Whole-genome analyses of DS-1-like human G2P[4] and G8P[4] rotavirus strains from Eastern, Western and Southern Africa

PubMed Central

Nyaga, Martin M.; Stucker, Karla M.; Esona, Mathew D.; Jere, Khuzwayo C.; Mwinyi, Bakari; Shonhai, Annie; Tsolenyanu, Enyonam; Mulindwa, Augustine; Chibumbya, Julia N.; Adolfine, Hokororo; Halpin, Rebecca A.; Roy, Sunando; Stockwell, Timothy B.; Berejena, Chipo; Seheri, Mapaseka L.; Mwenda, Jason M.; Steele, A. Duncan; Wentworth, David E.

2018-01-01

Group A rotaviruses (RVAs) with distinct G and P genotype combinations have been reported globally. We report the genome composition and possible origin of seven G8P[4] and five G2P[4] human RVA strains based on the genetic evolution of all 11 genome segments at the nucleotide level. Twelve RVA ELISA positive stool samples collected in the representative countries of Eastern, Southern and West Africa during the 2007–2012 surveillance seasons were subjected to sequencing using the Ion Torrent PGM and Illumina MiSeq platforms. A reference-based assembly was performed using CLC Bio’s clc_ref_assemble_long program, and full-genome consensus sequences were obtained. With the exception of the neutralising antigen, VP7, all study strains exhibited the DS-1-like genome constellation (P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) and clustered phylogenetically with reference strains having a DS-1-like genetic backbone. Comparison of the nucleotide and amino acid sequences with selected global cognate genome segments revealed nucleotide and amino acid sequence identities of 81.7–100 % and 90.6–100 %, respectively, with NSP4 gene segment showing the most diversity among the strains. Bayesian analyses of all gene sequences to estimate the time of divergence of the lineage indicated that divergence times ranged from 16 to 44 years, except for the NSP4 gene where the lineage seemed to arise in the more distant past at an estimated 203 years ago. However, the long-term effects of changes found within the NSP4 genome segment should be further explored, and thus we recommend continued whole-genome analyses from larger sample sets to determine the evolutionary mechanisms of the DS-1-like strains collected in Africa. PMID:24952422

The Echinococcus canadensis (G7) genome: a key knowledge of parasitic platyhelminth human diseases.

PubMed

Maldonado, Lucas L; Assis, Juliana; Araújo, Flávio M Gomes; Salim, Anna C M; Macchiaroli, Natalia; Cucher, Marcela; Camicia, Federico; Fox, Adolfo; Rosenzvit, Mara; Oliveira, Guilherme; Kamenetzky, Laura

2017-02-27

The parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools. We sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus. This is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high

Analysis of complete genome sequences of G9P[19] rotavirus strains from human and piglet with diarrhea provides evidence for whole-genome interspecies transmission of nonreassorted porcine rotavirus.

PubMed

Yodmeeklin, Arpaporn; Khamrin, Pattara; Chuchaona, Watchaporn; Kumthip, Kattareeya; Kongkaew, Aphisek; Vachirachewin, Ratchaya; Okitsu, Shoko; Ushijima, Hiroshi; Maneekarn, Niwat

2017-01-01

Whole genomes of G9P[19] human (RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19]) and porcine (RVA/Pig-wt/THA/CMP-015-12/2012/G9P[19]) rotaviruses concurrently detected in the same geographical area in northern Thailand were sequenced and analyzed for their genetic relationships using bioinformatic tools. The complete genome sequence of human rotavirus RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19] was most closely related to those of porcine rotavirus RVA/Pig-wt/THA/CMP-015-12/2012/G9P[19] and to those of porcine-like human and porcine rotaviruses reference strains than to those of human rotavirus reference strains. The genotype constellation of G9P[19] detected in human and piglet were identical and displayed as the G9-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotypes with the nucleotide sequence identities of VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4, and NSP5 at 99.0%, 99.5%, 93.2%, 97.7%, 97.7%, 85.6%, 89.5%, 93.2%, 92.9%, 94.0%, and 98.1%, respectively. The findings indicate that human rotavirus strain RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19] containing the genome segments of porcine genetic backbone is most likely a human rotavirus of porcine origin. Our data provide an evidence of interspecies transmission and whole-genome transmission of nonreassorted G9P[19] porcine RVA to human occurring in nature in northern Thailand. Copyright © 2016. Published by Elsevier B.V.

Entire nucleotide sequences of Gossypium raimondii and G. arboreum mitochondrial genomes revealed A-genome species as cytoplasmic donor of the allotetraploid species.

PubMed

Chen, Z; Nie, H; Grover, C E; Wang, Y; Li, P; Wang, M; Pei, H; Zhao, Y; Li, S; Wendel, J F; Hua, J

2017-05-01

Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A-G and K, and an allotetraploid genomic group, AD. Gossypium raimondii (D 5 ) and G. arboreum (A 2 ) are the putative contributors to the progenitor of G. hirsutum (AD 1 ), the economically important fibre-producing cotton species. Mitochondrial DNA from week-old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genomes were sequenced, assembled, annotated and analysed in orderly. Gossypium raimondii (D 5 ) and G. arboreum (A 2 ) mitochondrial genomes were provided in this study. The mitochondrial genomes of two diploid species harboured circular genome of 643,914 bp (D 5 ) and 687,482 bp (A 2 ), respectively. They differ in size and number of repeat sequences, both contain illuminating triplicate sequences with 7317 and 10,246 bp, respectively, demonstrating dynamic difference and rearranged genome organisations. Comparing the D 5 and A 2 mitogenomes with mitogenomes of tetraploid Gossypium species (AD 1 , G. hirsutum; AD 2 , G. barbadense), a shared 11 kbp fragment loss was detected in allotetraploid species, three regions shared by G. arboreum (A 2 ), G. hirsutum (AD 1 ) and G. barbadense (AD 2 ), while eight regions were specific to G. raimondii (D 5 ). The presence/absence variations and gene-based phylogeny supported that A-genome is a cytoplasmic donor to the progenitor of allotetraploid species G. hirsutum and G. barbadense. The results present structure variations and phylogeny of Gossypium mitochondrial genome evolution. © 2017 The Authors. Plant Biology published by John Wiley & Sons Ltd on behalf of German Botanical Society, Royal Dutch Botanical Society.

Genome Sequence of the Mesophilic Thermotogales Bacterium Mesotoga prima MesG1.Ag.4.2 Reveals the Largest Thermotogales Genome To Date

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

Zhaxybayeva, Olga; Swithers, Kristen S; Foght, Julia

2012-01-01

Here we describe the genome of Mesotoga prima MesG1.Ag4.2, the first genome of a mesophilic Thermotogales bacterium. Mesotoga prima was isolated from a polychlorinated biphenyl (PCB)-dechlorinating enrichment culture from Baltimore Harbor sediments. Its 2.97 Mb genome is considerably larger than any previously sequenced Thermotogales genomes, which range between 1.86 and 2.30 Mb. This larger size is due to both higher numbers of protein-coding genes and larger intergenic regions. In particular, the M. prima genome contains more genes for proteins involved in regulatory functions, for instance those involved in regulation of transcription. Together with its closest relative, Kosmotoga olearia, it alsomore » encodes different types of proteins involved in environmental and cell-cell interactions as compared with other Thermotogales bacteria. Amino acid composition analysis of M. prima proteins implies that this lineage has inhabited low-temperature environments for a long time. A large fraction of the M. prima genome has been acquired by lateral gene transfer (LGT): a DarkHorse analysis suggests that 766 (32%) of predicted protein-coding genes have been involved in LGT after Mesotoga diverged from the other Thermotogales lineages. A notable example of a lineage-specific LGT event is a reductive dehalogenase gene - a key enzyme in dehalorespiration, indicating M. prima may have a more active role in PCB dechlorination than was previously assumed.« less

Informed decision-making among students analyzing their personal genomes on a whole genome sequencing course: a longitudinal cohort study

PubMed Central

2013-01-01

Background Multiple laboratories now offer clinical whole genome sequencing (WGS). We anticipate WGS becoming routinely used in research and clinical practice. Many institutions are exploring how best to educate geneticists and other professionals about WGS. Providing students in WGS courses with the option to analyze their own genome sequence is one strategy that might enhance students’ engagement and motivation to learn about personal genomics. However, if this option is presented to students, it is vital they make informed decisions, do not feel pressured into analyzing their own genomes by their course directors or peers, and feel free to analyze a third-party genome if they prefer. We therefore developed a 26-hour introductory genomics course in part to help students make informed decisions about whether to receive personal WGS data in a subsequent advanced genomics course. In the advanced course, they had the option to receive their own personal genome data, or an anonymous genome, at no financial cost to them. Our primary aims were to examine whether students made informed decisions regarding analyzing their personal genomes, and whether there was evidence that the introductory course enabled the students to make a more informed decision. Methods This was a longitudinal cohort study in which students (N = 19) completed questionnaires assessing their intentions, informed decision-making, attitudes and knowledge before (T1) and after (T2) the introductory course, and before the advanced course (T3). Informed decision-making was assessed using the Decisional Conflict Scale. Results At the start of the introductory course (T1), most (17/19) students intended to receive their personal WGS data in the subsequent course, but many expressed conflict around this decision. Decisional conflict decreased after the introductory course (T2) indicating there was an increase in informed decision-making, and did not change before the advanced course (T3). This suggests

Variation, Evolution, and Correlation Analysis of C+G Content and Genome or Chromosome Size in Different Kingdoms and Phyla

PubMed Central

Li, Xiu-Qing; Du, Donglei

2014-01-01

C+G content (GC content or G+C content) is known to be correlated with genome/chromosome size in bacteria but the relationship for other kingdoms remains unclear. This study analyzed genome size, chromosome size, and base composition in most of the available sequenced genomes in various kingdoms. Genome size tends to increase during evolution in plants and animals, and the same is likely true for bacteria. The genomic C+G contents were found to vary greatly in microorganisms but were quite similar within each animal or plant subkingdom. In animals and plants, the C+G contents are ranked as follows: monocot plants>mammals>non-mammalian animals>dicot plants. The variation in C+G content between chromosomes within species is greater in animals than in plants. The correlation between average chromosome C+G content and chromosome length was found to be positive in Proteobacteria, Actinobacteria (but not in other analyzed bacterial phyla), Ascomycota fungi, and likely also in some plants; negative in some animals, insignificant in two protist phyla, and likely very weak in Archaea. Clearly, correlations between C+G content and chromosome size can be positive, negative, or not significant depending on the kingdoms/groups or species. Different phyla or species exhibit different patterns of correlation between chromosome-size and C+G content. Most chromosomes within a species have a similar pattern of variation in C+G content but outliers are common. The data presented in this study suggest that the C+G content is under genetic control by both trans- and cis- factors and that the correlation between C+G content and chromosome length can be positive, negative, or not significant in different phyla. PMID:24551092

Sequencing and phylogenetic analysis of the coding region of six common rotavirus strains: evidence for intragenogroup reassortment among co-circulating G1P[8] and G2P[4] strains from the United States.

PubMed

Bányai, K; Mijatovic-Rustempasic, S; Hull, J J; Esona, M D; Freeman, M M; Frace, A M; Bowen, M D; Gentsch, J R

2011-03-01

The segmented genome of rotaviruses provides an opportunity for rotavirus strains to generate a large genetic diversity through reassortment; however, this mechanism is considered to play little role in the generation of mosaic gene constellations between Wa-like and DS-1-like strains in genes other than the neutralization antigens. A pilot study was undertaken to analyze these two epidemiologically important strains at the genomic level in order to (i) identify intergenogroup reassortment and (ii) to make available additional reference genome sequences of G1P[8] and G2P[4] for future genomics analyses. The full or nearly complete coding region of all 11 genes for 3 G1P[8] (LB2719, LB2758, and LB2771) and 3 G2P[4] (LB2744, LB2764, and LB2772) strains isolated from children hospitalized with severe diarrhea in Long Beach, California, where these strains were circulating at comparable rates during 2005-2006 are described in this study. Based on the full-genome classification system, all G1P[8] strains had a conserved genomic constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1 and were mostly identical to the few Wa-like strains whose genome sequences have already been determined. Similarly, the genome sequences of the 3 G2P[4] strains were highly conserved: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-E2-H2 and displayed an overall lesser genetic divergence with reference DS-1-like strains. While intergenogroup reassortment was not seen between the G1P[8] and G2P[4] strains studied here, evidence for intragenogroup reassortment events was identified. Similar studies in the post-rotavirus genomic era will help uncover whether intergenogroup reassortment affecting the backbone genes could play a significant role in any potential vaccine breakthrough events by evading immunity of vaccinated children. Copyright © 2011 Wiley-Liss, Inc.

Genomic and Transcriptomic Associations Identify a New Insecticide Resistance Phenotype for the Selective Sweep at the Cyp6g1 Locus of Drosophila melanogaster.

PubMed

Battlay, Paul; Schmidt, Joshua M; Fournier-Level, Alexandre; Robin, Charles

2016-08-09

Scans of the Drosophila melanogaster genome have identified organophosphate resistance loci among those with the most pronounced signature of positive selection. In this study, the molecular basis of resistance to the organophosphate insecticide azinphos-methyl was investigated using the Drosophila Genetic Reference Panel, and genome-wide association. Recently released full transcriptome data were used to extend the utility of the Drosophila Genetic Reference Panel resource beyond traditional genome-wide association studies to allow systems genetics analyses of phenotypes. We found that both genomic and transcriptomic associations independently identified Cyp6g1, a gene involved in resistance to DDT and neonicotinoid insecticides, as the top candidate for azinphos-methyl resistance. This was verified by transgenically overexpressing Cyp6g1 using natural regulatory elements from a resistant allele, resulting in a 6.5-fold increase in resistance. We also identified four novel candidate genes associated with azinphos-methyl resistance, all of which are involved in either regulation of fat storage, or nervous system development. In Cyp6g1, we find a demonstrable resistance locus, a verification that transcriptome data can be used to identify variants associated with insecticide resistance, and an overlap between peaks of a genome-wide association study, and a genome-wide selective sweep analysis. Copyright © 2016 Battlay et al.

How do students react to analyzing their own genomes in a whole-genome sequencing course?: outcomes of a longitudinal cohort study.

PubMed

Sanderson, Saskia C; Linderman, Michael D; Zinberg, Randi; Bashir, Ali; Kasarskis, Andrew; Zweig, Micol; Suckiel, Sabrina; Shah, Hardik; Mahajan, Milind; Diaz, George A; Schadt, Eric E

2015-11-01

Health-care professionals need to be trained to work with whole-genome sequencing (WGS) in their practice. Our aim was to explore how students responded to a novel genome analysis course that included the option to analyze their own genomes. This was an observational cohort study. Questionnaires were administered before (T3) and after the genome analysis course (T4), as well as 6 months later (T5). In-depth interviews were conducted at T5. All students (n = 19) opted to analyze their own genomes. At T5, 12 of 15 students stated that analyzing their own genomes had been useful. Ten reported they had applied their knowledge in the workplace. Technical WGS knowledge increased (mean of 63.8% at T3, mean of 72.5% at T4; P = 0.005). In-depth interviews suggested that analyzing their own genomes may increase students' motivation to learn and their understanding of the patient experience. Most (but not all) of the students reported low levels of WGS results-related distress and low levels of regret about their decision to analyze their own genomes. Giving students the option of analyzing their own genomes may increase motivation to learn, but some students may experience personal WGS results-related distress and regret. Additional evidence is required before considering incorporating optional personal genome analysis into medical education on a large scale.

Mms1 is an assistant for regulating G-quadruplex DNA structures.

PubMed

Schwindt, Eike; Paeschke, Katrin

2018-06-01

The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

SOPanG: online text searching over a pan-genome.

PubMed

Cislak, Aleksander; Grabowski, Szymon; Holub, Jan

2018-06-22

The many thousands of high-quality genomes available nowadays imply a shift from single genome to pan-genomic analyses. A basic algorithmic building brick for such a scenario is online search over a collection of similar texts, a problem with surprisingly few solutions presented so far. We present SOPanG, a simple tool for exact pattern matching over an elastic-degenerate string, a recently proposed simplified model for the pan-genome. Thanks to bit-parallelism, it achieves pattern matching speeds above 400MB/s, more than an order of magnitude higher than of other software. SOPanG is available for free from: https://github.com/MrAlexSee/sopang. Supplementary data are available at Bioinformatics online.

Genomic sequence of Heliothis virescens ascovirus 3g isolated from Spodoptera exigua.

