences even in regions of low sequence similarity. There is a single N-glycosylation site in the mouse GM2 activator protein sequence (Asn151-Phe-Thr) which differs in its location from the single site reported in the human GM2 activator protein sequence (Asn63-Val-Thr). Images Figure 1 PMID:7689829

Short interspersed element (SINE) depletion and long interspersed element (LINE) abundance are not features universally required for imprinting.

PubMed

Cowley, Michael; de Burca, Anna; McCole, Ruth B; Chahal, Mandeep; Saadat, Ghazal; Oakey, Rebecca J; Schulz, Reiner

2011-04-20

Genomic imprinting is a form of gene dosage regulation in which a gene is expressed from only one of the alleles, in a manner dependent on the parent of origin. The mechanisms governing imprinted gene expression have been investigated in detail and have greatly contributed to our understanding of genome regulation in general. Both DNA sequence features, such as CpG islands, and epigenetic features, such as DNA methylation and non-coding RNAs, play important roles in achieving imprinted expression. However, the relative importance of these factors varies depending on the locus in question. Defining the minimal features that are absolutely required for imprinting would help us to understand how imprinting has evolved mechanistically. Imprinted retrogenes are a subset of imprinted loci that are relatively simple in their genomic organisation, being distinct from large imprinting clusters, and have the potential to be used as tools to address this question. Here, we compare the repeat element content of imprinted retrogene loci with non-imprinted controls that have a similar locus organisation. We observe no significant differences that are conserved between mouse and human, suggesting that the paucity of SINEs and relative abundance of LINEs at imprinted loci reported by others is not a sequence feature universally required for imprinting.

Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA.

PubMed

Hurrelbrink, R J; Nestorowicz, A; McMinn, P C

1999-12-01

An infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA into the low-copy-number plasmid vector pMC18. Designated pMVE-1-51, the clone consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing components of a cDNA library, in addition to cDNA of the 3' terminus derived by RT-PCR of poly(A)-tailed viral RNA. Upon comparison with other flavivirus sequences, the previously undetermined sequence of the 3' UTR was found to contain elements conserved throughout the genus FLAVIVIRUS: RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectious virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vitro. Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD(50) values and mortality profiles) were found to be identical in vivo in the mouse model. Through site-directed mutagenesis, the infectious clone should serve as a valuable tool for investigating the molecular determinants of virulence in MVE virus.

Identification of fungi in shotgun metagenomics datasets

PubMed Central

Donovan, Paul D.; Gonzalez, Gabriel; Higgins, Desmond G.

2018-01-01

Metagenomics uses nucleic acid sequencing to characterize species diversity in different niches such as environmental biomes or the human microbiome. Most studies have used 16S rRNA amplicon sequencing to identify bacteria. However, the decreasing cost of sequencing has resulted in a gradual shift away from amplicon analyses and towards shotgun metagenomic sequencing. Shotgun metagenomic data can be used to identify a wide range of species, but have rarely been applied to fungal identification. Here, we develop a sequence classification pipeline, FindFungi, and use it to identify fungal sequences in public metagenome datasets. We focus primarily on animal metagenomes, especially those from pig and mouse microbiomes. We identified fungi in 39 of 70 datasets comprising 71 fungal species. At least 11 pathogenic species with zoonotic potential were identified, including Candida tropicalis. We identified Pseudogymnoascus species from 13 Antarctic soil samples initially analyzed for the presence of bacteria capable of degrading diesel oil. We also show that Candida tropicalis and Candida loboi are likely the same species. In addition, we identify several examples where contaminating DNA was erroneously included in fungal genome assemblies. PMID:29444186

BAC sequencing using pooled methods.

PubMed

Saski, Christopher A; Feltus, F Alex; Parida, Laxmi; Haiminen, Niina

2015-01-01

Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly.

ESCAPE: database for integrating high-content published data collected from human and mouse embryonic stem cells.

PubMed

Xu, Huilei; Baroukh, Caroline; Dannenfelser, Ruth; Chen, Edward Y; Tan, Christopher M; Kou, Yan; Kim, Yujin E; Lemischka, Ihor R; Ma'ayan, Avi

2013-01-01

High content studies that profile mouse and human embryonic stem cells (m/hESCs) using various genome-wide technologies such as transcriptomics and proteomics are constantly being published. However, efforts to integrate such data to obtain a global view of the molecular circuitry in m/hESCs are lagging behind. Here, we present an m/hESC-centered database called Embryonic Stem Cell Atlas from Pluripotency Evidence integrating data from many recent diverse high-throughput studies including chromatin immunoprecipitation followed by deep sequencing, genome-wide inhibitory RNA screens, gene expression microarrays or RNA-seq after knockdown (KD) or overexpression of critical factors, immunoprecipitation followed by mass spectrometry proteomics and phosphoproteomics. The database provides web-based interactive search and visualization tools that can be used to build subnetworks and to identify known and novel regulatory interactions across various regulatory layers. The web-interface also includes tools to predict the effects of combinatorial KDs by additive effects controlled by sliders, or through simulation software implemented in MATLAB. Overall, the Embryonic Stem Cell Atlas from Pluripotency Evidence database is a comprehensive resource for the stem cell systems biology community. Database URL: http://www.maayanlab.net/ESCAPE

Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta

PubMed Central

Poulter, James A.; Murillo, Gina; Brookes, Steven J.; Smith, Claire E. L.; Parry, David A.; Silva, Sandra; Kirkham, Jennifer; Inglehearn, Chris F.; Mighell, Alan J.

2014-01-01

Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid. PMID:24858907

L1-mediated retrotransposition of murine B1 and B2 SINEs recapitulated in cultured cells.

PubMed

Dewannieux, Marie; Heidmann, Thierry

2005-06-03

SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.

Transcriptome sequencing and annotation for the Jamaican fruit bat (Artibeus jamaicensis).