PubMed

Huang, Guo-Hua; Wang, Yun-Sheng; Wang, Xing; Garretson, Tyler A; Dai, Liang-Ying; Zhang, Chuan-Xi; Cheng, Xiao-Wen

2012-11-01

Heliothis virescens ascovirus 3a (HvAV-3a), a member of the family Ascoviridae, has the highest diversity among ascovirus species that have been reported in Australia, Indonesia, China, and the United States. To understand the diversity and origin of this important ascovirus, the complete genome of the HvAV Indonesia strain (HvAV-3g), isolated from Spodoptera exigua, was determined to be 199,721 bp, with a G+C content of 45.9%. Therefore, HvAV-3g has the largest genome among the reported ascovirus genomes to date. There are 194 predicted open reading frames (ORFs) encoding proteins of 50 or more amino acid residues. In comparison to HvAV-3e reported from Australia, HvAV-3g has all the ORFs in HvAV-3e with 6 additional ORFs unique to HvAV-3g, including 1 peptidase C26 gene with the highest identity to Drosophila spp. and 2 gas vesicle protein U (GvpU) genes with identities to Bacillus megaterium. The five unique homologous regions (hrs) and 25 baculovirus repeat ORFs (bro) of HvAV-3g are highly variable.

Complete genome sequence of uropathogenic Escherichia coli isolate UPEC 26-1.

PubMed

Subhadra, Bindu; Kim, Dong Ho; Kim, Jaeseok; Woo, Kyungho; Sohn, Kyung Mok; Kim, Hwa-Jung; Han, Kyudong; Oh, Man Hwan; Choi, Chul Hee

2018-06-01

Urinary tract infections (UTIs) are among the most common infections in humans, predominantly caused by uropathogenic Escherichia coli (UPEC). The diverse genomes of UPEC strains mostly impede disease prevention and control measures. In this study, we comparatively analyzed the whole genome sequence of a highly virulent UPEC strain, namely UPEC 26-1, which was isolated from urine sample of a patient suffering from UTI in Korea. Whole genome analysis showed that the genome consists of one circular chromosome of 5,329,753 bp, comprising 5064 protein-coding genes, 122 RNA genes (94 tRNA, 22 rRNA and 6 ncRNA genes), and 100 pseudogenes, with an average G+C content of 50.56%. In addition, we identified 8 prophage regions comprising 5 intact, 2 incomplete and 1 questionable ones and 63 genomic islands, suggesting the possibility of horizontal gene transfer in this strain. Comparative genome analysis of UPEC 26-1 with the UPEC strain CFT073 revealed an average nucleotide identity of 99.7%. The genome comparison with CFT073 provides major differences in the genome of UPEC 26-1 that would explain its increased virulence and biofilm formation. Nineteen of the total GIs were unique to UPEC 26-1 compared to CFT073 and nine of them harbored unique genes that are involved in virulence, multidrug resistance, biofilm formation and bacterial pathogenesis. The data from this study will assist in future studies of UPEC strains to develop effective control measures.

Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells.

PubMed

Ikegami, Kohta; Iwatani, Misa; Suzuki, Masako; Tachibana, Makoto; Shinkai, Yoichi; Tanaka, Satoshi; Greally, John M; Yagi, Shintaro; Hattori, Naka; Shiota, Kunio

2007-01-01

In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a(-/-) embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a(-/-) cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a(-/-) cells. These data indicate that G9a site-selectively contributes to DNA methylation.

Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus.

PubMed

Zhang, Fengbo; Ma, Xiumin; Zhu, Yuejie; Wang, Hongying; Liu, Xianfei; Zhu, Min; Ma, Haimei; Wen, Hao; Fan, Haining; Ding, Jianbing

2014-01-01

This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus. Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively. EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%. Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity.

Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus

PubMed Central

Zhang, Fengbo; Ma, Xiumin; Zhu, Yuejie; Wang, Hongying; Liu, Xianfei; Zhu, Min; Ma, Haimei; Wen, Hao; Fan, Haining; Ding, Jianbing

2014-01-01

Objective: This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus. Methods: Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively. Results: EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%. Conclusion: Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity. PMID:25337206

Whole Genome Characterization, Phylogenetic and Genome Signature Analysis of Human Pandemic H1N1 Virus in Thailand, 2009–2012

PubMed Central

Makkoch, Jarika; Suwannakarn, Kamol; Payungporn, Sunchai; Prachayangprecha, Slinporn; Cheiocharnsin, Thaweesak; Linsuwanon, Piyada; Theamboonlers, Apiradee; Poovorawan, Yong

2012-01-01

Background Three waves of human pandemic influenza occurred in Thailand in 2009–2012. The genome signature features and evolution of pH1N1 need to be characterized to elucidate the aspects responsible for the multiple waves of pandemic. Methodology/Findings Forty whole genome sequences and 584 partial sequences of pH1N1 circulating in Thailand, divided into 1st, 2nd and 3rd wave and post-pandemic were characterized and 77 genome signatures were analyzed. Phylogenetic trees of concatenated whole genome and HA gene sequences were constructed calculating substitution rate and dN/dS of each gene. Phylogenetic analysis showed a distinct pattern of pH1N1 circulation in Thailand, with the first two isolates from May, 2009 belonging to clade 5 while clades 5, 6 and 7 co-circulated during the first wave of pH1N1 pandemic in Thailand. Clade 8 predominated during the second wave and different proportions of the pH1N1 viruses circulating during the third wave and post pandemic period belonged to clades 8, 11.1 and 11.2. The mutation analysis of pH1N1 revealed many adaptive mutations which have become the signature of each clade and may be responsible for the multiple pandemic waves in Thailand, especially with regard to clades 11.1 and 11.2 as evidenced with V731I, G154D of PB1 gene, PA I330V, HA A214T S160G and S202T. The substitution rate of pH1N1 in Thailand ranged from 2.53×10−3±0.02 (M2 genes) to 5.27×10−3±0.03 per site per year (NA gene). Conclusions All results suggested that this virus is still adaptive, maybe to evade the host's immune response and tends to remain in the human host although the dN/dS were under purifying selection in all 8 genes. Due to the gradual evolution of pH1N1 in Thailand, continuous monitoring is essential for evaluation and surveillance to be prepared for and able to control future influenza activities. PMID:23251479

Bionimbus: a cloud for managing, analyzing and sharing large genomics datasets

PubMed Central

Heath, Allison P; Greenway, Matthew; Powell, Raymond; Spring, Jonathan; Suarez, Rafael; Hanley, David; Bandlamudi, Chai; McNerney, Megan E; White, Kevin P; Grossman, Robert L

2014-01-01

Background As large genomics and phenotypic datasets are becoming more common, it is increasingly difficult for most researchers to access, manage, and analyze them. One possible approach is to provide the research community with several petabyte-scale cloud-based computing platforms containing these data, along with tools and resources to analyze it. Methods Bionimbus is an open source cloud-computing platform that is based primarily upon OpenStack, which manages on-demand virtual machines that provide the required computational resources, and GlusterFS, which is a high-performance clustered file system. Bionimbus also includes Tukey, which is a portal, and associated middleware that provides a single entry point and a single sign on for the various Bionimbus resources; and Yates, which automates the installation, configuration, and maintenance of the software infrastructure required. Results Bionimbus is used by a variety of projects to process genomics and phenotypic data. For example, it is used by an acute myeloid leukemia resequencing project at the University of Chicago. The project requires several computational pipelines, including pipelines for quality control, alignment, variant calling, and annotation. For each sample, the alignment step requires eight CPUs for about 12 h. BAM file sizes ranged from 5 GB to 10 GB for each sample. Conclusions Most members of the research community have difficulty downloading large genomics datasets and obtaining sufficient storage and computer resources to manage and analyze the data. Cloud computing platforms, such as Bionimbus, with data commons that contain large genomics datasets, are one choice for broadening access to research data in genomics. PMID:24464852

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs.

PubMed

Svarovskaia, Evguenia S; Xu, Hongzhan; Mbisa, Jean L; Barr, Rebekah; Gorelick, Robert J; Ono, Akira; Freed, Eric O; Hu, Wei-Shau; Pathak, Vinay K

2004-08-20

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30-40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained approximately 1% of the viral genomic RNA but approximately 30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.

G-Anchor: a novel approach for whole-genome comparative mapping utilizing evolutionary conserved DNA sequences.

PubMed

Lenis, Vasileios Panagiotis E; Swain, Martin; Larkin, Denis M

2018-05-01

Cross-species whole-genome sequence alignment is a critical first step for genome comparative analyses, ranging from the detection of sequence variants to studies of chromosome evolution. Animal genomes are large and complex, and whole-genome alignment is a computationally intense process, requiring expensive high-performance computing systems due to the need to explore extensive local alignments. With hundreds of sequenced animal genomes available from multiple projects, there is an increasing demand for genome comparative analyses. Here, we introduce G-Anchor, a new, fast, and efficient pipeline that uses a strictly limited but highly effective set of local sequence alignments to anchor (or map) an animal genome to another species' reference genome. G-Anchor makes novel use of a databank of highly conserved DNA sequence elements. We demonstrate how these elements may be aligned to a pair of genomes, creating anchors. These anchors enable the rapid mapping of scaffolds from a de novo assembled genome to chromosome assemblies of a reference species. Our results demonstrate that G-Anchor can successfully anchor a vertebrate genome onto a phylogenetically related reference species genome using a desktop or laptop computer within a few hours and with comparable accuracy to that achieved by a highly accurate whole-genome alignment tool such as LASTZ. G-Anchor thus makes whole-genome comparisons accessible to researchers with limited computational resources. G-Anchor is a ready-to-use tool for anchoring a pair of vertebrate genomes. It may be used with large genomes that contain a significant fraction of evolutionally conserved DNA sequences and that are not highly repetitive, polypoid, or excessively fragmented. G-Anchor is not a substitute for whole-genome aligning software but can be used for fast and accurate initial genome comparisons. G-Anchor is freely available and a ready-to-use tool for the pairwise comparison of two genomes.

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome

PubMed Central

De Nicola, Beatrice; Lech, Christopher J.; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N.; Phan, Anh Tuân

2016-01-01

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. PMID:27298260

Genome-wide colonization of gene regulatory elements by G4 DNA motifs

PubMed Central

Du, Zhuo; Zhao, Yiqiang; Li, Ning

2009-01-01

G-quadruplex (or G4 DNA), a stable four-stranded structure found in guanine-rich regions, is implicated in the transcriptional regulation of genes involved in growth and development. Previous studies on the role of G4 DNA in gene regulation mostly focused on genomic regions proximal to transcription start sites (TSSs). To gain a more comprehensive understanding of the regulatory role of G4 DNA, we examined the landscape of potential G4 DNA (PG4Ms) motifs in the human genome and found that G4 motifs, not restricted to those found in the TSS-proximal regions, are bias toward gene-associated regions. Significantly, analyses of G4 motifs in seven types of well-known gene regulatory elements revealed a constitutive enrichment pattern and the clusters of G4 motifs tend to be colocalized with regulatory elements. Considering our analysis from a genome evolutionary perspective, we found evidence that the occurrence and accumulation of certain progenitors and canonical G4 DNA motifs within regulatory regions were progressively favored by natural selection. Our results suggest that G4 DNA motifs are ‘colonized’ in regulatory regions, supporting a likely genome-wide role of G4 DNA in gene regulation. We hypothesize that G4 DNA is a regulatory apparatus situated in regulatory elements, acting as a molecular switch that can modulate the role of the host functional regions, by transition in DNA structure. PMID:19759215

Genome-wide DNA Methylation Profiling of CpG Islands in Hypospadias

PubMed Central

Choudhry, Shweta; Deshpande, Archana; Qiao, Liang; Beckman, Kenneth; Sen, Saunak; Baskin, Laurence S.

2013-01-01

Purpose Hypospadias is one of the most frequent genital malformations in the male newborn, and results from abnormal penile and urethral development. The etiology of hypospadias remains largely unknown despite intensive investigations. Fetal androgens have a crucial role in genital differentiation. Recent studies have suggested that molecular mechanisms that underlie the effects of androgens on the fetus may involve disruption of epigenetic programming of gene expression during development. We assessed whether epigenetic modification of DNA methylation is associated with hypospadias in a case-control study of 12 hypospadias and 8 control subjects. Materials and Methods Genome-wide DNA methylation profiling was performed on the study subjects using the Illumina Infinium® HumanMethylation450 Bead-Chip, which enables the direct investigation of methylation status of more than 485,000 individual CpG sites throughout the genome. The methylation level at each CpG site was compared between cases and controls using the t test and logistic regression. Results We identified 14 CpG sites that were associated with hypospadias with p <0.00001. These CpG sites were in or near the SCARB1, MYBPH, SORBS1, LAMA4, HOXD11, MYO1D, EGFL7, C10orf41, LMAN1L and SULF1 genes. Two CpG sites in SCARB1 and MYBPH genes remained statistically significant after correction for multiple testing (p = 2.61×10−09, pcorrected = 0.008; p = 3.06×10−08, pcorrected = 0.02, respectively). Conclusions To our knowledge this is the first study to investigate hypospadias using a unique and novel epigenetic approach. Our findings suggest DNA methylation patterns are useful in identifying new genes such as SCARB1 and MYBPH that may be involved in the etiology of hypospadias. PMID:22906644

GenPlay Multi-Genome, a tool to compare and analyze multiple human genomes in a graphical interface.

PubMed

Lajugie, Julien; Fourel, Nicolas; Bouhassira, Eric E

2015-01-01

Parallel visualization of multiple individual human genomes is a complex endeavor that is rapidly gaining importance with the increasing number of personal, phased and cancer genomes that are being generated. It requires the display of variants such as SNPs, indels and structural variants that are unique to specific genomes and the introduction of multiple overlapping gaps in the reference sequence. Here, we describe GenPlay Multi-Genome, an application specifically written to visualize and analyze multiple human genomes in parallel. GenPlay Multi-Genome is ideally suited for the comparison of allele-specific expression and functional genomic data obtained from multiple phased genomes in a graphical interface with access to multiple-track operation. It also allows the analysis of data that have been aligned to custom genomes rather than to a standard reference and can be used as a variant calling format file browser and as a tool to compare different genome assembly, such as hg19 and hg38. GenPlay is available under the GNU public license (GPL-3) from http://genplay.einstein.yu.edu. The source code is available at https://github.com/JulienLajugie/GenPlay. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Bionimbus: a cloud for managing, analyzing and sharing large genomics datasets.

PubMed

Heath, Allison P; Greenway, Matthew; Powell, Raymond; Spring, Jonathan; Suarez, Rafael; Hanley, David; Bandlamudi, Chai; McNerney, Megan E; White, Kevin P; Grossman, Robert L

2014-01-01

As large genomics and phenotypic datasets are becoming more common, it is increasingly difficult for most researchers to access, manage, and analyze them. One possible approach is to provide the research community with several petabyte-scale cloud-based computing platforms containing these data, along with tools and resources to analyze it. Bionimbus is an open source cloud-computing platform that is based primarily upon OpenStack, which manages on-demand virtual machines that provide the required computational resources, and GlusterFS, which is a high-performance clustered file system. Bionimbus also includes Tukey, which is a portal, and associated middleware that provides a single entry point and a single sign on for the various Bionimbus resources; and Yates, which automates the installation, configuration, and maintenance of the software infrastructure required. Bionimbus is used by a variety of projects to process genomics and phenotypic data. For example, it is used by an acute myeloid leukemia resequencing project at the University of Chicago. The project requires several computational pipelines, including pipelines for quality control, alignment, variant calling, and annotation. For each sample, the alignment step requires eight CPUs for about 12 h. BAM file sizes ranged from 5 GB to 10 GB for each sample. Most members of the research community have difficulty downloading large genomics datasets and obtaining sufficient storage and computer resources to manage and analyze the data. Cloud computing platforms, such as Bionimbus, with data commons that contain large genomics datasets, are one choice for broadening access to research data in genomics. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase

PubMed Central

Wallgren, Marcus; Mohammad, Jani B.; Yan, Kok-Phen; Pourbozorgi-Langroudi, Parham; Ebrahimi, Mahsa; Sabouri, Nasim

2016-01-01

Certain guanine-rich sequences have an inherent propensity to form G-quadruplex (G4) structures. G4 structures are e.g. involved in telomere protection and gene regulation. However, they also constitute obstacles during replication if they remain unresolved. To overcome these threats to genome integrity, organisms harbor specialized G4 unwinding helicases. In Schizosaccharomyces pombe, one such candidate helicase is Pfh1, an evolutionarily conserved Pif1 homolog. Here, we addressed whether putative G4 sequences in S. pombe can adopt G4 structures and, if so, whether Pfh1 can resolve them. We tested two G4 sequences, derived from S. pombe ribosomal and telomeric DNA regions, and demonstrated that they form inter- and intramolecular G4 structures, respectively. Also, Pfh1 was enriched in vivo at the ribosomal G4 DNA and telomeric sites. The nuclear isoform of Pfh1 (nPfh1) unwound both types of structure, and although the G4-stabilizing compound Phen-DC3 significantly enhanced their stability, nPfh1 still resolved them efficiently. However, stable G4 structures significantly inhibited adenosine triphosphate hydrolysis by nPfh1. Because ribosomal and telomeric DNA contain putative G4 regions conserved from yeasts to humans, our studies support the important role of G4 structure formation in these regions and provide further evidence for a conserved role for Pif1 helicases in resolving G4 structures. PMID:27185885

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation.