PubMed

Shaw, Timothy I; Srivastava, Anuj; Chou, Wen-Chi; Liu, Liang; Hawkinson, Ann; Glenn, Travis C; Adams, Rick; Schountz, Tony

2012-01-01

The Jamaican fruit bat (Artibeus jamaicensis) is one of the most common bats in the tropical Americas. It is thought to be a potential reservoir host of Tacaribe virus, an arenavirus closely related to the South American hemorrhagic fever viruses. We performed transcriptome sequencing and annotation from lung, kidney and spleen tissues using 454 and Illumina platforms to develop this species as an animal model. More than 100,000 contigs were assembled, with 25,000 genes that were functionally annotated. Of the remaining unannotated contigs, 80% were found within bat genomes or transcriptomes. Annotated genes are involved in a broad range of activities ranging from cellular metabolism to genome regulation through ncRNAs. Reciprocal BLAST best hits yielded 8,785 sequences that are orthologous to mouse, rat, cattle, horse and human. Species tree analysis of sequences from 2,378 loci was used to achieve 95% bootstrap support for the placement of bat as sister to the clade containing horse, dog, and cattle. Through substitution rate estimation between bat and human, 32 genes were identified with evidence for positive selection. We also identified 466 immune-related genes, which may be useful for studying Tacaribe virus infection of this species. The Jamaican fruit bat transcriptome dataset is a resource that should provide additional candidate markers for studying bat evolution and ecology, and tools for analysis of the host response and pathology of disease.

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed

Torrent, C; Gabus, C; Darlix, J L

1994-02-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

The Degradome database: mammalian proteases and diseases of proteolysis.

PubMed

Quesada, Víctor; Ordóñez, Gonzalo R; Sánchez, Luis M; Puente, Xose S; López-Otín, Carlos

2009-01-01

The degradome is defined as the complete set of proteases present in an organism. The recent availability of whole genomic sequences from multiple organisms has led us to predict the contents of the degradomes of several mammalian species. To ensure the fidelity of these predictions, our methods have included manual curation of individual sequences and, when necessary, direct cloning and sequencing experiments. The results of these studies in human, chimpanzee, mouse and rat have been incorporated into the Degradome database, which can be accessed through a web interface at http://degradome.uniovi.es. The annotations about each individual protease can be retrieved by browsing catalytic classes and families or by searching specific terms. This web site also provides detailed information about genetic diseases of proteolysis, a growing field of great importance for multiple users. Finally, the user can find additional information about protease structures, protease inhibitors, ancillary domains of proteases and differences between mammalian degradomes.

The Degradome database: mammalian proteases and diseases of proteolysis

PubMed Central

Quesada, Víctor; Ordóñez, Gonzalo R.; Sánchez, Luis M.; Puente, Xose S.; López-Otín, Carlos

2009-01-01

The degradome is defined as the complete set of proteases present in an organism. The recent availability of whole genomic sequences from multiple organisms has led us to predict the contents of the degradomes of several mammalian species. To ensure the fidelity of these predictions, our methods have included manual curation of individual sequences and, when necessary, direct cloning and sequencing experiments. The results of these studies in human, chimpanzee, mouse and rat have been incorporated into the Degradome database, which can be accessed through a web interface at http://degradome.uniovi.es. The annotations about each individual protease can be retrieved by browsing catalytic classes and families or by searching specific terms. This web site also provides detailed information about genetic diseases of proteolysis, a growing field of great importance for multiple users. Finally, the user can find additional information about protease structures, protease inhibitors, ancillary domains of proteases and differences between mammalian degradomes. PMID:18776217

Epigenetic functions enriched in transcription factors binding to mouse recombination hotspots.

PubMed

Wu, Min; Kwoh, Chee-Keong; Przytycka, Teresa M; Li, Jing; Zheng, Jie

2012-06-21

The regulatory mechanism of recombination is a fundamental problem in genomics, with wide applications in genome-wide association studies, birth-defect diseases, molecular evolution, cancer research, etc. In mammalian genomes, recombination events cluster into short genomic regions called "recombination hotspots". Recently, a 13-mer motif enriched in hotspots is identified as a candidate cis-regulatory element of human recombination hotspots; moreover, a zinc finger protein, PRDM9, binds to this motif and is associated with variation of recombination phenotype in human and mouse genomes, thus is a trans-acting regulator of recombination hotspots. However, this pair of cis and trans-regulators covers only a fraction of hotspots, thus other regulators of recombination hotspots remain to be discovered. In this paper, we propose an approach to predicting additional trans-regulators from DNA-binding proteins by comparing their enrichment of binding sites in hotspots. Applying this approach on newly mapped mouse hotspots genome-wide, we confirmed that PRDM9 is a major trans-regulator of hotspots. In addition, a list of top candidate trans-regulators of mouse hotspots is reported. Using GO analysis we observed that the top genes are enriched with function of histone modification, highlighting the epigenetic regulatory mechanisms of recombination hotspots.

Epigenetic functions enriched in transcription factors binding to mouse recombination hotspots

PubMed Central

2012-01-01

The regulatory mechanism of recombination is a fundamental problem in genomics, with wide applications in genome-wide association studies, birth-defect diseases, molecular evolution, cancer research, etc. In mammalian genomes, recombination events cluster into short genomic regions called "recombination hotspots". Recently, a 13-mer motif enriched in hotspots is identified as a candidate cis-regulatory element of human recombination hotspots; moreover, a zinc finger protein, PRDM9, binds to this motif and is associated with variation of recombination phenotype in human and mouse genomes, thus is a trans-acting regulator of recombination hotspots. However, this pair of cis and trans-regulators covers only a fraction of hotspots, thus other regulators of recombination hotspots remain to be discovered. In this paper, we propose an approach to predicting additional trans-regulators from DNA-binding proteins by comparing their enrichment of binding sites in hotspots. Applying this approach on newly mapped mouse hotspots genome-wide, we confirmed that PRDM9 is a major trans-regulator of hotspots. In addition, a list of top candidate trans-regulators of mouse hotspots is reported. Using GO analysis we observed that the top genes are enriched with function of histone modification, highlighting the epigenetic regulatory mechanisms of recombination hotspots. PMID:22759569

Structure and expression of the human thymocyte antigens CD1a, CD1b, and CD1c

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, L.H.; Calabi, F.; Lefebvre, F.A.

1987-12-01

The CD1 human antigens are a family of at least three components, CD1a, CD1b, and CD1c, that are characteristic of the cortical stage of thymocyte maturation. CD1a was originally named HTA1 or T6 and thought to be the human equivalent of mouse Tla. The genes coding for all three have not been identified by transfection into mouse cells. The transfectants express the surface antigens that can then be recognized by the corresponding cluster of monoclonal antibodies used to define the three members of CD1. The full sequence of the genomic DNA is described for all three. The intron-exon structure ofmore » CD1a is deduced by comparison with a near-full-length cDNA clone. Similar structures are proposed for the other two, largely based on sequence homology. An unusually long 5'-untranslated exon (280 bases long) is highly conserved between the three genes, suggesting an important but unknown function. CD1c has a duplicated form of this exon that is thought to be spliced out. The major homology between the three antigens is in the ..beta../sub 2/-microglobulin-binding-domain. The general relatedness to major histocompatibility complex class I and class II molecules is significant but low, with no section of higher homology to mouse Tla.« less

Cowpox virus encodes a fifth member of the tumor necrosis factor receptor family: A soluble, secreted CD30 homologue

PubMed Central

Panus, Joanne Fanelli; Smith, Craig A.; Ray, Caroline A.; Smith, Terri Davis; Patel, Dhavalkumar D.; Pickup, David J.

2002-01-01

Cowpox virus (Brighton Red strain) possesses one of the largest genomes in the Orthopoxvirus genus. Sequence analysis of a region of the genome that is type-specific for cowpox virus identified a gene, vCD30, encoding a soluble, secreted protein that is the fifth member of the tumor necrosis factor receptor family known to be encoded by cowpox virus. The vCD30 protein contains 110 aa, including a 21-residue signal peptide, a potential O-linked glycosylation site, and a 58-aa sequence sharing 51–59% identity with highly conserved extracellular segments of both mouse and human CD30. A vCD30Fc fusion protein binds CD153 (CD30 ligand) specifically, and it completely inhibits CD153/CD30 interactions. Although the functions of CD30 are not well understood, the existence of vCD30 suggests that the cellular receptor plays a significant role in normal immune responses. Viral inhibition of CD30 also lends support to the potential therapeutic value of targeting CD30 in human inflammatory and autoimmune diseases. PMID:12034885

Regulatory Features for Odorant Receptor Genes in the Mouse Genome.

PubMed

Degl'Innocenti, Andrea; D'Errico, Anna

2017-01-01

The odorant receptor genes, seven transmembrane receptor genes constituting the vastest mammalian gene multifamily, are expressed monogenically and monoallelicaly in each sensory neuron in the olfactory epithelium. This characteristic, often referred to as the one neuron-one receptor rule, is driven by mostly uncharacterized molecular dynamics, generally named odorant receptor gene choice . Much attention has been paid by the scientific community to the identification of sequences regulating the expression of odorant receptor genes within their loci , where related genes are usually arranged in genomic clusters. A number of studies identified transcription factor binding sites on odorant receptor promoter sequences. Similar binding sites were also found on a number of enhancers that regulate in cis their transcription, but have been proposed to form interchromosomal networks. Odorant receptor gene choice seems to occur via the local removal of strongly repressive epigenetic markings, put in place during the maturation of the sensory neuron on each odorant receptor locus . Here we review the fast-changing state of art for the study of regulatory features for odorant receptor genes.

The high-level expression of human tissue plasminogen activator in the milk of transgenic mice with hybrid gene locus strategy.

PubMed

Zhou, Yanrong; Lin, Yanli; Wu, Xiaojie; Xiong, Fuyin; Lv, Yuemeng; Zheng, Tao; Huang, Peitang; Chen, Hongxing

2012-02-01

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.

Self-Cloning CRISPR.

PubMed

Arbab, Mandana; Sherwood, Richard I

2016-08-17

CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR-based addition of short homology arms, we achieve efficient site-specific knock-in of transgenes such as GFP without traditional plasmid cloning or genome-integrated selection cassette (2% to 4% knock-in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Discovering transcription factor binding sites in highly repetitive regions of genomes with multi-read analysis of ChIP-Seq data.

PubMed

Chung, Dongjun; Kuan, Pei Fen; Li, Bo; Sanalkumar, Rajendran; Liang, Kun; Bresnick, Emery H; Dewey, Colin; Keleş, Sündüz

2011-07-01

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is rapidly replacing chromatin immunoprecipitation combined with genome-wide tiling array analysis (ChIP-chip) as the preferred approach for mapping transcription-factor binding sites and chromatin modifications. The state of the art for analyzing ChIP-seq data relies on using only reads that map uniquely to a relevant reference genome (uni-reads). This can lead to the omission of up to 30% of alignable reads. We describe a general approach for utilizing reads that map to multiple locations on the reference genome (multi-reads). Our approach is based on allocating multi-reads as fractional counts using a weighted alignment scheme. Using human STAT1 and mouse GATA1 ChIP-seq datasets, we illustrate that incorporation of multi-reads significantly increases sequencing depths, leads to detection of novel peaks that are not otherwise identifiable with uni-reads, and improves detection of peaks in mappable regions. We investigate various genome-wide characteristics of peaks detected only by utilization of multi-reads via computational experiments. Overall, peaks from multi-read analysis have similar characteristics to peaks that are identified by uni-reads except that the majority of them reside in segmental duplications. We further validate a number of GATA1 multi-read only peaks by independent quantitative real-time ChIP analysis and identify novel target genes of GATA1. These computational and experimental results establish that multi-reads can be of critical importance for studying transcription factor binding in highly repetitive regions of genomes with ChIP-seq experiments.

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.

PubMed

Amaral, Paulo P; Leonardi, Tommaso; Han, Namshik; Viré, Emmanuelle; Gascoigne, Dennis K; Arias-Carrasco, Raúl; Büscher, Magdalena; Pandolfini, Luca; Zhang, Anda; Pluchino, Stefano; Maracaja-Coutinho, Vinicius; Nakaya, Helder I; Hemberg, Martin; Shiekhattar, Ramin; Enright, Anton J; Kouzarides, Tony

2018-03-15

The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

COSMOS: accurate detection of somatic structural variations through asymmetric comparison between tumor and normal samples.