PubMed

Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C; Gorelick, Robert J; Keele, Brandon F; Hu, Wei-Shau; Pathak, Vinay K

2016-05-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10-21 and1 × 10-11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

PubMed

Juergens, Hannes; Varela, Javier A; Gorter de Vries, Arthur R; Perli, Thomas; Gast, Veronica J M; Gyurchev, Nikola Y; Rajkumar, Arun S; Mans, Robert; Pronk, Jack T; Morrissey, John P; Daran, Jean-Marc G

2018-05-01

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.

PubMed

De Nicola, Beatrice; Lech, Christopher J; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N; Phan, Anh Tuân

2016-07-27

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genomic organization of the neurofibromatosis 1 gene (NF1)

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

Li, Y.; O`Connell, P.; Huntsman Breidenbach, H.

Neurofibromatosis 1 maps to chromosome band 17q11.2, and the NF1 locus has been partially characterized. Even though the full-length NF1 cDNA has been sequenced, the complete genomic structure of the NF1 gene has not been elucidated. The 5{prime} end of NF1 is embedded in a CpG island containing a NotI restriction site, and the remainder of the gene lies in the adjacent 350-kb NotI fragment. In our efforts to develop a comprehensive screen for NF1 mutations, we have isolated genomic DNA clones that together harbor the entire NF1 cDNA sequence. We have identified all intron-exon boundaries of the coding regionmore » and established that it is composed of 59 exons. Furthermore, we have defined the 3{prime}-untranslated region (3{prime}-UTR) of the NF1 gene; it spans approximately 3.5 kb of genomic DNA sequence and is continuous with the stop codon. Oligonucleotide primer pairs synthesized from exon-flanking DNA sequences were used in the polymerase chain reaction with cloned, chromosome 17-specific genomic DNA as template to amplify NF1 exons 1 through 27b and the exon containing the 3{prime}-UTR separately. This information should be useful for implementing a comprehensive NF1 mutation screen using genomic DNA as template. 41 refs., 3 figs., 2 tabs.« less

G1P[8] species A rotavirus over 27 years--pre- and post-vaccination eras--in Brazil: full genomic constellation analysis and no evidence for selection pressure by Rotarix® vaccine.

PubMed

da Silva, Marcelle Figueira Marques; Rose, Tatiana Lundgren; Gómez, Mariela Martínez; Carvalho-Costa, Filipe Anibal; Fialho, Alexandre Madi; de Assis, Rosane Maria Santos; de Andrade, Juliana da Silva Ribeiro; Volotão, Eduardo de Mello; Leite, José Paulo Gagliardi

2015-03-01

Epidemiological data on species A rotavirus (RVA) infections have demonstrated the genetic diversity of strains circulating worldwide. Many G and P genotype combinations have been described over the years, varying regionally and temporally, especially in developing countries. However, the most common G and P genotype combinations identified in RVA human strains worldwide are G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. RVA genotype G1P[8] strains are responsible for more than 50% of child infections worldwide and component of the two vaccines (Rotarix® [RV1] and RotaTeq® [RV5]) licensed globally. For a better understanding of the evolutionary mechanisms of this genotype in Brazil, phylogenetic analyses based on the 11 RVA genome segments (genomic constellation) from 90 G1P[8] RVA strains collected in two eras - (i) pre-vaccination with RV1 (1996-February 2006); (ii) post-vaccination (March 2006-2013) - in different Brazilian states were performed. The results showed the Wa-like genomic constellation of the Brazilian G1P[8] strains with a I1-R1-C1-M1-A1-N1-T1-E1-H1 specificity, except for two strains (rj14055-07 and ba19030-10) that belong to a I1-R1-C1-M1-A1-N1-T3-E1-H1 genomic constellation, evidencing the occurrence of reassortment (Wa-like×AU-1-like) of the NSP3 gene. Reassortment events were also demonstrated between Brazilian G1P[8] strains and the RV1 vaccine strain in some genes in vaccinated and unvaccinated children. VP7 and VP8* antigenic site analysis showed that the amino acid substitutions observed in samples collected after the introduction of RV1 in Brazil were already detected in samples collected in the 1980s and 1990s, suggesting that mass Brazilian RV1 vaccination had no impact on the diversity observed inside antigenic sites for these two proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

Cpf1-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cpf1.

PubMed

Park, Jeongbin; Bae, Sangsu

2018-03-15

Following the type II CRISPR-Cas9 system, type V CRISPR-Cpf1 endonucleases have been found to be applicable for genome editing in various organisms in vivo. However, there are as yet no web-based tools capable of optimally selecting guide RNAs (gRNAs) among all possible genome-wide target sites. Here, we present Cpf1-Database, a genome-wide gRNA library design tool for LbCpf1 and AsCpf1, which have DNA recognition sequences of 5'-TTTN-3' at the 5' ends of target sites. Cpf1-Database provides a sophisticated but simple way to design gRNAs for AsCpf1 nucleases on the genome scale. One can easily access the data using a straightforward web interface, and using the powerful collections feature one can easily design gRNAs for thousands of genes in short time. Free access at http://www.rgenome.net/cpf1-database/. sangsubae@hanyang.ac.kr.

Systematic CpT (ApG) depletion and CpG excess are unique genomic signatures of large DNA viruses infecting invertebrates.

PubMed

Upadhyay, Mohita; Sharma, Neha; Vivekanandan, Perumal

2014-01-01

Differences in the relative abundance of dinucleotides, if any may provide important clues on host-driven evolution of viruses. We studied dinucleotide frequencies of large DNA viruses infecting vertebrates (n = 105; viruses infecting mammals = 99; viruses infecting aves = 6; viruses infecting reptiles = 1) and invertebrates (n = 88; viruses infecting insects = 84; viruses infecting crustaceans = 4). We have identified systematic depletion of CpT(ApG) dinucleotides and over-representation of CpG dinucleotides as the unique genomic signature of large DNA viruses infecting invertebrates. Detailed investigation of this unique genomic signature suggests the existence of invertebrate host-induced pressures specifically targeting CpT(ApG) and CpG dinucleotides. The depletion of CpT dinucleotides among large DNA viruses infecting invertebrates is at least in part, explained by non-canonical DNA methylation by the infected host. Our findings highlight the role of invertebrate host-related factors in shaping virus evolution and they also provide the necessary framework for future studies on evolution, epigenetics and molecular biology of viruses infecting this group of hosts.

Systematic CpT (ApG) Depletion and CpG Excess Are Unique Genomic Signatures of Large DNA Viruses Infecting Invertebrates

PubMed Central

Upadhyay, Mohita; Sharma, Neha; Vivekanandan, Perumal

2014-01-01

Differences in the relative abundance of dinucleotides, if any may provide important clues on host-driven evolution of viruses. We studied dinucleotide frequencies of large DNA viruses infecting vertebrates (n = 105; viruses infecting mammals = 99; viruses infecting aves = 6; viruses infecting reptiles = 1) and invertebrates (n = 88; viruses infecting insects = 84; viruses infecting crustaceans = 4). We have identified systematic depletion of CpT(ApG) dinucleotides and over-representation of CpG dinucleotides as the unique genomic signature of large DNA viruses infecting invertebrates. Detailed investigation of this unique genomic signature suggests the existence of invertebrate host-induced pressures specifically targeting CpT(ApG) and CpG dinucleotides. The depletion of CpT dinucleotides among large DNA viruses infecting invertebrates is at least in part, explained by non-canonical DNA methylation by the infected host. Our findings highlight the role of invertebrate host-related factors in shaping virus evolution and they also provide the necessary framework for future studies on evolution, epigenetics and molecular biology of viruses infecting this group of hosts. PMID:25369195

Significant contribution of subtype G to HIV-1 genetic complexity in Nigeria identified by a newly developed subtyping assay specific for subtype G and CRF02_AG

PubMed Central

Heipertz, Richard A.; Ayemoba, Ojor; Sanders-Buell, Eric; Poltavee, Kultida; Pham, Phuc; Kijak, Gustavo H.; Lei, Esther; Bose, Meera; Howell, Shana; O'Sullivan, Anne Marie; Bates, Adam; Cervenka, Taylor; Kuroiwa, Janelle; Akintunde, Akindiran; Ibezim, Onyekachukwu; Alabi, Abraham; Okoye, Obumneke; Manak, Mark; Malia, Jennifer; Peel, Sheila; Maisaka, Mohammed; Singer, Darrell; O’Connell, Robert J.; Robb, Merlin L.; Kim, Jerome H.; Michael, Nelson L.; Njoku, Ogbonnaya; Tovanabutra, Sodsai

2016-01-01

Abstract While abundant sequence information is available from human immunodeficiency virus type 1 (HIV-1) subtypes A, B, C and CRF01_AE for HIV-1 vaccine design, sequences from West Africa are less represented. We sought to augment our understanding of HIV-1 variants circulating in 6 Nigerian cities as a step to subsequent HIV-1 vaccine development. The G/CRF02_AG multi-region hybridization assay (MHA) was developed to differentiate subtype G, CRF02_AG and their recombinants from other subtypes based on 7 HIV-1 segments. Plasma from 224 HIV-1 infected volunteers enrolled in a cohort examining HIV-1 prevalence, risk factor, and subtype from Makurdi (30), Abuja (18), Enugu (11), Kaduna (12), Tafa (95), and Ojo/Lagos (58) was analyzed using MHA. HIV-1 genomes from 42 samples were sequenced to validate the MHA and fully explore the recombinant structure of G and CRF02_AG variants. The sensitivity and specificity of MHA varied between 73–100% and 90–100%, respectively. The subtype distribution as identified by MHA among 224 samples revealed 38% CRF02_AG, 28% G, and 26% G/CRF02_AG recombinants while 8% remained nontypeable strains. In envelope (env) gp120, 38.84% of the samples reacted to a G probe while 31.25% reacted to a CRF02 (subtype A) probe. Full genome characterization of 42 sequences revealed the complexity of Nigerian HIV-1 variants. CRF02_AG, subtype G, and their recombinants were the major circulating HIV-1 variants in 6 Nigerian cities. High proportions of samples reacted to a G probe in env gp120 confirms that subtype G infections are abundant and should be considered in strategies for global HIV-1 vaccine development. PMID:27512845

G2S: a web-service for annotating genomic variants on 3D protein structures.

PubMed

Wang, Juexin; Sheridan, Robert; Sumer, S Onur; Schultz, Nikolaus; Xu, Dong; Gao, Jianjiong

2018-06-01

Accurately mapping and annotating genomic locations on 3D protein structures is a key step in structure-based analysis of genomic variants detected by recent large-scale sequencing efforts. There are several mapping resources currently available, but none of them provides a web API (Application Programming Interface) that supports programmatic access. We present G2S, a real-time web API that provides automated mapping of genomic variants on 3D protein structures. G2S can align genomic locations of variants, protein locations, or protein sequences to protein structures and retrieve the mapped residues from structures. G2S API uses REST-inspired design and it can be used by various clients such as web browsers, command terminals, programming languages and other bioinformatics tools for bringing 3D structures into genomic variant analysis. The webserver and source codes are freely available at https://g2s.genomenexus.org. g2s@genomenexus.org. Supplementary data are available at Bioinformatics online.

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation

PubMed Central

Nikolaitchik, Olga A.; Burdick, Ryan C.; Gorelick, Robert J.; Keele, Brandon F.; Hu, Wei-Shau; Pathak, Vinay K.

2016-01-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10−5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10−21 and1 × 10−11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic

LINE1 family member is negative regulator of HLA-G expression.

PubMed

Ikeno, Masashi; Suzuki, Nobutaka; Kamiya, Megumi; Takahashi, Yuji; Kudoh, Jun; Okazaki, Tsuneko

2012-11-01

Class Ia molecules of human leucocyte antigen (HLA-A, -B and -C) are widely expressed and play a central role in the immune system by presenting peptides derived from the lumen of the endoplasmic reticulum. In contrast, class Ib molecules such as HLA-G serve novel functions. The distribution of HLA-G is mostly limited to foetal trophoblastic tissues and some tumour tissues. The mechanism required for the tissue-specific regulation of the HLA-G gene has not been well understood. Here, we investigated the genomic regulation of HLA-G by manipulating one copy of a genomic DNA fragment on a human artificial chromosome. We identified a potential negative regulator of gene expression in a sequence upstream of HLA-G that overlapped with the long interspersed element (LINE1); silencing of HLA-G involved a DNA secondary structure generated in LINE1. The presence of a LINE1 gene silencer may explain the limited expression of HLA-G compared with other class I genes.

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination

PubMed Central

Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A.; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S.; Lan, Li

2015-01-01

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome. PMID:26100862

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination.

PubMed

Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S; Lan, Li

2015-07-07

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.

Whole-genome sequencing of Aspergillus tubingensis G131 and overview of its secondary metabolism potential.

PubMed

Choque, Elodie; Klopp, Christophe; Valiere, Sophie; Raynal, José; Mathieu, Florence

2018-03-15

Black Aspergilli represent one of the most important fungal resources of primary and secondary metabolites for biotechnological industry. Having several black Aspergilli sequenced genomes should allow targeting the production of certain metabolites with bioactive properties. In this study, we report the draft genome of a black Aspergilli, A. tubingensis G131, isolated from a French Mediterranean vineyard. This 35 Mb genome includes 10,994 predicted genes. A genomic-based discovery identifies 80 secondary metabolites biosynthetic gene clusters. Genomic sequences of these clusters were blasted on 3 chosen black Aspergilli genomes: A. tubingensis CBS 134.48, A. niger CBS 513.88 and A. kawachii IFO 4308. This comparison highlights different levels of clusters conservation between the four strains. It also allows identifying seven unique clusters in A. tubingensis G131. Moreover, the putative secondary metabolites clusters for asperazine and naphtho-gamma-pyrones production were proposed based on this genomic analysis. Key biosynthetic genes required for the production of 2 mycotoxins, ochratoxin A and fumonisin, are absent from this draft genome. Even if intergenic sequences of these mycotoxins biosynthetic pathways are present, this could not lead to the production of those mycotoxins by A. tubingensis G131. Functional and bioinformatics analyses of A. tubingensis G131 genome highlight its potential for metabolites production in particular for TAN-1612, asperazine and naphtho-gamma-pyrones presenting antioxidant, anticancer or antibiotic properties.

Mitochondrial genome data confirm that yaks can serve as the intermediate host of Echinococcus canadensis (G10) on the Tibetan Plateau.

PubMed

Wu, Yantao; Li, Li; Zhu, Guoqiang; Li, Wenhui; Zhang, Nianzhang; Li, Shuangnan; Yao, Gang; Tian, Wenjun; Fu, Baoquan; Yin, Hong; Zhu, Xingquan; Yan, Hongbin; Jia, Wanzhong

2018-03-09

Cervids used to be considered the only animal intermediate hosts of the G10 genotype of Echinococcus canadensis. Yaks are often herded in the Qinghai-Tibet Plateau, China, where echinococcosis remains prevalent. However, no E. canadensis G10 cases have been recorded in yaks until now. The aim of our study was to identify causative agents of echinococcosis in yaks in this region. Total genomic DNA was extracted from the germinal layer of one hydatid using a Blood and Tissue Kit. Full-length mitochondrial (mt) cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes were amplified by PCR. All purified PCR products were directly sequenced in both directions. Then seven pairs of overlap primers were designed to amplify the entire mt genome sequence of a suspected E. canadensis G10 isolate. Phylogenetic analyses were performed based on concatenated nucleotides from the 12 protein-coding genes of mt genomes of Echinococcus species in a Bayesian framework using MrBayes v3.1 and implementing the GTR + I + G model. Hydatids were found in yaks (n = 129) when organs were inspected at the slaughterhouse in Maqu county, Gannan Tibetan Autonomous Prefecture, Gansu Province, China in October 2016. Of these, 33 (25.6%) harbored up to a dozen hydatid cysts. One cyst from each yak was characterized by sequencing its mitochondrial (mt) cox1 and nad1 genes. On the basis of these sequence data, 32 cysts were identified as Echinococcus granulosus (sensu stricto) (G1-G3) and the remaining one was identified as the G10 genotype of E. canadensis. Its mt genome was then fully sequenced and compared with that of the G10 genotype in GenBank (AB745463). Phylogenetic analysis using complete mt genomes confirmed the Chinese cyst as belonging to the G10 genotype. To our knowledge, this is the first report globally of E. canadensis (G10) from yaks in China, which suggests that the G10 genotype has a wider geographical distribution and broader host range than

Full genome analysis of rotavirus G9P[8] strains identified in acute gastroenteritis cases reveals genetic diversity: Pune, western India.