PubMed

Yamagata, Koichi; Yamanishi, Ayako; Kokubu, Chikara; Takeda, Junji; Sese, Jun

2016-05-05

An important challenge in cancer genomics is precise detection of structural variations (SVs) by high-throughput short-read sequencing, which is hampered by the high false discovery rates of existing analysis tools. Here, we propose an accurate SV detection method named COSMOS, which compares the statistics of the mapped read pairs in tumor samples with isogenic normal control samples in a distinct asymmetric manner. COSMOS also prioritizes the candidate SVs using strand-specific read-depth information. Performance tests on modeled tumor genomes revealed that COSMOS outperformed existing methods in terms of F-measure. We also applied COSMOS to an experimental mouse cell-based model, in which SVs were induced by genome engineering and gamma-ray irradiation, followed by polymerase chain reaction-based confirmation. The precision of COSMOS was 84.5%, while the next best existing method was 70.4%. Moreover, the sensitivity of COSMOS was the highest, indicating that COSMOS has great potential for cancer genome analysis. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Polymorphic human (CTAT)n microsatellite provides a conserved linkage marker for mouse mutants causing cleft palate, vestibular defects, obesity and ataxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffith, A.J.; Burgess, D.L.; Kohrman, D.

1994-09-01

The Twirler mutation (Tw) causing cleft palate {plus_minus} cleft lip, vestibular defects and obesity is located within 0.5 cM of an ataxia locus (ax) on mouse chromosome 18. We identified a transgene-induced insertional mutation with vestibular and craniofacial defects that appears to be a new allele of Twirler. Mouse DNA flanking the transgene insertion site was isolated from a cosmid library. An evolutionarily conserved, zoo blot positive cosmid subclone was used to probe a human {lambda} genomic library. From the sequence of a highly homologous human {lambda} clone, we designed STS primers and screened a human P1 library. DNA frommore » two positive P1 clones was hybridized with simple sequence probes, and a (CTAT){sub 12} repeat was detected. Analysis of 62 CEPH parents with primers flanking the repeat identified six alleles containing 9 to 14 copies of the repeat, at frequencies of 0.17, 0.17, 0.17, 0.27, 0.15 and 0.07, respectively. The observed heterozygosity was 49/62 with a calculated PIC value of 0.76. This polymorphic microsatellite marker, designated Umi3, was mapped to the predicted conserved human linkage group by analysis of somatic cell hybrid panels. The anticipated short distance between Umi3 and the disease genes will facilitate detection of linkage in small families. We would like to type appropriate human pedigrees with Umi3 in order to identify patients with inherited disorders homologous to the mouse mutations Twirler and ataxia.« less

Evaluation of potential models for imprinted and nonimprinted components of human chromosome 15q11-q13 syndromes by fine-structure homology mapping in the mouse.

PubMed Central

Nicholls, R D; Gottlieb, W; Russell, L B; Davda, M; Horsthemke, B; Rinchik, E M

1993-01-01

Prader-Willi and Angelman syndromes are complex neurobehavioral contiguous gene syndromes whose expression depends on the unmasking of genomic imprinting for different genetic loci in human chromosome 15q11-q13. The homologous chromosomal region in the mouse genome has been fine-mapped by using interspecific (Mus spretus) crosses and overlapping, radiation-induced deletions to evaluate potential animal models for both imprinted and nonimprinted components of these syndromes. Four evolutionarily conserved sequences from human 15q11-q13, including two cDNAs from fetal brain (DN10, D15S12h; DN34, D15S9h-1), a microdissected clone (MN7; D15F37S1h) expressed in mouse brain, and the gene for the beta 3 subunit of the gamma-aminobutyric acid type A receptor (Gabrb3), were mapped in mouse chromosome 7 by analysis of deletions at the pink-eyed dilution (p) locus. Three of these loci are deleted in pre- and postnatally lethal p-locus mutations, which extend up to 5.5 +/- 1.7 centimorgans (cM) proximal to p; D15S9h-1, which maps 1.1 +/- 0.8 cM distal to p and is the mouse homolog of the human gene D15S9 (which shows a DNA methylation imprint), is not deleted in any of the p-locus deletion series. A transcript from the Gabrb3 gene, but not the transcript detected by MN7 at the D15F37S1h locus, is expressed in mice homozygous for the p6H deletion, which have an abnormal neurological phenotype. Furthermore, the Gabrb3 transcript is expressed equally well from the maternal or paternal chromosome 7 and, therefore, its expression is not imprinted in mouse brain. Deletions at the mouse p locus should serve as intermediate genetic reagents and models with which to analyze the genetics and etiology of individual components of human 15q11-q13 disorders. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8095339

Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles.

PubMed

McMeel, O M; Hoey, E M; Ferguson, A

2001-01-01

The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.

Comprehensive genomic profiles of small cell lung cancer

PubMed Central

George, Julie; Lim, Jing Shan; Jang, Se Jin; Cun, Yupeng; Ozretić, Luka; Kong, Gu; Leenders, Frauke; Lu, Xin; Fernández-Cuesta, Lynnette; Bosco, Graziella; Müller, Christian; Dahmen, Ilona; Jahchan, Nadine S.; Park, Kwon-Sik; Yang, Dian; Karnezis, Anthony N.; Vaka, Dedeepya; Torres, Angela; Wang, Maia Segura; Korbel, Jan O.; Menon, Roopika; Chun, Sung-Min; Kim, Deokhoon; Wilkerson, Matt; Hayes, Neil; Engelmann, David; Pützer, Brigitte; Bos, Marc; Michels, Sebastian; Vlasic, Ignacija; Seidel, Danila; Pinther, Berit; Schaub, Philipp; Becker, Christian; Altmüller, Janine; Yokota, Jun; Kohno, Takashi; Iwakawa, Reika; Tsuta, Koji; Noguchi, Masayuki; Muley, Thomas; Hoffmann, Hans; Schnabel, Philipp A.; Petersen, Iver; Chen, Yuan; Soltermann, Alex; Tischler, Verena; Choi, Chang-min; Kim, Yong-Hee; Massion, Pierre P.; Zou, Yong; Jovanovic, Dragana; Kontic, Milica; Wright, Gavin M.; Russell, Prudence A.; Solomon, Benjamin; Koch, Ina; Lindner, Michael; Muscarella, Lucia A.; la Torre, Annamaria; Field, John K.; Jakopovic, Marko; Knezevic, Jelena; Castaños-Vélez, Esmeralda; Roz, Luca; Pastorino, Ugo; Brustugun, Odd-Terje; Lund-Iversen, Marius; Thunnissen, Erik; Köhler, Jens; Schuler, Martin; Botling, Johan; Sandelin, Martin; Sanchez-Cespedes, Montserrat; Salvesen, Helga B.; Achter, Viktor; Lang, Ulrich; Bogus, Magdalena; Schneider, Peter M.; Zander, Thomas; Ansén, Sascha; Hallek, Michael; Wolf, Jürgen; Vingron, Martin; Yatabe, Yasushi; Travis, William D.; Nürnberg, Peter; Reinhardt, Christian; Perner, Sven; Heukamp, Lukas; Büttner, Reinhard; Haas, Stefan A.; Brambilla, Elisabeth; Peifer, Martin; Sage, Julien; Thomas, Roman K.

2016-01-01

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer. PMID:26168399

Identification and characterisation of the angiotensin converting enzyme-3 (ACE3) gene: a novel mammalian homologue of ACE

PubMed Central

Rella, Monika; Elliot, Joann L; Revett, Timothy J; Lanfear, Jerry; Phelan, Anne; Jackson, Richard M; Turner, Anthony J; Hooper, Nigel M

2007-01-01

Background Mammalian angiotensin converting enzyme (ACE) plays a key role in blood pressure regulation. Although multiple ACE-like proteins exist in non-mammalian organisms, to date only one other ACE homologue, ACE2, has been identified in mammals. Results Here we report the identification and characterisation of the gene encoding a third homologue of ACE, termed ACE3, in several mammalian genomes. The ACE3 gene is located on the same chromosome downstream of the ACE gene. Multiple sequence alignment and molecular modelling have been employed to characterise the predicted ACE3 protein. In mouse, rat, cow and dog, the predicted protein has mutations in some of the critical residues involved in catalysis, including the catalytic Glu in the HEXXH zinc binding motif which is Gln, and ESTs or reverse-transcription PCR indicate that the gene is expressed. In humans, the predicted ACE3 protein has an intact HEXXH motif, but there are other deletions and insertions in the gene and no ESTs have been identified. Conclusion In the genomes of several mammalian species there is a gene that encodes a novel, single domain ACE-like protein, ACE3. In mouse, rat, cow and dog ACE3, the catalytic Glu is replaced by Gln in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, no evidence was found that the ACE3 gene is expressed and the presence of deletions and insertions in the sequence indicate that ACE3 is a pseudogene. PMID:17597519

Equalizer reduces SNP bias in Affymetrix microarrays.

PubMed

Quigley, David

2015-07-30

Gene expression microarrays measure the levels of messenger ribonucleic acid (mRNA) in a sample using probe sequences that hybridize with transcribed regions. These probe sequences are designed using a reference genome for the relevant species. However, most model organisms and all humans have genomes that deviate from their reference. These variations, which include single nucleotide polymorphisms, insertions of additional nucleotides, and nucleotide deletions, can affect the microarray's performance. Genetic experiments comparing individuals bearing different population-associated single nucleotide polymorphisms that intersect microarray probes are therefore subject to systemic bias, as the reduction in binding efficiency due to a technical artifact is confounded with genetic differences between parental strains. This problem has been recognized for some time, and earlier methods of compensation have attempted to identify probes affected by genome variants using statistical models. These methods may require replicate microarray measurement of gene expression in the relevant tissue in inbred parental samples, which are not always available in model organisms and are never available in humans. By using sequence information for the genomes of organisms under investigation, potentially problematic probes can now be identified a priori. However, there is no published software tool that makes it easy to eliminate these probes from an annotation. I present equalizer, a software package that uses genome variant data to modify annotation files for the commonly used Affymetrix IVT and Gene/Exon platforms. These files can be used by any microarray normalization method for subsequent analysis. I demonstrate how use of equalizer on experiments mapping germline influence on gene expression in a genetic cross between two divergent mouse species and in human samples significantly reduces probe hybridization-induced bias, reducing false positive and false negative findings. The equalizer package reduces probe hybridization bias from experiments performed on the Affymetrix microarray platform, allowing accurate assessment of germline influence on gene expression.

The NS2 proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells.

PubMed

Eichwald, Virginie; Daeffler, Laurent; Klein, Michèle; Rommelaere, Jean; Salomé, Nathalie

2002-10-01

The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses.

The NS2 Proteins of Parvovirus Minute Virus of Mice Are Required for Efficient Nuclear Egress of Progeny Virions in Mouse Cells

PubMed Central

Eichwald, Virginie; Daeffler, Laurent; Klein, Michèle; Rommelaere, Jean; Salomé, Nathalie

2002-01-01

The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses. PMID:12239307

Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments.

PubMed

Yanik, Mert; Ponnam, Surya Prakash Goud; Wimmer, Tobias; Trimborn, Lennart; Müller, Carina; Gambert, Isabel; Ginsberg, Johanna; Janise, Annabella; Domicke, Janina; Wende, Wolfgang; Lorenz, Birgit; Stieger, Knut

2018-06-01

Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

PubMed Central

Siggs, Owen M.; Miosge, Lisa A.; Roots, Carla M.; Enders, Anselm; Bertram, Edward M.; Crockford, Tanya L.; Whittle, Belinda; Potter, Paul K.; Simon, Michelle M.; Mallon, Ann-Marie; Brown, Steve D. M.; Beutler, Bruce; Goodnow, Christopher C.; Lunter, Gerton; Cornall, Richard J.

2013-01-01

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis. PMID:23382690

Use of in vivo Expression Technology for the Identification of Putative Host Adaptation Factors of the Lyme Disease Spirochete.

PubMed

Casselli, Timothy; Bankhead, Troy

2015-01-01

The causative agent of Lyme disease, Borrelia burgdorferi, is an obligate parasite that requires either a tick vector or a mammalian host for survival. Identification of the bacterial genes that are specifically expressed during infection of the mammalian host could provide targets for novel therapeutics and vaccines. In vivo expression technology (IVET) is a reporter-based promoter trap system that utilizes selectable markers to identify promoters of bacterial host-specific genes. Using previously characterized genes for in vivo and in vitro selection, this study utilized an IVET system that allows for selection of B. burgdorferi sequences that act as active promoters only during murine infection. This promoter trap system was able to successfully distinguish active promoter sequences both in vivo and in vitro from control sequences and a library of cloned B. burgdorferi genomic fragments. However, a bottleneck effect during the experimental mouse infection limited the utility for genome-wide promoter screening. Overall, IVET was demonstrated as a tool for the identification of in vivo-induced promoter elements of B. burgdorferi, and the observed infection bottleneck apparent using a polyclonal infection pool provides insight into the dynamics of experimental infection with B. burgdorferi. © 2015 S. Karger AG, Basel.