PubMed

Tatte, Vaishali S; Chaphekar, Deepa; Gopalkrishna, Varanasi

2017-08-01

Group A rotaviruses (RVA) are the major enteric etiological agents of severe acute gastroenteritis among children globally. As G9 RVA now represents as one of the major human RVA genotypes, studies on full genome of this particular genotype are being carried out worldwide. So far, no such studies on G9P[8] RVAs have been reported from Pune, western part of India. Keeping in view of this, the study was undertaken to understand the degree of genetic diversity of the commonly circulating G9P[8] RVA strains. Rotavirus surveillance studies carried out earlier during the years 2009-2011 showed increase in the prevalence of G9P[8] RVAs. Representative G9P[8] RVA strains from the years 2009, 2010, and 2011 were selected for the study. In general, all the G9 RVA strains showed clustering in the globally circulating sublineage of the VP7 gene and showed nucleotide/amino acid identities of 96.8-99.7%/96.9-99.8% with global G9 RV strains. Full genome analysis, of all three RVAs in this study indicated Wa-like genotype constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Within the strains nucleotide/amino acid divergence of 0.1-3.4%/0.0-4.1% was noted in all the RVA structural and non-structural genes. In conclusion, the present study highlights intra-genotypic variations throughout the RVA genome. The study further emphasizes the need for surveillance and analysis of the whole genomic constellation of the commonly circulating RVA strains of other regions in the country for understanding to a greater degree of the impact of rotavirus vaccination recently introduced in India. © 2017 Wiley Periodicals, Inc.

Complete Genome Sequence of EtG, the First Phage Sequenced from Erwinia tracheiphila.

PubMed

Andrade-Domínguez, Andrés; Kolter, Roberto; Shapiro, Lori R

2018-02-22

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits. Here, we report the genome sequence of the temperate phage EtG, which was isolated from an E. tracheiphila -infected cucumber plant. Phage EtG has a linear 30,413-bp double-stranded DNA genome with cohesive ends and 45 predicted open reading frames. Copyright © 2018 Andrade-Domínguez et al.

A Hybrid Approach for CpG Island Detection in the Human Genome.

PubMed

Yang, Cheng-Hong; Lin, Yu-Da; Chiang, Yi-Cheng; Chuang, Li-Yeh

2016-01-01

CpG islands have been demonstrated to influence local chromatin structures and simplify the regulation of gene activity. However, the accurate and rapid determination of CpG islands for whole DNA sequences remains experimentally and computationally challenging. A novel procedure is proposed to detect CpG islands by combining clustering technology with the sliding-window method (PSO-based). Clustering technology is used to detect the locations of all possible CpG islands and process the data, thus effectively obviating the need for the extensive and unnecessary processing of DNA fragments, and thus improving the efficiency of sliding-window based particle swarm optimization (PSO) search. This proposed approach, named ClusterPSO, provides versatile and highly-sensitive detection of CpG islands in the human genome. In addition, the detection efficiency of ClusterPSO is compared with eight CpG island detection methods in the human genome. Comparison of the detection efficiency for the CpG islands in human genome, including sensitivity, specificity, accuracy, performance coefficient (PC), and correlation coefficient (CC), ClusterPSO revealed superior detection ability among all of the test methods. Moreover, the combination of clustering technology and PSO method can successfully overcome their respective drawbacks while maintaining their advantages. Thus, clustering technology could be hybridized with the optimization algorithm method to optimize CpG island detection. The prediction accuracy of ClusterPSO was quite high, indicating the combination of CpGcluster and PSO has several advantages over CpGcluster and PSO alone. In addition, ClusterPSO significantly reduced implementation time.

Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems

PubMed Central

2011-01-01

Background The generation and analysis of high-throughput sequencing data are becoming a major component of many studies in molecular biology and medical research. Illumina's Genome Analyzer (GA) and HiSeq instruments are currently the most widely used sequencing devices. Here, we comprehensively evaluate properties of genomic HiSeq and GAIIx data derived from two plant genomes and one virus, with read lengths of 95 to 150 bases. Results We provide quantifications and evidence for GC bias, error rates, error sequence context, effects of quality filtering, and the reliability of quality values. By combining different filtering criteria we reduced error rates 7-fold at the expense of discarding 12.5% of alignable bases. While overall error rates are low in HiSeq data we observed regions of accumulated wrong base calls. Only 3% of all error positions accounted for 24.7% of all substitution errors. Analyzing the forward and reverse strands separately revealed error rates of up to 18.7%. Insertions and deletions occurred at very low rates on average but increased to up to 2% in homopolymers. A positive correlation between read coverage and GC content was found depending on the GC content range. Conclusions The errors and biases we report have implications for the use and the interpretation of Illumina sequencing data. GAIIx and HiSeq data sets show slightly different error profiles. Quality filtering is essential to minimize downstream analysis artifacts. Supporting previous recommendations, the strand-specificity provides a criterion to distinguish sequencing errors from low abundance polymorphisms. PMID:22067484

CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

PubMed Central

Jiang, Yu; Qian, Fenghui; Yang, Junjie; Liu, Yingmiao; Dong, Feng; Xu, Chongmao; Sun, Bingbing; Chen, Biao; Xu, Xiaoshu; Li, Yan; Wang, Renxiao; Yang, Sheng

2017-01-01

Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86–100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of FnCpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3N+4 or 3N+2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L-proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system. PMID:28469274

Arabidopsis thaliana population analysis reveals high plasticity of the genomic region spanning MSH2, AT3G18530 and AT3G18535 genes and provides evidence for NAHR-driven recurrent CNV events occurring in this location.

PubMed

Zmienko, Agnieszka; Samelak-Czajka, Anna; Kozlowski, Piotr; Szymanska, Maja; Figlerowicz, Marek

2016-11-08

Intraspecies copy number variations (CNVs), defined as unbalanced structural variations of specific genomic loci, ≥1 kb in size, are present in the genomes of animals and plants. A growing number of examples indicate that CNVs may have functional significance and contribute to phenotypic diversity. In the model plant Arabidopsis thaliana at least several hundred protein-coding genes might display CNV; however, locus-specific genotyping studies in this plant have not been conducted. We analyzed the natural CNVs in the region overlapping MSH2 gene that encodes the DNA mismatch repair protein, and AT3G18530 and AT3G18535 genes that encode poorly characterized proteins. By applying multiplex ligation-dependent probe amplification and droplet digital PCR we genotyped those genes in 189 A. thaliana accessions. We found that AT3G18530 and AT3G18535 were duplicated (2-14 times) in 20 and deleted in 101 accessions. MSH2 was duplicated in 12 accessions (up to 12-14 copies) but never deleted. In all but one case, the MSH2 duplications were associated with those of AT3G18530 and AT3G18535. Considering the structure of the CNVs, we distinguished 5 genotypes for this region, determined their frequency and geographical distribution. We defined the CNV breakpoints in 35 accessions with AT3G18530 and AT3G18535 deletions and tandem duplications and showed that they were reciprocal events, resulting from non-allelic homologous recombination between 99 %-identical sequences flanking these genes. The widespread geographical distribution of the deletions supported by the SNP and linkage disequilibrium analyses of the genomic sequence confirmed the recurrent nature of this CNV. We characterized in detail for the first time the complex multiallelic CNV in Arabidopsis genome. The region encoding MSH2, AT3G18530 and AT3G18535 genes shows enormous variation of copy numbers among natural ecotypes, being a remarkable example of high Arabidopsis genome plasticity. We provided the molecular

Complete Genome Sequence and Updated Annotation of Desulfovibrio alaskensis G20

DOE PAGES

Hauser, Loren J.; Land, Miriam L.; Brown, Steven D.; ...

2011-06-17

Desulfovibrio alaskensis G20 (formerly desulfuricans G20) is a Gram-negative mesophilic sulfate-reducing bacterium (SRB), known to corrode ferrous metals and to reduce toxic radionuclides and metals such as uranium and chromium to sparingly soluble and less toxic forms. We present the 3.7 Mb genome sequence to provide insights into its physiology.

Comparative genomic analysis of single-molecule sequencing and hybrid approaches for finishing the Clostridium autoethanogenum JA1-1 strain DSM 10061 genome

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

Brown, Steven D; Nagaraju, Shilpa; Utturkar, Sagar M

Background Clostridium autoethanogenum strain JA1-1 (DSM 10061) is an acetogen capable of fermenting CO, CO2 and H2 (e.g. from syngas or waste gases) into biofuel ethanol and commodity chemicals such as 2,3-butanediol. A draft genome sequence consisting of 100 contigs has been published. Results A closed, high-quality genome sequence for C. autoethanogenum DSM10061 was generated using only the latest single-molecule DNA sequencing technology and without the need for manual finishing. It is assigned to the most complex genome classification based upon genome features such as repeats, prophage, nine copies of the rRNA gene operons. It has a low G +more » C content of 31.1%. Illumina, 454, Illumina/454 hybrid assemblies were generated and then compared to the draft and PacBio assemblies using summary statistics, CGAL, QUAST and REAPR bioinformatics tools and comparative genomic approaches. Assemblies based upon shorter read DNA technologies were confounded by the large number repeats and their size, which in the case of the rRNA gene operons were ~5 kb. CRISPR (Clustered Regularly Interspaced Short Paloindromic Repeats) systems among biotechnologically relevant Clostridia were classified and related to plasmid content and prophages. Potential associations between plasmid content and CRISPR systems may have implications for historical industrial scale Acetone-Butanol-Ethanol (ABE) fermentation failures and future large scale bacterial fermentations. While C. autoethanogenum contains an active CRISPR system, no such system is present in the closely related Clostridium ljungdahlii DSM 13528. A common prophage inserted into the Arg-tRNA shared between the strains suggests a common ancestor. However, C. ljungdahlii contains several additional putative prophages and it has more than double the amount of prophage DNA compared to C. autoethanogenum. Other differences include important metabolic genes for central metabolism (as an additional hydrogenase and the absence of a

Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation

PubMed Central

Soto-Rifo, Ricardo; Limousin, Taran; Rubilar, Paulina S.; Ricci, Emiliano P.; Décimo, Didier; Moncorgé, Olivier; Trabaud, Mary-Anne; André, Patrice; Cimarelli, Andrea; Ohlmann, Théophile

2012-01-01

The 5′-untranslated region (5′-UTR) of the genomic RNA of human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5′-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5′-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which was much inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens. PMID:22121214

Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens

PubMed Central

Lassalle, Florent; Campillo, Tony; Vial, Ludovic; Baude, Jessica; Costechareyre, Denis; Chapulliot, David; Shams, Malek; Abrouk, Danis; Lavire, Céline; Oger-Desfeux, Christine; Hommais, Florence; Guéguen, Laurent; Daubin, Vincent; Muller, Daniel; Nesme, Xavier

2011-01-01

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species. PMID:21795751

Whole-genome sequence of “Candidatus Liberibacter solanacearum” strain R1 from California

USDA-ARS?s Scientific Manuscript database

The draft whole-genome sequence of “Candidatus Liberibacter solanacearum” strain R1, isolated from a tomato plant in California, United States, is reported. The R1 strain genome is 1,204,257 bp in size (G+C content of 35.3%), encoding 1,101 open reading frames and 57 RNA genes....

Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

NASA Astrophysics Data System (ADS)

Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

2016-01-01

The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks.

PubMed

Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

2016-01-22

The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named "DeepMethyl" to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites

PubMed Central

Piazza, Aurèle; Adrian, Michael; Samazan, Frédéric; Heddi, Brahim; Hamon, Florian; Serero, Alexandre; Lopes, Judith; Teulade-Fichou, Marie-Paule; Phan, Anh Tuân; Nicolas, Alain

2015-01-01

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or functionin vivo are unknown. InS. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with thein vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved inC. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequencesin vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to formin vivo. PMID:25956747

Endonuclease G promotes mitochondrial genome cleavage and replication

PubMed Central

Wiehe, Rahel Stefanie; Gole, Boris; Chatre, Laurent; Walther, Paul; Calzia, Enrico; Ricchetti, Miria; Wiesmüller, Lisa

2018-01-01

Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis. PMID:29719607

Complete mitochondrial genome of the aluminum-tolerant fungus Rhodotorula taiwanensis RS1 and comparative analysis of Basidiomycota mitochondrial genomes

PubMed Central

Zhao, Xue Qiang; Aizawa, Tomoko; Schneider, Jessica; Wang, Chao; Shen, Ren Fang; Sunairi, Michio

2013-01-01

The complete mitochondrial genome of Rhodotorula taiwanensis RS1, an aluminum-tolerant Basidiomycota fungus, was determined and compared with the known mitochondrial genomes of 12 Basidiomycota species. The mitochondrial genome of R. taiwanensis RS1 is a circular DNA molecule of 40,392 bp and encodes the typical 15 mitochondrial proteins, 23 tRNAs, and small and large rRNAs as well as 10 intronic open reading frames. These genes are apparently transcribed in two directions and do not show syntenies in gene order with other investigated Basidiomycota species. The average G+C content (41%) of the mitochondrial genome of R. taiwanensis RS1 is the highest among the Basidiomycota species. Two introns were detected in the sequence of the atp9 gene of R. taiwanensis RS1, but not in that of other Basidiomycota species. Rhodotorula taiwanensis is the first species of the genus Rhodotorula whose full mitochondrial genome has been sequenced; and the data presented here supply valuable information for understanding the evolution of fungal mitochondrial genomes and researching the mechanism of aluminum tolerance in microorganisms. PMID:23427135

G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures.

PubMed

Solomon, Oz; Kunik, Vered; Simon, Amos; Kol, Nitzan; Barel, Ortal; Lev, Atar; Amariglio, Ninette; Somech, Raz; Rechavi, Gidi; Eyal, Eran

2016-08-26

Evaluation of the possible implications of genomic variants is an increasingly important task in the current high throughput sequencing era. Structural information however is still not routinely exploited during this evaluation process. The main reasons can be attributed to the partial structural coverage of the human proteome and the lack of tools which conveniently convert genomic positions, which are the frequent output of genomic pipelines, to proteins and structure coordinates. We present G23D, a tool for conversion of human genomic coordinates to protein coordinates and protein structures. G23D allows mapping of genomic positions/variants on evolutionary related (and not only identical) protein three dimensional (3D) structures as well as on theoretical models. By doing so it significantly extends the space of variants for which structural insight is feasible. To facilitate interpretation of the variant consequence, pathogenic variants, functional sites and polymorphism sites are displayed on protein sequence and structure diagrams alongside the input variants. G23D also provides modeling of the mutant structure, analysis of intra-protein contacts and instant access to functional predictions and predictions of thermo-stability changes. G23D is available at http://www.sheba-cancer.org.il/G23D . G23D extends the fraction of variants for which structural analysis is applicable and provides better and faster accessibility for structural data to biologists and geneticists who routinely work with genomic information.

The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes.

PubMed

Hijri, Mohamed; Sanders, Ian R

2004-02-01

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.

Deep Sequencing Reveals the Complete Genome Sequence of Sweet potato virus G from East Timor

PubMed Central

Maina, Solomon; Edwards, Owain R.; Barbetti, Martin J.; de Almeida, Luis; Ximenes, Abel

2016-01-01

We present the first complete Sweet potato virus G (SPVG) genome from sweet potato in East Timor and compare it with seven complete SPVG genomes from South Korea (three), Taiwan (two), Argentina (one), and the United States (one). It most resembles the genomes from the United States and South Korea. PMID:27609925

AB036. Analysis of human mitochondrial genome mutations of Vietnamese patients tentatively diagnosed with encephalomyopathy

PubMed Central

Nghia, Phan Tuan; Thai, Trinh Hong; Hue, Truong Thi; Van Minh, Nguyen; Khanh, Phung Bao; Hiep, Tran Duc; Anh, Tran Kieu; Loan, Nguyen Thi Hong; Van, Nguyen Thi Hong; Anh, Pham Van; Hung, Cao Vu; Anh, Le Ngoc

2015-01-01

Human mitochondrial genome consists of 16,569 bp, and replicates independently from the nuclear genome. Mutations in mitochondrial genome are usually causative factors of various metabolic disorders, especially those of encephalomyopathy. DNA analysis is the most reliable method for detection of mitochondrial genome mutations, and accordingly an excellent diagnostic tool for mitochondrial mutation-related diseases. In this study, 19 different mitochondrial genome mutations including A3243G, A3251G, T3271C and T3291C (MELAS); A8344G, T8356C and G8363A (MERRF); G3460A, G11778A and T14484C (LHON); T8993G/C and T9176G (Leigh); A1555G (deafness) and A4225G, G4298A, T10010C, T14727C, T14728C, T14709C (encephalomyopathy in general) were analyzed using PCR-RFLP in combination with DNA sequencing. In addition, a real-time PCR method using locked nucleic acid (LNA) Taqman probe was set up for heteroplasmy determination. Screening of 283 tentatively diagnosed encephalomyopathy patients revealed 7 cases of A3243G, 1 case of G11778A, 1 case of A1555G, 1 case of A4225G, 1 case G4298A, and 1 case of 6 bp (ACTCCT/CTCCTA) deletion. Using the LNA Taqman probe real-time PCR, the heteroplasmy of some point mutations was determined and the results support a potential relationship between heteroplasmy level and severity of the disease.

Bipyrimidine Signatures as a Photoprotective Genome Strategy in G + C-rich Halophilic Archaea.

PubMed

Jones, Daniel L; Baxter, Bonnie K

2016-09-02

Halophilic archaea experience high levels of ultraviolet (UV) light in their environments and demonstrate resistance to UV irradiation. DNA repair systems and carotenoids provide UV protection but do not account for the high resistance observed. Herein, we consider genomic signatures as an additional photoprotective strategy. The predominant forms of UV-induced DNA damage are cyclobutane pyrimidine dimers, most notoriously thymine dimers (T^Ts), which form at adjacent Ts. We tested whether the high G + C content seen in halophilic archaea serves a photoprotective function through limiting T nucleotides, and thus T^T lesions. However, this speculation overlooks the other bipyrimidine sequences, all of which capable of forming photolesions to varying degrees. Therefore, we designed a program to determine the frequencies of the four bipyrimidine pairs (5' to 3': TT, TC, CT, and CC) within genomes of halophilic archaea and four other randomized sample groups for comparison. The outputs for each sampled genome were weighted by the intrinsic photoreactivities of each dinucleotide pair. Statistical methods were employed to investigate intergroup differences. Our findings indicate that the UV-resistance seen in halophilic archaea can be attributed in part to a genomic strategy: high G + C content and the resulting bipyrimidine signature reduces the genomic photoreactivity.

Bipyrimidine Signatures as a Photoprotective Genome Strategy in G + C-rich Halophilic Archaea

PubMed Central

Jones, Daniel L.; Baxter, Bonnie K.

2016-01-01

Halophilic archaea experience high levels of ultraviolet (UV) light in their environments and demonstrate resistance to UV irradiation. DNA repair systems and carotenoids provide UV protection but do not account for the high resistance observed. Herein, we consider genomic signatures as an additional photoprotective strategy. The predominant forms of UV-induced DNA damage are cyclobutane pyrimidine dimers, most notoriously thymine dimers (T^Ts), which form at adjacent Ts. We tested whether the high G + C content seen in halophilic archaea serves a photoprotective function through limiting T nucleotides, and thus T^T lesions. However, this speculation overlooks the other bipyrimidine sequences, all of which capable of forming photolesions to varying degrees. Therefore, we designed a program to determine the frequencies of the four bipyrimidine pairs (5’ to 3’: TT, TC, CT, and CC) within genomes of halophilic archaea and four other randomized sample groups for comparison. The outputs for each sampled genome were weighted by the intrinsic photoreactivities of each dinucleotide pair. Statistical methods were employed to investigate intergroup differences. Our findings indicate that the UV-resistance seen in halophilic archaea can be attributed in part to a genomic strategy: high G + C content and the resulting bipyrimidine signature reduces the genomic photoreactivity. PMID:27598206

Survey of genome sequences in a wild sweet potato, Ipomoea trifida (H. B. K.) G. Don

PubMed Central

Hirakawa, Hideki; Okada, Yoshihiro; Tabuchi, Hiroaki; Shirasawa, Kenta; Watanabe, Akiko; Tsuruoka, Hisano; Minami, Chiharu; Nakayama, Shinobu; Sasamoto, Shigemi; Kohara, Mitsuyo; Kishida, Yoshie; Fujishiro, Tsunakazu; Kato, Midori; Nanri, Keiko; Komaki, Akiko; Yoshinaga, Masaru; Takahata, Yasuhiro; Tanaka, Masaru; Tabata, Satoshi; Isobe, Sachiko N.

2015-01-01

Ipomoea trifida (H. B. K.) G. Don. is the most likely diploid ancestor of the hexaploid sweet potato, I. batatas (L.) Lam. To assist in analysis of the sweet potato genome, de novo whole-genome sequencing was performed with two lines of I. trifida, namely the selfed line Mx23Hm and the highly heterozygous line 0431-1, using the Illumina HiSeq platform. We classified the sequences thus obtained as either ‘core candidates’ (common to the two lines) or ‘line specific’. The total lengths of the assembled sequences of Mx23Hm (ITR_r1.0) was 513 Mb, while that of 0431-1 (ITRk_r1.0) was 712 Mb. Of the assembled sequences, 240 Mb (Mx23Hm) and 353 Mb (0431-1) were classified into core candidate sequences. A total of 62,407 (62.4 Mb) and 109,449 (87.2 Mb) putative genes were identified, respectively, in the genomes of Mx23Hm and 0431-1, of which 11,823 were derived from core sequences of Mx23Hm, while 28,831 were from the core candidate sequence of 0431-1. There were a total of 1,464,173 single-nucleotide polymorphisms and 16,682 copy number variations (CNVs) in the two assembled genomic sequences (under the condition of log2 ratio of >1 and CNV size >1,000 bases). The results presented here are expected to contribute to the progress of genomic and genetic studies of I. trifida, as well as studies of the sweet potato and the genus Ipomoea in general. PMID:25805887

Draft Genome Sequence of Pseudomonas sp. Strain B1, Isolated from a Contaminated Sediment

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

Pathak, Ashish; Jaswal, Rajneesh; Stothard, Paul

ABSTRACT The draft genome sequence of Pseudomonas sp. strain B1, isolated from a contaminated soil, is reported. The genome comprises 6,706,934 bases, 6,059 coding sequences, and 70 RNAs and has a G+C content of 60.3%. A suite of biodegradative genes, many located on genomic islands, were identified from strain B1, further enhancing our understanding of the versatile pseudomonads.

Draft Genome Sequence of Pseudomonas sp. Strain B1, Isolated from a Contaminated Sediment

DOE PAGES

Pathak, Ashish; Jaswal, Rajneesh; Stothard, Paul; ...

2018-06-21

ABSTRACT The draft genome sequence of Pseudomonas sp. strain B1, isolated from a contaminated soil, is reported. The genome comprises 6,706,934 bases, 6,059 coding sequences, and 70 RNAs and has a G+C content of 60.3%. A suite of biodegradative genes, many located on genomic islands, were identified from strain B1, further enhancing our understanding of the versatile pseudomonads.

Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution

USDA-ARS?s Scientific Manuscript database

Genetic and genomic analyses of Upland cotton (Gossypium hirsutum) are difficult because it has a complex allotetraploid (AADD; 2n = 4x = 52) genome. Here we sequenced, assembled and analyzed the world's most important cultivated cotton genome with 246.2 gigabase (Gb) clean data obtained using whol...

Genome-wide association studies suggest that APOL1-environment interactions more likely trigger kidney disease in African Americans with nondiabetic nephropathy than strong APOL1-second gene interactions.

PubMed

Langefeld, Carl D; Comeau, Mary E; Ng, Maggie C Y; Guan, Meijian; Dimitrov, Latchezar; Mudgal, Poorva; Spainhour, Mitzie H; Julian, Bruce A; Edberg, Jeffrey C; Croker, Jennifer A; Divers, Jasmin; Hicks, Pamela J; Bowden, Donald W; Chan, Gary C; Ma, Lijun; Palmer, Nicholette D; Kimberly, Robert P; Freedman, Barry I

2018-06-06

African Americans carrying two apolipoprotein L1 gene (APOL1) renal risk variants have a high risk for nephropathy. However, only a minority develops end-stage renal disease (ESRD). Hence, modifying factors likely contribute to initiation of kidney disease such as endogenous (HIV infection) or exogenous (interferon treatment) environmental modifiers. In this report, genome-wide association studies and a meta-analysis were performed to identify novel loci for nondiabetic ESRD in African Americans and to detect genetic modifiers in APOL1-associated nephropathy. Two African American cohorts were analyzed, 1749 nondiabetic ESRD cases and 1136 controls from Wake Forest and 901 lupus nephritis (LN)-ESRD cases and 520 controls with systemic lupus erythematosus but lacking nephropathy from the LN-ESRD Consortium. Association analyses adjusting for APOL1 G1/G2 renal-risk variants were completed and stratified by APOL1 risk genotype status. Individual genome-wide association studies and meta-analysis results of all 2650 ESRD cases and 1656 controls did not detect significant genome-wide associations with ESRD beyond APOL1. Similarly, no single nucleotide polymorphism showed significant genome-wide evidence of an interaction with APOL1 risk variants. Thus, although variants with small individual effects cannot be ruled out and are likely to exist, our results suggest that APOL1-environment interactions may be of greater clinical importance in triggering nephropathy in African Americans than APOL1 interactions with other single nucleotide polymorphisms. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Comparative genomic analysis of Genlisea (corkscrew plants—Lentibulariaceae) chloroplast genomes reveals an increasing loss of the ndh genes

PubMed Central

Silva, Saura R.; Michael, Todd P.; Meer, Elliott J.; Pinheiro, Daniel G.; Miranda, Vitor F. O.

2018-01-01

In the carnivorous plant family Lentibulariaceae, all three genome compartments (nuclear, chloroplast, and mitochondria) have some of the highest rates of nucleotide substitutions across angiosperms. While the genera Genlisea and Utricularia have the smallest known flowering plant nuclear genomes, the chloroplast genomes (cpDNA) are mostly structurally conserved except for deletion and/or pseudogenization of the NAD(P)H-dehydrogenase complex (ndh) genes known to be involved in stress conditions of low light or CO2 concentrations. In order to determine how the cpDNA are changing, and to better understand the evolutionary history within the Genlisea genus, we sequenced, assembled and analyzed complete cpDNA from six species (G. aurea, G. filiformis, G. pygmaea, G. repens, G. tuberosa and G. violacea) together with the publicly available G. margaretae cpDNA. In general, the cpDNA structure among the analyzed Genlisea species is highly similar. However, we found that the plastidial ndh genes underwent a progressive process of degradation similar to the other terrestrial Lentibulariaceae cpDNA analyzed to date, but in contrast to the aquatic species. Contrary to current thinking that the terrestrial environment is a more stressful environment and thus requiring the ndh genes, we provide evidence that in the Lentibulariaceae the terrestrial forms have progressive loss while the aquatic forms have the eleven plastidial ndh genes intact. Therefore, the Lentibulariaceae system provides an important opportunity to understand the evolutionary forces that govern the transition to an aquatic environment and may provide insight into how plants manage water stress at a genome scale. PMID:29293597

Comparative genomic analysis of Genlisea (corkscrew plants-Lentibulariaceae) chloroplast genomes reveals an increasing loss of the ndh genes.

PubMed

Silva, Saura R; Michael, Todd P; Meer, Elliott J; Pinheiro, Daniel G; Varani, Alessandro M; Miranda, Vitor F O

2018-01-01

In the carnivorous plant family Lentibulariaceae, all three genome compartments (nuclear, chloroplast, and mitochondria) have some of the highest rates of nucleotide substitutions across angiosperms. While the genera Genlisea and Utricularia have the smallest known flowering plant nuclear genomes, the chloroplast genomes (cpDNA) are mostly structurally conserved except for deletion and/or pseudogenization of the NAD(P)H-dehydrogenase complex (ndh) genes known to be involved in stress conditions of low light or CO2 concentrations. In order to determine how the cpDNA are changing, and to better understand the evolutionary history within the Genlisea genus, we sequenced, assembled and analyzed complete cpDNA from six species (G. aurea, G. filiformis, G. pygmaea, G. repens, G. tuberosa and G. violacea) together with the publicly available G. margaretae cpDNA. In general, the cpDNA structure among the analyzed Genlisea species is highly similar. However, we found that the plastidial ndh genes underwent a progressive process of degradation similar to the other terrestrial Lentibulariaceae cpDNA analyzed to date, but in contrast to the aquatic species. Contrary to current thinking that the terrestrial environment is a more stressful environment and thus requiring the ndh genes, we provide evidence that in the Lentibulariaceae the terrestrial forms have progressive loss while the aquatic forms have the eleven plastidial ndh genes intact. Therefore, the Lentibulariaceae system provides an important opportunity to understand the evolutionary forces that govern the transition to an aquatic environment and may provide insight into how plants manage water stress at a genome scale.

Complete mitochondrial genome of the aluminum-tolerant fungus Rhodotorula taiwanensis RS1 and comparative analysis of Basidiomycota mitochondrial genomes.

PubMed

Zhao, Xue Qiang; Aizawa, Tomoko; Schneider, Jessica; Wang, Chao; Shen, Ren Fang; Sunairi, Michio

2013-04-01

The complete mitochondrial genome of Rhodotorula taiwanensis RS1, an aluminum-tolerant Basidiomycota fungus, was determined and compared with the known mitochondrial genomes of 12 Basidiomycota species. The mitochondrial genome of R. taiwanensis RS1 is a circular DNA molecule of 40,392 bp and encodes the typical 15 mitochondrial proteins, 23 tRNAs, and small and large rRNAs as well as 10 intronic open reading frames. These genes are apparently transcribed in two directions and do not show syntenies in gene order with other investigated Basidiomycota species. The average G+C content (41%) of the mitochondrial genome of R. taiwanensis RS1 is the highest among the Basidiomycota species. Two introns were detected in the sequence of the atp9 gene of R. taiwanensis RS1, but not in that of other Basidiomycota species. Rhodotorula taiwanensis is the first species of the genus Rhodotorula whose full mitochondrial genome has been sequenced; and the data presented here supply valuable information for understanding the evolution of fungal mitochondrial genomes and researching the mechanism of aluminum tolerance in microorganisms. © 2013 The Authors. Published by Blackwell Publishing Ltd.

TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

PubMed Central

Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

2015-01-01

Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis. PMID:26283799

Evaluation of the utility of the new rainbow trout genome assembly for analyzing RNA-seq data from stress response experiments

USDA-ARS?s Scientific Manuscript database

The newly released rainbow trout genome assembly in NCBI RefSeq has greatly expanded our abilities for analyzing rainbow trout sequencing data. In this poster, we evaluate the utility of this genome assembly for analyzing RNA sequencing (RNA-seq) data of rainbow trout responses to various stressors,...

TeachEnG: a Teaching Engine for Genomics.

PubMed

Kim, Minji; Kim, Yeonsung; Qian, Lei; Song, Jun S

2017-10-15

Bioinformatics is a rapidly growing field that has emerged from the synergy of computer science, statistics and biology. Given the interdisciplinary nature of bioinformatics, many students from diverse fields struggle with grasping bioinformatic concepts only from classroom lectures. Interactive tools for helping students reinforce their learning would be thus desirable. Here, we present an interactive online educational tool called TeachEnG (acronym for Teaching Engine for Genomics) for reinforcing key concepts in sequence alignment and phylogenetic tree reconstruction. Our instructional games allow students to align sequences by hand, fill out the dynamic programming matrix in the Needleman-Wunsch global sequence alignment algorithm, and reconstruct phylogenetic trees via the maximum parsimony, Unweighted Pair Group Method with Arithmetic mean (UPGMA) and Neighbor-Joining algorithms. With an easily accessible interface and instant visual feedback, TeachEnG will help promote active learning in bioinformatics. TeachEnG is freely available at http://teacheng.illinois.edu. The source code is available from https://github.com/KnowEnG/TeachEnG under the Artistic License 2.0. It is written in JavaScript and compatible with Firefox, Safari, Chrome and Microsoft Edge. songj@illinois.edu. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Toward the 1,000 dollars human genome.