Menzerath-Altmann law in mammalian exons reflects the dynamics of gene structure evolution.

PubMed

Nikolaou, Christoforos

2014-12-01

Genomic sequences exhibit self-organization properties at various hierarchical levels. One such is the gene structure of higher eukaryotes with its complex exon/intron arrangement. Exon sizes and exon numbers in genes have been shown to conform to a law derived from statistical linguistics and formulated by Menzerath and Altmann, according to which the mean size of the constituents of an entity is inversely related to the number of these constituents. We herein perform a detailed analysis of this property in the complete exon set of the mouse genome in correlation to the sequence conservation of each exon and the transcriptional complexity of each gene locus. We show that extensive linear fits, representative of accordance to Menzerath-Altmann law are restricted to a particular subset of genes that are formed by exons under low or intermediate sequence constraints and have a small number of alternative transcripts. Based on this observation we propose a hypothesis for the law of Menzerath-Altmann in mammalian genes being predominantly due to genes that are more versatile in function and thus, more prone to undergo changes in their structure. To this end we demonstrate one test case where gene categories of different functionality also show differences in the extent of conformity to Menzerath-Altmann law. Copyright © 2014 Elsevier Ltd. All rights reserved.

Whole-genome sequencing for comparative genomics and de novo genome assembly.

PubMed

Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

2015-01-01

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

Cloning and genomic characterization of sytdep, a new synaptotagmin XIV-related gene.

PubMed

Herrero-Turrión, M Javier; Fukuda, Mitsunori; Mollinedo, Faustino

2006-02-10

We have identified a new human gene coined sytdep (synaptotagmin XIV-derived protein) in human neutrophils. Sytdep encodes a 188-amino acid sequence with a 21.435kDa deduced molecular mass, showing 75% identity to human synaptotagmin (syt) XIV. Human neutrophils express sytdep, but not syt XIV. Sytdep was upregulated during HL-60 neutrophil differentiation. Sytdep gene is located in human chromosome 4 and contains a unique exon, whereas syt XIV gene, located in chromosome 1, comprises 10 exons with 9 introns. Mouse genome did not contain sytdep. The N-terminal region of sytdep shows no homology with any known protein and, unlike synaptotagmin XIV isoforms, sytdep shows a unique C-terminal C2B domain. Polyclonal antibodies against the C2B domain of syt XIV recognized sytdep as a 27-kDa protein in human neutrophils. Genomic analyses suggest that human sytdep could derive from a retrotranslocation of a syt XIV transcript into chromosome 4.

Non-B DB v2.0: a database of predicted non-B DNA-forming motifs and its associated tools.

PubMed

Cer, Regina Z; Donohue, Duncan E; Mudunuri, Uma S; Temiz, Nuri A; Loss, Michael A; Starner, Nathan J; Halusa, Goran N; Volfovsky, Natalia; Yi, Ming; Luke, Brian T; Bacolla, Albino; Collins, Jack R; Stephens, Robert M

2013-01-01

The non-B DB, available at http://nonb.abcc.ncifcrf.gov, catalogs predicted non-B DNA-forming sequence motifs, including Z-DNA, G-quadruplex, A-phased repeats, inverted repeats, mirror repeats, direct repeats and their corresponding subsets: cruciforms, triplexes and slipped structures, in several genomes. Version 2.0 of the database revises and re-implements the motif discovery algorithms to better align with accepted definitions and thresholds for motifs, expands the non-B DNA-forming motifs coverage by including short tandem repeats and adds key visualization tools to compare motif locations relative to other genomic annotations. Non-B DB v2.0 extends the ability for comparative genomics by including re-annotation of the five organisms reported in non-B DB v1.0, human, chimpanzee, dog, macaque and mouse, and adds seven additional organisms: orangutan, rat, cow, pig, horse, platypus and Arabidopsis thaliana. Additionally, the non-B DB v2.0 provides an overall improved graphical user interface and faster query performance.

Mechanistic basis of infertility of mouse intersubspecific hybrids

PubMed Central

Bhattacharyya, Tanmoy; Gregorova, Sona; Mihola, Ondrej; Anger, Martin; Sebestova, Jaroslava; Denny, Paul; Simecek, Petr; Forejt, Jiri

2013-01-01

According to the Dobzhansky–Muller model, hybrid sterility is a consequence of the independent evolution of related taxa resulting in incompatible genomic interactions of their hybrids. The model implies that the incompatibilities evolve randomly, unless a particular gene or nongenic sequence diverges much faster than the rest of the genome. Here we propose that asynapsis of heterospecific chromosomes in meiotic prophase provides a recurrently evolving trigger for the meiotic arrest of interspecific F1 hybrids. We observed extensive asynapsis of chromosomes and disturbance of the sex body in >95% of pachynemas of Mus m. musculus × Mus m. domesticus sterile F1 males. Asynapsis was not preceded by a failure of double-strand break induction, and the rate of meiotic crossing over was not affected in synapsed chromosomes. DNA double-strand break repair was delayed or failed in unsynapsed autosomes, and misexpression of chromosome X and chromosome Y genes was detected in single pachynemas and by genome-wide expression profiling. Oocytes of F1 hybrid females showed the same kind of synaptic problems but with the incidence reduced to half. Most of the oocytes with pachytene asynapsis were eliminated before birth. We propose the heterospecific pairing of homologous chromosomes as a preexisting condition of asynapsis in interspecific hybrids. The asynapsis may represent a universal mechanistic basis of F1 hybrid sterility manifested by pachytene arrest. It is tempting to speculate that a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis may be the culprit. PMID:23329330

Mechanistic basis of infertility of mouse intersubspecific hybrids.