PubMed

Bennett, Simon T; Barnes, Colin; Cox, Anthony; Davies, Lisa; Brown, Clive

2005-06-01

Revolutionary new technologies, capable of transforming the economics of sequencing, are providing an unparalleled opportunity to analyze human genetic variation comprehensively at the whole-genome level within a realistic timeframe and at affordable costs. Current estimates suggest that it would cost somewhere in the region of 30 million US dollars to sequence an entire human genome using Sanger-based sequencing, and on one machine it would take about 60 years. Solexa is widely regarded as a company with the necessary disruptive technology to be the first to achieve the ultimate goal of the so-called 1,000 dollars human genome - the conceptual cost-point needed for routine analysis of individual genomes. Solexa's technology is based on completely novel sequencing chemistry capable of sequencing billions of individual DNA molecules simultaneously, a base at a time, to enable highly accurate, low cost analysis of an entire human genome in a single experiment. When applied over a large enough genomic region, these new approaches to resequencing will enable the simultaneous detection and typing of known, as well as unknown, polymorphisms, and will also offer information about patterns of linkage disequilibrium in the population being studied. Technological progress, leading to the advent of single-molecule-based approaches, is beginning to dramatically drive down costs and increase throughput to unprecedented levels, each being several orders of magnitude better than that which is currently available. A new sequencing paradigm based on single molecules will be faster, cheaper and more sensitive, and will permit routine analysis at the whole-genome level.

The Genomic HyperBrowser: an analysis web server for genome-scale data

PubMed Central

Sandve, Geir K.; Gundersen, Sveinung; Johansen, Morten; Glad, Ingrid K.; Gunathasan, Krishanthi; Holden, Lars; Holden, Marit; Liestøl, Knut; Nygård, Ståle; Nygaard, Vegard; Paulsen, Jonas; Rydbeck, Halfdan; Trengereid, Kai; Clancy, Trevor; Drabløs, Finn; Ferkingstad, Egil; Kalaš, Matúš; Lien, Tonje; Rye, Morten B.; Frigessi, Arnoldo; Hovig, Eivind

2013-01-01

The immense increase in availability of genomic scale datasets, such as those provided by the ENCODE and Roadmap Epigenomics projects, presents unprecedented opportunities for individual researchers to pose novel falsifiable biological questions. With this opportunity, however, researchers are faced with the challenge of how to best analyze and interpret their genome-scale datasets. A powerful way of representing genome-scale data is as feature-specific coordinates relative to reference genome assemblies, i.e. as genomic tracks. The Genomic HyperBrowser (http://hyperbrowser.uio.no) is an open-ended web server for the analysis of genomic track data. Through the provision of several highly customizable components for processing and statistical analysis of genomic tracks, the HyperBrowser opens for a range of genomic investigations, related to, e.g., gene regulation, disease association or epigenetic modifications of the genome. PMID:23632163

The Genomic HyperBrowser: an analysis web server for genome-scale data.

PubMed

Sandve, Geir K; Gundersen, Sveinung; Johansen, Morten; Glad, Ingrid K; Gunathasan, Krishanthi; Holden, Lars; Holden, Marit; Liestøl, Knut; Nygård, Ståle; Nygaard, Vegard; Paulsen, Jonas; Rydbeck, Halfdan; Trengereid, Kai; Clancy, Trevor; Drabløs, Finn; Ferkingstad, Egil; Kalas, Matús; Lien, Tonje; Rye, Morten B; Frigessi, Arnoldo; Hovig, Eivind

2013-07-01

The immense increase in availability of genomic scale datasets, such as those provided by the ENCODE and Roadmap Epigenomics projects, presents unprecedented opportunities for individual researchers to pose novel falsifiable biological questions. With this opportunity, however, researchers are faced with the challenge of how to best analyze and interpret their genome-scale datasets. A powerful way of representing genome-scale data is as feature-specific coordinates relative to reference genome assemblies, i.e. as genomic tracks. The Genomic HyperBrowser (http://hyperbrowser.uio.no) is an open-ended web server for the analysis of genomic track data. Through the provision of several highly customizable components for processing and statistical analysis of genomic tracks, the HyperBrowser opens for a range of genomic investigations, related to, e.g., gene regulation, disease association or epigenetic modifications of the genome.

Differential genomic effects on canonical signaling pathways by two different CeO2 nanoparticles in HepG2 cells

EPA Science Inventory

Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells. Sheau-Fung Thai1, Kathleen A. Wallace1, Carlton P. Jones1, Hongzu Ren2, Benjamin T. Castellon1, James Crooks2, Kirk T. Kitchin1. 1Integrated Systems Toxicology Divison, 2Resea...

The Genomic Impact of DNA CpG Methylation on Gene Expression; Relationships in Prostate Cancer.

PubMed

Long, Mark D; Smiraglia, Dominic J; Campbell, Moray J

2017-02-14

The process of DNA CpG methylation has been extensively investigated for over 50 years and revealed associations between changing methylation status of CpG islands and gene expression. As a result, DNA CpG methylation is implicated in the control of gene expression in developmental and homeostasis processes, as well as being a cancer-driver mechanism. The development of genome-wide technologies and sophisticated statistical analytical approaches has ushered in an era of widespread analyses, for example in the cancer arena, of the relationships between altered DNA CpG methylation, gene expression, and tumor status. The remarkable increase in the volume of such genomic data, for example, through investigators from the Cancer Genome Atlas (TCGA), has allowed dissection of the relationships between DNA CpG methylation density and distribution, gene expression, and tumor outcome. In this manner, it is now possible to test that the genome-wide correlations are measurable between changes in DNA CpG methylation and gene expression. Perhaps surprisingly is that these associations can only be detected for hundreds, but not thousands, of genes, and the direction of the correlations are both positive and negative. This, perhaps, suggests that CpG methylation events in cancer systems can act as disease drivers but the effects are possibly more restricted than suspected. Additionally, the positive and negative correlations suggest direct and indirect events and an incomplete understanding. Within the prostate cancer TCGA cohort, we examined the relationships between expression of genes that control DNA methylation, known targets of DNA methylation and tumor status. This revealed that genes that control the synthesis of S -adenosyl-l-methionine (SAM) associate with altered expression of DNA methylation targets in a subset of aggressive tumors.

Unexpected effects of different genetic backgrounds on identification of genomic rearrangements via whole-genome next generation sequencing.

PubMed

Chen, Zhangguo; Gowan, Katherine; Leach, Sonia M; Viboolsittiseri, Sawanee S; Mishra, Ameet K; Kadoishi, Tanya; Diener, Katrina; Gao, Bifeng; Jones, Kenneth; Wang, Jing H

2016-10-21

Whole genome next generation sequencing (NGS) is increasingly employed to detect genomic rearrangements in cancer genomes, especially in lymphoid malignancies. We recently established a unique mouse model by specifically deleting a key non-homologous end-joining DNA repair gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in germinal center B cells. This mouse model spontaneously develops mature B cell lymphomas (termed G1XP lymphomas). Here, we attempt to employ whole genome NGS to identify novel structural rearrangements, in particular inter-chromosomal translocations (CTXs), in these G1XP lymphomas. We sequenced six lymphoma samples, aligned our NGS data with mouse reference genome (in C57BL/6J (B6) background) and identified CTXs using CREST algorithm. Surprisingly, we detected widespread CTXs in both lymphomas and wildtype control samples, majority of which were false positive and attributable to different genetic backgrounds. In addition, we validated our NGS pipeline by sequencing multiple control samples from distinct tissues of different genetic backgrounds of mouse (B6 vs non-B6). Lastly, our studies showed that widespread false positive CTXs can be generated by simply aligning sequences from different genetic backgrounds of mouse. We conclude that mapping and alignment with reference genome might not be a preferred method for analyzing whole-genome NGS data obtained from a genetic background different from reference genome. Given the complex genetic background of different mouse strains or the heterogeneity of cancer genomes in human patients, in order to minimize such systematic artifacts and uncover novel CTXs, a preferred method might be de novo assembly of personalized normal control genome and cancer cell genome, instead of mapping and aligning NGS data to mouse or human reference genome. Thus, our studies have critical impact on the manner of data analysis for cancer genomics.

Sequential replication-coupled destruction at G1/S ensures genome stability

PubMed Central

Coleman, Kate E.; Grant, Gavin D.; Haggerty, Rachel A.; Brantley, Kristen; Shibata, Etsuko; Workman, Benjamin D.; Dutta, Anindya; Varma, Dileep; Purvis, Jeremy E.; Cook, Jeanette Gowen

2015-01-01

Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4Cdt2, which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4Cdt2 targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNADNA) and recruits CRL4Cdt2. Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNADNA. Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability. PMID:26272819

The Association of Plasminogen Activator Inhibitor Type 1 (PAI-1) Level and PAI-1 4G/5G Gene Polymorphism with the Formation and the Grade of Endometrial Cancer.

PubMed

Yıldırım, Malik Ejder; Karakuş, Savas; Kurtulgan, Hande Küçük; Kılıçgün, Hasan; Erşan, Serpil; Bakır, Sevtap

2017-08-01

Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (Serpine 1), and it inhibits both tissue plasminogen activator and urokinase plasminogen activator which are important in fibrinolysis. We aimed to find whether there is a possible association between PAI-1 level, PAI-1 4G/5G polymorphism, and endometrial cancer. PAI-1 levels in peripheral blood were determined in 82 patients with endometrial carcinoma and 76 female healthy controls using an enzyme-linked immunoassay (ELISA). Then, the genomic DNA was extracted and screened by reverse hybridization procedure (Strip assay) to detect PAI 1 4G/5G polymorphism. The levels of PAI-1 in the patients were higher statistically in comparison to controls (P < 0.001). The distribution of PAI-1 4G/5G polymorphism was quite different between patients and controls (P = 0.008), and 4G allelic frequency was significantly higher in the patients of endometrial cancer than in controls (P = 0.026). We found significant difference between Grade 1 and Grade 2+3 patients in terms of the PAI-1 levels (P = 0.047). There was no association between PAI-1 4G/5G polymorphism and the grades of endometrial cancer (P = 0.993). Our data suggest that the level of PAI-1 and PAI-1 4G/5G gene polymorphism are effective in the formation of endometrial cancer. PAI-1 levels are also associated with the grades of endometrial cancer.

Genome-wide prediction of vaccine targets for human herpes simplex viruses using Vaxign reverse vaccinology

PubMed Central

2013-01-01

Herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2) are the most common infectious agents of humans. No safe and effective HSV vaccines have been licensed. Reverse vaccinology is an emerging and revolutionary vaccine development strategy that starts with the prediction of vaccine targets by informatics analysis of genome sequences. Vaxign (http://www.violinet.org/vaxign) is the first web-based vaccine design program based on reverse vaccinology. In this study, we used Vaxign to analyze 52 herpesvirus genomes, including 3 HSV-1 genomes, one HSV-2 genome, 8 other human herpesvirus genomes, and 40 non-human herpesvirus genomes. The HSV-1 strain 17 genome that contains 77 proteins was used as the seed genome. These 77 proteins are conserved in two other HSV-1 strains (strain F and strain H129). Two envelope glycoproteins gJ and gG do not have orthologs in HSV-2 or 8 other human herpesviruses. Seven HSV-1 proteins (including gJ and gG) do not have orthologs in all 40 non-human herpesviruses. Nineteen proteins are conserved in all human herpesviruses, including capsid scaffold protein UL26.5 (NP_044628.1). As the only HSV-1 protein predicted to be an adhesin, UL26.5 is a promising vaccine target. The MHC Class I and II epitopes were predicted by the Vaxign Vaxitop prediction program and IEDB prediction programs recently installed and incorporated in Vaxign. Our comparative analysis found that the two programs identified largely the same top epitopes but also some positive results predicted from one program might not be positive from another program. Overall, our Vaxign computational prediction provides many promising candidates for rational HSV vaccine development. The method is generic and can also be used to predict other viral vaccine targets. PMID:23514126

G protein-coupled oestrogen receptor 1 (GPER1)/GPR30: a new player in cardiovascular and metabolic oestrogenic signalling.

PubMed

Nilsson, Bengt-Olof; Olde, Björn; Leeb-Lundberg, L M Fredrik

2011-07-01

Oestrogens are important sex hormones central to health and disease in both genders that have protective effects on the cardiovascular and metabolic systems. These hormones act in complex ways via both genomic and non-genomic mechanisms. The genomic mechanisms are relatively well characterized, whereas the non-genomic ones are only beginning to be explored. Two oestrogen receptors (ER), ERα and ERβ, have been described that act as nuclear transcription factors but can also associate with the plasma membrane and influence cytosolic signalling. ERα has been shown to mediate both anti-atherogenic effects and pro-survival effects in pancreatic β-cells. In recent years, a third membrane-bound ER has emerged, G protein-coupled receptor 30 or G protein-coupled oestrogen receptor 1 (GPER1), which mediates oestrogenic responses in cardiovascular and metabolic regulation. Both GPER1 knock-out models and pharmacological agents are now available to study GPER1 function. These tools have revealed that GPER1 activation may have several beneficial effects in the cardiovascular system including vasorelaxation, inhibition of smooth muscle cell proliferation, and protection of the myocardium against ischaemia/reperfusion injury, and in the metabolic system including stimulation of insulin release and protection against pancreatic β-cell apoptosis. Thus, GPER1 is emerging as a candidate therapeutic target in both cardiovascular and metabolic disease. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Flipping the Switch from G1 to S Phase with E3 Ubiquitin Ligases

PubMed Central

Rizzardi, Lindsay F.

2012-01-01

The cell cycle ensures genome maintenance by coordinating the processes of DNA replication and chromosome segregation. Of particular importance is the irreversible transition from the G1 phase of the cell cycle to S phase. This transition marks the switch from preparing chromosomes for replication (“origin licensing”) to active DNA synthesis (“origin firing”). Ubiquitin-mediated proteolysis is essential for restricting DNA replication to only once per cell cycle and is the major mechanism regulating the G1 to S phase transition. Although some changes in protein levels are attributable to regulated mRNA abundance, protein degradation elicits very rapid changes in protein abundance and is critical for the sharp and irreversible transition from one cell cycle stage to the next. Not surprisingly, regulation of the G1-to-S phase transition is perturbed in most cancer cells, and deregulation of key molecular events in G1 and S phase drives not only cell proliferation but also genome instability. In this review we focus on the mechanisms by which E3 ubiquitin ligases control the irreversible transition from G1 to S phase in mammalian cells. PMID:23634252

Draft genome sequence of Xylella fastidiosa supsp. multiplex strain Griffin-1 from Quercus rubra in Georgia

USDA-ARS?s Scientific Manuscript database

The draft genome sequence of Xylella fastidiosa subsp. multiplex Strain Griffin-1 isolated from a red oak tree (Quercus rubra) in Georgia, U.S.A. is reported. The bacterium has a genome size of 2,387,314 bp with 51.7% G+C content and comprises 2,903 predicted open reading frames (ORFs), and 50 RNA g...

Effects of 2.0-g 1.75-g and 1.5-g Hypergravity on Pregnancy Outcome in Rats (Rattus norvegicus)

NASA Technical Reports Server (NTRS)

Mills, Nicole A.; Baer, Lisa A.; Ronca, April E.

2001-01-01

In 1995, ten pregnant female rats were launched on the Space Shuttle (STS-70) on Gestational day(G) 11 of their 22-day pregnancy as part of the NASA/NIH.Rodent (R)2 Experiment. Following landing on G20, fetuses were harvested from half of the dams, while the remaining five dams underwent birth. Spaceflight did not interrupt pregnancy, alter litter sizes, or affect body weights or gender ratios of the fetuses or neonates. In the present study we used the NASA/NIH.R2 experimental paradigm to analyze the effects of hypergravity on pregnancy outcome. On G10, time-bred Sprague-Dawley rat dams were assigned to either G20 or Birth conditions, then further assigned to Hypergravity (HG) 2.0-g, HG 1.75-g, HG 1.5-g, Rotational Control (RC, 1.03), or Stationary Control (SC, 1.0-g) treatments. Dams were exposed to continuous centrifugation from G11 through G20, with brief daily stops for animal health checks and maintenance. For both the G20 and Birth dams, comparable litter sizes and litter gender ratios were observed across gravity conditions. However, centrifugation-exposed (HG and RC) fetuses and neonates showed significantly lower body masses (p less than 0.05) relative to SC offspring. HG 2.0-g offspring weighed significantly less than those in all other gravity conditions (p less than 0.05). The observed reductions in offspring body mass at 1.5-g and 1.75-g, can be attributed to the rotational component of centrifugation, rather than to increased gravitational load, whereas 2.0-g hypergravity exposure further exacerbated the gravity centrifugation effect on offspring body mass. Pregnant dams exposed to centrifugation weighed significantly less than SC dams (p less than 0.05), suggesting that centrifugation effects on maternal body mass may contribute to reduced size of the developing offspring. These findings are consistent with previous reports of non-pregnant adult animals suggesting that, whereas spaceflight has virtually no effect on body mass, centrifugation is

BμG@Sbase—a microbial gene expression and comparative genomic database

PubMed Central

Witney, Adam A.; Waldron, Denise E.; Brooks, Lucy A.; Tyler, Richard H.; Withers, Michael; Stoker, Neil G.; Wren, Brendan W.; Butcher, Philip D.; Hinds, Jason

2012-01-01

The reducing cost of high-throughput functional genomic technologies is creating a deluge of high volume, complex data, placing the burden on bioinformatics resources and tool development. The Bacterial Microarray Group at St George's (BμG@S) has been at the forefront of bacterial microarray design and analysis for over a decade and while serving as a hub of a global network of microbial research groups has developed BμG@Sbase, a microbial gene expression and comparative genomic database. BμG@Sbase (http://bugs.sgul.ac.uk/bugsbase/) is a web-browsable, expertly curated, MIAME-compliant database that stores comprehensive experimental annotation and multiple raw and analysed data formats. Consistent annotation is enabled through a structured set of web forms, which guide the user through the process following a set of best practices and controlled vocabulary. The database currently contains 86 expertly curated publicly available data sets (with a further 124 not yet published) and full annotation information for 59 bacterial microarray designs. The data can be browsed and queried using an explorer-like interface; integrating intuitive tree diagrams to present complex experimental details clearly and concisely. Furthermore the modular design of the database will provide a robust platform for integrating other data types beyond microarrays into a more Systems analysis based future. PMID:21948792

BμG@Sbase--a microbial gene expression and comparative genomic database.

PubMed

Witney, Adam A; Waldron, Denise E; Brooks, Lucy A; Tyler, Richard H; Withers, Michael; Stoker, Neil G; Wren, Brendan W; Butcher, Philip D; Hinds, Jason

2012-01-01

The reducing cost of high-throughput functional genomic technologies is creating a deluge of high volume, complex data, placing the burden on bioinformatics resources and tool development. The Bacterial Microarray Group at St George's (BμG@S) has been at the forefront of bacterial microarray design and analysis for over a decade and while serving as a hub of a global network of microbial research groups has developed BμG@Sbase, a microbial gene expression and comparative genomic database. BμG@Sbase (http://bugs.sgul.ac.uk/bugsbase/) is a web-browsable, expertly curated, MIAME-compliant database that stores comprehensive experimental annotation and multiple raw and analysed data formats. Consistent annotation is enabled through a structured set of web forms, which guide the user through the process following a set of best practices and controlled vocabulary. The database currently contains 86 expertly curated publicly available data sets (with a further 124 not yet published) and full annotation information for 59 bacterial microarray designs. The data can be browsed and queried using an explorer-like interface; integrating intuitive tree diagrams to present complex experimental details clearly and concisely. Furthermore the modular design of the database will provide a robust platform for integrating other data types beyond microarrays into a more Systems analysis based future.

Tet1 is required for Rb phosphorylation during G1/S phase transition

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

Huang, Shengsong; Zhu, Ziqi; Wang, Yiqin

2013-05-03

Highlights: •Tet1 was required for NIT3T3 proliferation. •Tet1 depletion inhibited G1-S entry. •Cyclin D1 accumulation and Rb phosphorylation was blocked by Tet1 knockdown. -- Abstract: DNA methylation plays an important role in many biological processes, including regulation of gene expression, maintenance of chromatin conformation and genomic stability. TET-family proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which indicates that these enzymes may participate in DNA demethylation. The function of TET1 has not yet been well characterized in somatic cells. Here, we show that depletion of Tet1 in NIH3T3 cells inhibits cell growth. Furthermore, Tet1 knockdown blocks cyclin D1 accumulation in G1more » phase, inhibits Rb phosphorylation and consequently delays entrance to G1/S phase. Taken together, this study demonstrates that Tet1 is required for cell proliferation and that this process is mediated through the Rb pathway.« less

in silico Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de novo Genome Sequencing Studies

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

Zhou, Xiaofan; Peris, David; Kominek, Jacek

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in nonmodel organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimentalmore » design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects, and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.« less

in silico Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de novo Genome Sequencing Studies

DOE PAGES

Zhou, Xiaofan; Peris, David; Kominek, Jacek; ...

2016-09-16

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in nonmodel organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimentalmore » design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects, and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.« less

The Temporal Regulation of S Phase Proteins During G1

PubMed Central

Grant, Gavin D.; Cook, Jeanette G.

2018-01-01

Successful DNA replication requires intimate coordination with cell cycle progression. Prior to DNA replication initiation in S phase, a series of essential preparatory events in G1 phase ensures timely, complete, and precise genome duplication. Among the essential molecular processes are regulated transcriptional upregulation of genes that encode replication proteins, appropriate post-transcriptional control of replication factor abundance and activity, and the assembly of DNA-loaded protein complexes to license replication origins. In this chapter we describe these critical G1 events necessary for DNA replication and their regulation in the context of both cell cycle entry and cell cycle progression. PMID:29357066

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

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

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

2011-07-01

Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, theirmore » exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.« less

Complete genome sequence of Cupriavidus basilensis 4G11, isolated from the Oak Ridge Field Research Center site

DOE PAGES

Ray, Jayashree; Waters, R. Jordan; Skerker, Jeffrey M.; ...

2015-05-14

Cupriavidus basilensis 4G11 was isolated from groundwater at the Oak Ridge Field Research Center (FRC) site. Here, we report the complete genome sequence and annotation of Cupriavidus basilensis 4G11. The genome contains 8,421,483 bp, 7,661 predicted protein-coding genes, and a total GC content of 64.4%.

A Genome-Wide Integrative Genomic Study Localizes Genetic Factors Influencing Antibodies against Epstein-Barr Virus Nuclear Antigen 1 (EBNA-1)

PubMed Central

Rubicz, Rohina; Yolken, Robert; Drigalenko, Eugene; Carless, Melanie A.; Dyer, Thomas D.; Bauman, Lara; Melton, Phillip E.; Kent, Jack W.; Harley, John B.; Curran, Joanne E.; Johnson, Matthew P.; Cole, Shelley A.; Almasy, Laura; Moses, Eric K.; Dhurandhar, Nikhil V.; Kraig, Ellen; Blangero, John; Leach, Charles T.; Göring, Harald H. H.

2013-01-01

Infection with Epstein-Barr virus (EBV) is highly prevalent worldwide, and it has been associated with infectious mononucleosis and severe diseases including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal lymphoma, and lymphoproliferative disorders. Although EBV has been the focus of extensive research, much still remains unknown concerning what makes some individuals more sensitive to infection and to adverse outcomes as a result of infection. Here we use an integrative genomics approach in order to localize genetic factors influencing levels of Epstein Barr virus (EBV) nuclear antigen-1 (EBNA-1) IgG antibodies, as a measure of history of infection with this pathogen, in large Mexican American families. Genome-wide evidence of both significant linkage and association was obtained on chromosome 6 in the human leukocyte antigen (HLA) region and replicated in an independent Mexican American sample of large families (minimum p-value in combined analysis of both datasets is 1.4×10−15 for SNPs rs477515 and rs2516049). Conditional association analyses indicate the presence of at least two separate loci within MHC class II, and along with lymphocyte expression data suggest genes HLA-DRB1 and HLA-DQB1 as the best candidates. The association signals are specific to EBV and are not found with IgG antibodies to 12 other pathogens examined, and therefore do not simply reveal a general HLA effect. We investigated whether SNPs significantly associated with diseases in which EBV is known or suspected to play a role (namely nasopharyngeal lymphoma, Hodgkin lymphoma, systemic lupus erythematosus, and multiple sclerosis) also show evidence of associated with EBNA-1 antibody levels, finding an overlap only for the HLA locus, but none elsewhere in the genome. The significance of this work is that a major locus related to EBV infection has been identified, which may ultimately reveal the underlying mechanisms by which the immune system regulates infection with this pathogen

Prognostic value of the PAI-1 4G/5G polymorphism in invasive ductal carcinoma of the breast.

PubMed

Yagmurdur, M C; Atac, F B; Tutar, N U; Verdi, H; Isiklar, I; Ozdemir, B H; Ozbek, N; Karakayali, H; Haberal, M

2008-01-01

The study group was derived from the archive materials of 55 invasive ductal breast cancer (IDC) patients who had undergone breast-preserving surgery (partial mastectomy/ axillary dissection). All patients included in the study had clinically T(1)-2, N0-M0 invasive ductal carcinoma. Genomic DNA species were extracted from paraffin-embedded blocks, and plasminogen activator inhibitor type-1 (PAI-1) gene 4G/5G genotyping was done by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Patient demographics, axillary metastasis status, metastatic lymph nodi/total dissected lymph nodes from axilla, histopathologic characteristics of tumors, local recurrences, and survival ratio were assessed. PAI-1 4G/5G genotype frequencies were 4G/4G (64%), 4G/5G (31%), and 5G/5G (5%) in the patient group. According to the results based on frequencies, the demographics were not different. Five-year local recurrence rate of 4G/5G patients was the lowest (2/17, 12%) (P = 0.02). Also five-year distant metastases ratio of 4G/5G patients was the highest (18%) (P = 0.01). Five- and 10-year disease-free survival rates for the 4G/4G, 4G/5G, and 5G/5G groups were 97% and 94%, 82% and 77%, and 100% and 94%, respectively (P = 0.004). The results of this study indicate that the 4G allele in the PAI 1 gene had a negative impact on local recurrence and disease-free survival of patients with clinical T(1)-2N0M0 IDC.

Analyzing large scale genomic data on the cloud with Sparkhit

PubMed Central

Huang, Liren; Krüger, Jan

2018-01-01

Abstract Motivation The increasing amount of next-generation sequencing data poses a fundamental challenge on large scale genomic analytics. Existing tools use different distributed computational platforms to scale-out bioinformatics workloads. However, the scalability of these tools is not efficient. Moreover, they have heavy run time overheads when pre-processing large amounts of data. To address these limitations, we have developed Sparkhit: a distributed bioinformatics framework built on top of the Apache Spark platform. Results Sparkhit integrates a variety of analytical methods. It is implemented in the Spark extended MapReduce model. It runs 92–157 times faster than MetaSpark on metagenomic fragment recruitment and 18–32 times faster than Crossbow on data pre-processing. We analyzed 100 terabytes of data across four genomic projects in the cloud in 21 h, which includes the run times of cluster deployment and data downloading. Furthermore, our application on the entire Human Microbiome Project shotgun sequencing data was completed in 2 h, presenting an approach to easily associate large amounts of public datasets with reference data. Availability and implementation Sparkhit is freely available at: https://rhinempi.github.io/sparkhit/. Contact asczyrba@cebitec.uni-bielefeld.de Supplementary information Supplementary data are available at Bioinformatics online. PMID:29253074

Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication.

PubMed

Trávníček, Pavel; Ponert, Jan; Urfus, Tomáš; Jersáková, Jana; Vrána, Jan; Hřibová, Eva; Doležel, Jaroslav; Suda, Jan

2015-10-01

Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for ∼10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species. © 2015 International Society for Advancement of Cytometry.

Genome analysis of Listeria ivanovii strain G770 that caused a deadly aortic prosthesis infection

PubMed Central

Beye, M.; Gouriet, F.; Michelle, C.; Casalta, J.-P.; Habib, G.; Raoult, D.; Fournier, P.-E.

2016-01-01

We sequenced the genome of Listeria ivanovii strain G770, which caused a deadly infection of the thoracic aortic prosthesis of a 78-year-old man. The 2.9 Mb genome exhibited 21 specific genes among L. ivanovii strains, including five genes encoding a type I restriction modification system and one glycopeptide resistance gene. PMID:26933501

G-quadruplex prediction in E. coli genome reveals a conserved putative G-quadruplex-Hairpin-Duplex switch.

PubMed

Kaplan, Oktay I; Berber, Burak; Hekim, Nezih; Doluca, Osman

2016-11-02

Many studies show that short non-coding sequences are widely conserved among regulatory elements. More and more conserved sequences are being discovered since the development of next generation sequencing technology. A common approach to identify conserved sequences with regulatory roles relies on topological changes such as hairpin formation at the DNA or RNA level. G-quadruplexes, non-canonical nucleic acid topologies with little established biological roles, are increasingly considered for conserved regulatory element discovery. Since the tertiary structure of G-quadruplexes is strongly dependent on the loop sequence which is disregarded by the generally accepted algorithm, we hypothesized that G-quadruplexes with similar topology and, indirectly, similar interaction patterns, can be determined using phylogenetic clustering based on differences in the loop sequences. Phylogenetic analysis of 52 G-quadruplex forming sequences in the Escherichia coli genome revealed two conserved G-quadruplex motifs with a potential regulatory role. Further analysis revealed that both motifs tend to form hairpins and G quadruplexes, as supported by circular dichroism studies. The phylogenetic analysis as described in this work can greatly improve the discovery of functional G-quadruplex structures and may explain unknown regulatory patterns. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cas9 gRNA engineering for genome editing, activation and repression

DOE PAGES

Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; ...

2015-09-07

Here we demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Demonstration of IgG Subclass (IgG1 and IgG3) in Immuno-Related Hemocytopenia.

PubMed

Shao, Yuanyuan; Qi, Xiao; Fu, Rong; Liu, Hui; Wang, Yihao; Ding, Shaoxue; Wang, Huaquan; Li, Lijuan; Shao, Zonghong

2018-06-01

Immuno-related hemocytopenia (IRH) is defined as idiopathic cytopenia of undetermined significance (ICUS) patients with autoantibodies. In our previous studies, we found that IgG1 levels were increased in IRH patients and might cause the destruction of hematopoietic cells. In this study, we analyzed IgG subclasses in 30 IRH patients (male:female = 13:17, median age 32 years, range 18 - 56), 15 IRH remission patients (IRH-R) (male:female = 6:9, median age 34, range 20 - 52) and 20 normal controls (male:female = 8:12, median age 27, range 24 - 36) by Cytometric Bead Array, Flow Cytometry and Immunohistochemical staining. Levels of IgG1/IgG3 in the bone marrow supernatant of IRH patents, as well as the proportion of CD5+ B lymphocytes and Th2 cells (CD3+CD8-IL-4+) were higher than those of IRH-R patients and normal controls, and IgG1 levels had a positive correlation with the proportion of Th2 cells. In IRH patients, IgG1 and IgG3 were positive on nucleated erythrocytes and granulocytes, which were negative in IRH-R patients and healthy controls and had inverse correlations with hematopoietic function. Using immunohistochemical staining, IgG1 were also detected on bone marrow biopsies of IRH patients. The results indicated that IgG1 and IgG3 autoantibodies in IRH patients might play a key role in the IRH pathogenesis and in the abnormal immune function of IRH patients.

Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1).

PubMed

de la Torre, Andrea; Metivier, Aisha; Chu, Frances; Laurens, Lieve M L; Beck, David A C; Pienkos, Philip T; Lidstrom, Mary E; Kalyuzhnaya, Marina G

2015-11-25

Methane-utilizing bacteria (methanotrophs) are capable of growth on methane and are attractive systems for bio-catalysis. However, the application of natural methanotrophic strains to large-scale production of value-added chemicals/biofuels requires a number of physiological and genetic alterations. An accurate metabolic model coupled with flux balance analysis can provide a solid interpretative framework for experimental data analyses and integration. A stoichiometric flux balance model of Methylomicrobium buryatense strain 5G(B1) was constructed and used for evaluating metabolic engineering strategies for biofuels and chemical production with a methanotrophic bacterium as the catalytic platform. The initial metabolic reconstruction was based on whole-genome predictions. Each metabolic step was manually verified, gapfilled, and modified in accordance with genome-wide expression data. The final model incorporates a total of 841 reactions (in 167 metabolic pathways). Of these, up to 400 reactions were recruited to produce 118 intracellular metabolites. The flux balance simulations suggest that only the transfer of electrons from methanol oxidation to methane oxidation steps can support measured growth and methane/oxygen consumption parameters, while the scenario employing NADH as a possible source of electrons for particulate methane monooxygenase cannot. Direct coupling between methane oxidation and methanol oxidation accounts for most of the membrane-associated methane monooxygenase activity. However the best fit to experimental results is achieved only after assuming that the efficiency of direct coupling depends on growth conditions and additional NADH input (about 0.1-0.2 mol of incremental NADH per one mol of methane oxidized). The additional input is proposed to cover loss of electrons through inefficiency and to sustain methane oxidation at perturbations or support uphill electron transfer. Finally, the model was used for testing the carbon conversion

Novel mutations of MYO7A and USH1G in Israeli Arab families with Usher syndrome type 1.