PubMed

Bhattacharyya, Tanmoy; Gregorova, Sona; Mihola, Ondrej; Anger, Martin; Sebestova, Jaroslava; Denny, Paul; Simecek, Petr; Forejt, Jiri

2013-02-05

According to the Dobzhansky-Muller model, hybrid sterility is a consequence of the independent evolution of related taxa resulting in incompatible genomic interactions of their hybrids. The model implies that the incompatibilities evolve randomly, unless a particular gene or nongenic sequence diverges much faster than the rest of the genome. Here we propose that asynapsis of heterospecific chromosomes in meiotic prophase provides a recurrently evolving trigger for the meiotic arrest of interspecific F1 hybrids. We observed extensive asynapsis of chromosomes and disturbance of the sex body in >95% of pachynemas of Mus m. musculus × Mus m. domesticus sterile F1 males. Asynapsis was not preceded by a failure of double-strand break induction, and the rate of meiotic crossing over was not affected in synapsed chromosomes. DNA double-strand break repair was delayed or failed in unsynapsed autosomes, and misexpression of chromosome X and chromosome Y genes was detected in single pachynemas and by genome-wide expression profiling. Oocytes of F1 hybrid females showed the same kind of synaptic problems but with the incidence reduced to half. Most of the oocytes with pachytene asynapsis were eliminated before birth. We propose the heterospecific pairing of homologous chromosomes as a preexisting condition of asynapsis in interspecific hybrids. The asynapsis may represent a universal mechanistic basis of F1 hybrid sterility manifested by pachytene arrest. It is tempting to speculate that a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis may be the culprit.

iRAGu: A Novel Inducible and Reversible Mouse Model for Ubiquitous Recombinase Activity

PubMed Central

Bonnet, Marie; Sarmento, Leonor Morais; Martins, Ana C.; Sobral, Daniel; Silva, Joana; Demengeot, Jocelyne

2017-01-01

Developing lymphocytes express the recombination activating genes (RAGs) 1 and 2 products that form a site specific recombinase complex (RAG), introducing double strand DNA breaks (DSBs) at recombination signal sequences (RSSs) flanking the V, D, and J gene segments in the antigen receptor loci. The subsequent steps in the reaction consist in the ligation of DSBs by ubiquitous enzymes of the non-homologous end joining DNA repair pathway. This mutagenesis process is responsible for the generation of the very large clonal diversity of T and B lymphocytes, itself allowing the recognition of a virtually open-ended antigenic universe. Sequences resembling RSS are found at high frequency all over the genome, and involved in RAG mediated illegitimate recombination and translocations. Hence, natural and induced ectopic activity of RAG is a threat to the genome only recently underscored. Here, we report and characterize a novel mouse transgenic system for which ubiquitous expression of the recombinase is inducible. In this system, the RAG1 protein is constitutively expressed and functional, while the RAG2 protein, coupled to the estrogen receptor, becomes functionally active upon 4-hydroxytamoxifen (TAM) administration. We describe two transgenic lines. The first one, when introgressed into an endogenous Rag2−/− genetic background is faithfully recapitulating lymphocyte development, repertoire dynamics and cryptic rearrangements, in a TAM-dependent manner. In this model, deprivation of TAM is followed by lymphocyte development arrest, evidencing the reversibility of the system. The second transgenic line is leaky, as the transgenes promote lymphocyte differentiation in absence of TAM treatment. Upon TAM-induction defects in lymphocytes composition and global health reveals the deleterious effect of uncontrolled RAG activity. Overall, this novel transgenic model provides a tool where RAG activity can be specifically manipulated to assess the dynamics of lymphocyte differentiation and the challenges imposed by the recombinase on the vertebrate genome. PMID:29176980

Expression profiles of urbilaterian genes uniquely shared between honey bee and vertebrates

PubMed Central

Matsui, Toshiaki; Yamamoto, Toshiyuki; Wyder, Stefan; Zdobnov, Evgeny M; Kadowaki, Tatsuhiko

2009-01-01

Background Large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of Bilateria (Urbilateria) is lost in each animal lineage. This event could be one of the underlying mechanisms involved in generating metazoan diversity. However, the present functions of these ancient genes have not been addressed extensively. To understand the functions and evolutionary mechanisms of such ancient Urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes (honey bee-vertebrate specific, HVS genes) as a model. Results We identified 30 honey bee and 55 mouse HVS genes. Many HVS genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee HVS genes was found to be brain enriched, and 24% of mouse HVS genes were highly expressed in either or both the brain and testis. Moreover, a minimum of 38% of mouse HVS genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. Furthermore, gene ontology (GO) analysis of HVS genes predicted that 35% of genes are associated with DNA transcription and RNA processing. Conclusion These results suggest that HVS genes include genes that are biased towards expression in the brain and gonads. They also demonstrate that at least some of Urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes. PMID:19138430

Expression profiles of urbilaterian genes uniquely shared between honey bee and vertebrates.

PubMed

Matsui, Toshiaki; Yamamoto, Toshiyuki; Wyder, Stefan; Zdobnov, Evgeny M; Kadowaki, Tatsuhiko

2009-01-12

Large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of Bilateria (Urbilateria) is lost in each animal lineage. This event could be one of the underlying mechanisms involved in generating metazoan diversity. However, the present functions of these ancient genes have not been addressed extensively. To understand the functions and evolutionary mechanisms of such ancient Urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes (honey bee-vertebrate specific, HVS genes) as a model. We identified 30 honey bee and 55 mouse HVS genes. Many HVS genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee HVS genes was found to be brain enriched, and 24% of mouse HVS genes were highly expressed in either or both the brain and testis. Moreover, a minimum of 38% of mouse HVS genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. Furthermore, gene ontology (GO) analysis of HVS genes predicted that 35% of genes are associated with DNA transcription and RNA processing. These results suggest that HVS genes include genes that are biased towards expression in the brain and gonads. They also demonstrate that at least some of Urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes.

Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation.

PubMed

Chakraborty, Anirban; Wakamiya, Maki; Venkova-Canova, Tatiana; Pandita, Raj K; Aguilera-Aguirre, Leopoldo; Sarker, Altaf H; Singh, Dharmendra Kumar; Hosoki, Koa; Wood, Thomas G; Sharma, Gulshan; Cardenas, Victor; Sarkar, Partha S; Sur, Sanjiv; Pandita, Tej K; Boldogh, Istvan; Hazra, Tapas K

2015-10-09

Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural analysis of the 5{prime} region of mouse and human Huntington disease genes reveals conservation of putative promoter region and Di- and trinucleotide polymorphisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Biaoyang; Nasir, J.; Kalchman, M.A.

1995-02-10

We have previously cloned and characterized the murine homologue of the Huntington disease (HD) gene and shown that it maps to mouse chromosome 5 within a region of conserved synteny with human chromosome 4p16.3. Here we present a detailed comparison of the sequence of the putative promoter and the organization of the 5{prime} genomic region of the murine (Hdh) and human HD genes encompassing the first five exons. We show that in this region these two genes share identical exon boundaries, but have different-size introns. Two dinucleotide (CT) and one trinucleotide intronic polymorphism in Hdh and an intronic CA polymorphismmore » in the HD gene were identified. Comparison of 940-bp sequence 5{prime} to the putative translation start site reveals a highly conserved region (78.8% nucleotide identity) between Hdh and the HD gene from nucleotide -56 to -206 (of Hdh). Neither Hdh nor the HD gene have typical TATA or CCAAT elements, but both show one putative AP2 binding site and numerous potential Sp1 binding sites. The high sequence identity between Hdh and the HD gene for approximately 200 bp 5{prime} to the putative translation start site indicates that these sequences may play a role in regulating expression of the Huntington disease gene. 30 refs., 4 figs., 2 tabs.« less

Sequence analysis, expression patterns and transcriptional regulation of mouse Ifrg15 during preimplantation embryonic development.

PubMed

Wu, Feng-Rui; Ding, Biao; Qi, Bin; Shang, Ming-Bao; Yang, Xun-Xun; Liu, Yong; Li, Wen-Yong

2012-10-10

Ifrg15 is a newly identified interferon alpha responsive gene and is implicated in a wide variety of physiological roles in mammals. In the present study, multiple alignments of the deduced amino acids of 10 eutherian mammalian IFRG15/Ifrg15s isolated from open genomic database revealed that they were highly conserved. Real-time PCR showed that mouse Ifrg15 mRNA was expressed in MII stage oocytes and preimplantation embryos, and its highest value peaked at the stage of mouse blastocysts. To understand the effect of three development-related genes on the promoter activity of mouse Ifrg15, promoter analysis using luciferase assays in COS-7 cells were performed. The results showed that the transcription of mouse Ifrg15 was suppressed by Oct4 and Nanog when transfected with the longest Ifrg15 promoter reporter gene. After the relatively shorter promoters were co-transfected with Oct4, c-Myc and Nanog, the relative luciferase activities of Ifrg15 were gradually increased. These in vitro results data and expression profiles of Ifrg15 as revealed by real-time PCR partly indicated that Ifrg15 transcription might be either potentially regulated or dependent on the post-transcriptional effects of IFN-α mediated by the three genes indirectly. Our data suggested that the mouse Ifrg15 might interact with these key development-related genes and play significant roles on the mouse preimplantation embryos development, especially for the development of mouse blastocysts. Copyright © 2012 Elsevier B.V. All rights reserved.

New consensus nomenclature for mammalian keratins

PubMed Central

Schweizer, Jürgen; Bowden, Paul E.; Coulombe, Pierre A.; Langbein, Lutz; Lane, E. Birgitte; Magin, Thomas M.; Maltais, Lois; Omary, M. Bishr; Parry, David A.D.; Rogers, Michael A.; Wright, Mathew W.

2006-01-01

Keratins are intermediate filament–forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells. In 1982, a nomenclature was devised to name the keratin proteins that were known at that point. The systematic sequencing of the human genome in recent years uncovered the existence of several novel keratin genes and their encoded proteins. Their naming could not be adequately handled in the context of the original system. We propose a new consensus nomenclature for keratin genes and proteins that relies upon and extends the 1982 system and adheres to the guidelines issued by the Human and Mouse Genome Nomenclature Committees. This revised nomenclature accommodates functional genes and pseudogenes, and although designed specifically for the full complement of human keratins, it offers the flexibility needed to incorporate additional keratins from other mammalian species. PMID:16831889

GRID-seq reveals the global RNA-chromatin interactome

PubMed Central

Li, Xiao; Zhou, Bing; Chen, Liang; Gou, Lan-Tao; Li, Hairi; Fu, Xiang-Dong

2017-01-01

Higher eukaryotic genomes are bound by a large number of coding and non-coding RNAs, but approaches to comprehensively map the identity and binding sites of these RNAs are lacking. Here we report a method to in situ capture global RNA interactions with DNA by deep sequencing (GRID-seq), which enables the comprehensive identification of the entire repertoire of chromatin-interacting RNAs and their respective binding sites. In human, mouse and Drosophila cells, we detected a large set of tissue-specific coding and non-coding RNAs that are bound to active promoters and enhancers, especially super-enhancers. Assuming that most mRNA-chromatin interactions indicate the physical proximity of a promoter and an enhancer, we constructed a three-dimensional global connectivity map of promoters and enhancers, revealing transcription activity-linked genomic interactions in the nucleus. PMID:28922346

An efficient approach to BAC based assembly of complex genomes.

PubMed

Visendi, Paul; Berkman, Paul J; Hayashi, Satomi; Golicz, Agnieszka A; Bayer, Philipp E; Ruperao, Pradeep; Hurgobin, Bhavna; Montenegro, Juan; Chan, Chon-Kit Kenneth; Staňková, Helena; Batley, Jacqueline; Šimková, Hana; Doležel, Jaroslav; Edwards, David

2016-01-01

There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

DNA sequence chromatogram browsing using JAVA and CORBA.

PubMed

Parsons, J D; Buehler, E; Hillier, L

1999-03-01

DNA sequence chromatograms (traces) are the primary data source for all large-scale genomic and expressed sequence tags (ESTs) sequencing projects. Access to the sequencing trace assists many later analyses, for example contig assembly and polymorphism detection, but obtaining and using traces is problematic. Traces are not collected and published centrally, they are much larger than the base calls derived from them, and viewing them requires the interactivity of a local graphical client with local data. To provide efficient global access to DNA traces, we developed a client/server system based on flexible Java components integrated into other applications including an applet for use in a WWW browser and a stand-alone trace viewer. Client/server interaction is facilitated by CORBA middleware which provides a well-defined interface, a naming service, and location independence. [The software is packaged as a Jar file available from the following URL: http://www.ebi.ac.uk/jparsons. Links to working examples of the trace viewers can be found at http://corba.ebi.ac.uk/EST. All the Washington University mouse EST traces are available for browsing at the same URL.