PubMed

Rizel, Leah; Safieh, Christine; Shalev, Stavit A; Mezer, Eedy; Jabaly-Habib, Haneen; Ben-Neriah, Ziva; Chervinsky, Elena; Briscoe, Daniel; Ben-Yosef, Tamar

2011-01-01

This study investigated the genetic basis for Usher syndrome type 1 (USH1) in four consanguineous Israeli Arab families. Haplotype analysis for all known USH1 loci was performed in each family. In families for which haplotype analysis was inconclusive, we performed genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array. For mutation analysis, specific primers were used to PCR amplify the coding exons of the MYO7A, USH1C, and USH1G genes including intron-exon boundaries. Mutation screening was performed with direct sequencing. A combination of haplotype analysis and genome-wide homozygosity mapping indicated linkage to the USH1B locus in two families, USH1C in one family and USH1G in another family. Sequence analysis of the relevant genes (MYO7A, USH1C, and USH1G) led to the identification of pathogenic mutations in all families. Two of the identified mutations are novel (c.1135-1147dup in MYO7A and c.206-207insC in USH1G). USH1 is a genetically heterogenous condition. Of the five USH1 genes identified to date, USH1C and USH1G are the rarest contributors to USH1 etiology worldwide. It is therefore interesting that two of the four Israeli Arab families reported here have mutations in these two genes. This finding further demonstrates the unique genetic structure of the Israeli population in general, and the Israeli Arab population in particular, which due to high rates of consanguinity segregates many rare autosomal recessive genetic conditions.

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

PubMed

Macaulay, Iain C; Teng, Mabel J; Haerty, Wilfried; Kumar, Parveen; Ponting, Chris P; Voet, Thierry

2016-11-01

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

Complete genomic sequence of the Lactobacillus temperate phage LF1.

PubMed

Yoon, Bo Hyun; Chang, Hyo Ihl

2011-10-01

Bacteriophage LF1, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild type Lactobacillus fermentum, was found to contain a double-strand DNA of 42,606 base pairs (bp) with a G+C content of 45%. Bioinformatic analysis of the phage genome revealed 57 putative open reading frames (ORFs). The predicted protein products of ORFs were determined and described. According to morphological analysis by transmission electron microscopy (TEM), LF1 has an isometric head and a non-contractile tail, indicating that it belongs to the family Siphoviridae. The temperate phage LF1 has a good genetic mosaic relationship with ΦPYB5 in the packaging module. To our knowledge, this is first report of genomic sequencing and characterization of temperate phage LF1 from wild-type L. fermentum isolated from Kimchi in Korea.

Mitochondrial genomic analysis of late onset Alzheimer's disease reveals protective haplogroups H6A1A/H6A1B: the Cache County Study on Memory in Aging.

PubMed

Ridge, Perry G; Maxwell, Taylor J; Corcoran, Christopher D; Norton, Maria C; Tschanz, Joann T; O'Brien, Elizabeth; Kerber, Richard A; Cawthon, Richard M; Munger, Ronald G; Kauwe, John S K

2012-01-01

Alzheimer's disease (AD) is the most common cause of dementia and AD risk clusters within families. Part of the familial aggregation of AD is accounted for by excess maternal vs. paternal inheritance, a pattern consistent with mitochondrial inheritance. The role of specific mitochondrial DNA (mtDNA) variants and haplogroups in AD risk is uncertain. We determined the complete mitochondrial genome sequence of 1007 participants in the Cache County Study on Memory in Aging, a population-based prospective cohort study of dementia in northern Utah. AD diagnoses were made with a multi-stage protocol that included clinical examination and review by a panel of clinical experts. We used TreeScanning, a statistically robust approach based on haplotype networks, to analyze the mtDNA sequence data. Participants with major mitochondrial haplotypes H6A1A and H6A1B showed a reduced risk of AD (p=0.017, corrected for multiple comparisons). The protective haplotypes were defined by three variants: m.3915G>A, m.4727A>G, and m.9380G>A. These three variants characterize two different major haplogroups. Together m.4727A>G and m.9380G>A define H6A1, and it has been suggested m.3915G>A defines H6A. Additional variants differentiate H6A1A and H6A1B; however, none of these variants had a significant relationship with AD case-control status. Our findings provide evidence of a reduced risk of AD for individuals with mtDNA haplotypes H6A1A and H6A1B. These findings are the results of the largest study to date with complete mtDNA genome sequence data, yet the functional significance of the associated haplotypes remains unknown and replication in others studies is necessary.

Upsurge and spread of G3 rotaviruses in Eastern India (2014-2016): Full genome analyses reveals heterogeneity within Wa-like genomic constellation.

PubMed

Banerjee, Anindita; Lo, Mahadeb; Indwar, Pallavi; Deb, Alok K; Das, Santasabuj; Manna, Byomkesh; Dutta, Shanta; Bhadra, Uchhal K; Bhattacharya, Mala; Okamoto, Keinosuke; Chawla-Sarkar, Mamta

2018-05-26

Advent of new strains and shift in predominantly circulating genotypes are characteristics of group- A rotavirus (RVA), one of the major causes of childhood gastroenteritis. During diarrheal disease surveillance at Kolkata, India (2014-2016), a shift in circulating RVA strains from G1P[8] to G3P[8] was seen. Stool samples from children (n = 3048) with acute gastroenteritis were tested of which 38.7% were RVA positive. G1 was the predominant strain (65.3%) in 2014-2015 whereas in late 2015 and 2016, G3 became the preponderant strain (44.6%). In the past decade G3 strains were not observed in this region, we conducted whole genome sequencing of representative strains to gain insight into the phenomenon of emergence and genetic constellation of these circulating human G3 strains. The analyses revealed intergenogroup reassortment in G3P[4] strains (among Wa and DS-1-like genogroup) whereas G3P[8] strains were authentic Wa-like. Phylogenetic analysis revealed Kolkata G3 strains as polymorphic and thus they formed two sub-clusters due to antigenic differences in their VP7 protein. One of the sub-clusters had the wild-type threonine at 87 amino acid position while another sub-cluster had an isoleucine mutation. Presence of additional N-linked glycosylation site at amino acid 283 of VP7 glycoprotein suggests that the major neutralizing epitope on the VP7 (G3) of RotaTeq vaccine differs from the currently circulating G3 strains. The study is important as efficiency of rotavirus vaccine depends on the circulating heterogeneous genotype constellations. Continuous monitoring of circulating RVA strains in endemic settings like India is therefore important in pre- and post-vaccination period to monitor the emergence of new reassortant genotypes in addition to assessing vaccine efficacy. Copyright © 2018. Published by Elsevier B.V.

Genetic traits of avascular necrosis of the femoral head analyzed by array comparative genomic hybridization and real-time polymerase chain reaction.

PubMed

Hwang, Jung-Taek; Baik, Seung-Ho; Choi, Jin-Soo; Lee, Kweon-Haeng; Rhee, Seung-Koo

2011-01-03

In an attempt to observe the genetic traits of avascular necrosis of the femoral head, we analyzed the genomic alterations in blood samples of 18 patients with avascular necrosis of the femoral head (9 idiopathic and 9 alcoholic cases) using the array comparative genomic hybridization method and real-time polymerase chain reaction. Several candidate genes were identified that may induce avascular necrosis of the femoral head, and we investigated their role in the pathomechanism of osteonecrosis of bone. The frequency of each candidate gene over all the categories of avascular necrosis of the femoral head was also calculated by real-time polymerase chain reaction. The highest frequency specific genes in each category were FLJ40296, CYP27C1, and CTDP1. FLJ40296 and CYP27C1 had the highest frequency (55.6%) in the idiopathic category. FLJ40296 had a high frequency (44.4%) in the alcoholic category, but CYP27C1 had a relatively low frequency (33.3%) in the alcoholic category. However, CTDP1 showed a significantly high frequency (55.6%) in the alcoholic category and a low frequency (22.2%) in the idiopathic category. Although we statistically analyzed the frequency of each gene with Fisher's exact test, we could not prove statistical significance due to the small number of samples. Further studies are needed with larger sample numbers. If the causal genes of avascular necrosis of the femoral head are found, they may be used for early detection, prognosis prediction, and genomic treatment of avascular necrosis of the femoral head in the future. Copyright 2011, SLACK Incorporated.

Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs.

PubMed

Ranganathan, Vinod; Wahlin, Karl; Maruotti, Julien; Zack, Donald J

2014-08-08

The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.

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

Viral Genome DataBase: storing and analyzing genes and proteins from complete viral genomes.

PubMed

Hiscock, D; Upton, C

2000-05-01

The Viral Genome DataBase (VGDB) contains detailed information of the genes and predicted protein sequences from 15 completely sequenced genomes of large (&100 kb) viruses (2847 genes). The data that is stored includes DNA sequence, protein sequence, GenBank and user-entered notes, molecular weight (MW), isoelectric point (pI), amino acid content, A + T%, nucleotide frequency, dinucleotide frequency and codon use. The VGDB is a mySQL database with a user-friendly JAVA GUI. Results of queries can be easily sorted by any of the individual parameters. The software and additional figures and information are available at http://athena.bioc.uvic.ca/genomes/index.html .

Mitochondrial Genomic Analysis of Late Onset Alzheimer’s Disease Reveals Protective Haplogroups H6A1A/H6A1B: The Cache County Study on Memory in Aging

PubMed Central

Ridge, Perry G.; Maxwell, Taylor J.; Corcoran, Christopher D.; Norton, Maria C.; Tschanz, JoAnn T.; O’Brien, Elizabeth; Kerber, Richard A.; Cawthon, Richard M.; Munger, Ronald G.; Kauwe, John S. K.

2012-01-01

Background Alzheimer’s disease (AD) is the most common cause of dementia and AD risk clusters within families. Part of the familial aggregation of AD is accounted for by excess maternal vs. paternal inheritance, a pattern consistent with mitochondrial inheritance. The role of specific mitochondrial DNA (mtDNA) variants and haplogroups in AD risk is uncertain. Methodology/Principal Findings We determined the complete mitochondrial genome sequence of 1007 participants in the Cache County Study on Memory in Aging, a population-based prospective cohort study of dementia in northern Utah. AD diagnoses were made with a multi-stage protocol that included clinical examination and review by a panel of clinical experts. We used TreeScanning, a statistically robust approach based on haplotype networks, to analyze the mtDNA sequence data. Participants with major mitochondrial haplotypes H6A1A and H6A1B showed a reduced risk of AD (p = 0.017, corrected for multiple comparisons). The protective haplotypes were defined by three variants: m.3915G>A, m.4727A>G, and m.9380G>A. These three variants characterize two different major haplogroups. Together m.4727A>G and m.9380G>A define H6A1, and it has been suggested m.3915G>A defines H6A. Additional variants differentiate H6A1A and H6A1B; however, none of these variants had a significant relationship with AD case-control status. Conclusions/Significance Our findings provide evidence of a reduced risk of AD for individuals with mtDNA haplotypes H6A1A and H6A1B. These findings are the results of the largest study to date with complete mtDNA genome sequence data, yet the functional significance of the associated haplotypes remains unknown and replication in others studies is necessary. PMID:23028804

A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome.

PubMed

Wang, Hui-Yun; Luo, Minjie; Tereshchenko, Irina V; Frikker, Danielle M; Cui, Xiangfeng; Li, James Y; Hu, Guohong; Chu, Yi; Azaro, Marco A; Lin, Yong; Shen, Li; Yang, Qifeng; Kambouris, Manousos E; Gao, Richeng; Shih, Weichung; Li, Honghua

2005-02-01

A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system. The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.

The association between PAI-1 -675 4G/5G polymorphism and type 2 diabetes mellitus.

PubMed

Chen, L; Li, S-Y; Liu, M

2017-08-15

In this study, we aimed to analyze the association between plasminogen activator inhibitor 1 (PAI-1) -675 4G/5G polymorphism and type 2 diabetes mellitus (T2DM) risk. We included in 187 T2DM patients and 186 heathy controls between 2014 and 2017 from Tianjin Gong An Hospital, China. All patients and controls were ethnically Chinese Han population. The primers and polymerase chain reaction (PCR) conditions were performed. Results from this case-control study suggested that PAI-1 -675 4G/5G polymorphism was not associated with T2DM risk in four genetic models. Additionally, PAI-1 -675 4G/5G polymorphism was not associated with clinical and laboratory characteristics, such as age, gender, body mass index, systolic blood pressure, diastolic blood pressure, total cholesterol, triglycerides, and HbA1c. In conclusion, this case-control study suggested that PAI-1 -675 4G/5G polymorphism was not associated with T2DM risk in this population.

Topoisomerase IIα maintains genomic stability through decatenation G2 checkpoint signaling

PubMed Central

Bower, Jacquelyn J.; Karaca, Gamze F.; Zhou, Yingchun; Simpson, Dennis A.; Cordeiro-Stone, Marila; Kaufmann, William K.

2010-01-01

Topoisomerase IIα (topoIIα) is an essential mammalian enzyme that topologically modifies DNA and is required for chromosome segregation during mitosis. Previous research suggests that inhibition of topoII decatenatory activity triggers a G2 checkpoint response, which delays mitotic entry due to insufficient decatenation of daughter chromatids. Here we examine the effects of both topoIIα and topoIIβ on decatenatory activity in cell extracts, DNA damage and decatenation G2 checkpoint function, and the frequencies of p16INK4A allele loss and gain. In diploid human fibroblast lines, depletion of topoIIα by siRNA was associated with severely reduced decatenatory activity, delayed progression from G2 into mitosis, and insensitivity to G2 arrest induced by the topoII catalytic inhibitor ICRF-193. Furthermore, interphase nuclei of topoIIα-depleted cells displayed increased frequencies of losses and gains of the tumor suppressor genetic locus p16INK4A. This study demonstrates that the topoIIα protein is required for decatenation G2 checkpoint function, and inactivation of decatenation and the decatenation G2 checkpoint leads to abnormal chromosome segregation and genomic instability. PMID:20562910

Interaction of Hb South Florida (codon 1; GTG-->ATG) and HbE, with beta-thalassemia (IVS1-1; G-->A): expression of different clinical phenotypes.

PubMed

Tan, Jin-Ai Mary Anne; Tan, Kim-Lian; Omar, Khairul Zaman; Chan, Lee-Lee; Wee, Yong-Chui; George, Elizabeth

2009-09-01

Interactions of different hemoglobin variants with thalassemia alleles can result in various clinical phenotypes. HbE-beta-thalassemia generally manifests with severe anemia where individuals exhibit beta-thalassemia major with regular blood transfusions or beta-thalassemia intermedia with periodic blood transfusions. This study presents a unique Malay family with three beta-globin gene defects-HbE, Hb South Florida, and IVS1-1 (G-->A). HbE activates a cryptic splice site that produces non-functional mRNAs. Hb South Florida is a rare beta-hemoglobin variant, and its interactions with other beta-thalassemia alleles have not been reported. IVS1-1 is a Mediterranean mutation that affects mRNA processing giving rise to beta(o)-thalassemia. Fifteen mutations along the beta-globin gene complex were analyzed using the amplification refractory mutation system. Hb South Florida was identified by direct sequencing using genomic DNA. The affected child with HbE/IVS1-1 produced a beta-thalassemia major phenotype. Compound heterozygosity for Hb South Florida/IVS1-1 produced a beta-thalassemia carrier phenotype in the mother.

Genome-wide CpG island methylation and intergenic demethylation propensities vary among different tumor sites.

PubMed

Lee, Seung-Tae; Wiemels, Joseph L

2016-02-18

The epigenetic landscape of cancer includes both focal hypermethylation and broader hypomethylation in a genome-wide manner. By means of a comprehensive genomic analysis on 6637 tissues of 21 tumor types, we here show that the degrees of overall methylation in CpG island (CGI) and demethylation in intergenic regions, defined as 'backbone', largely vary among different tumors. Depending on tumor type, both CGI methylation and backbone demethylation are often associated with clinical, epidemiological and biological features such as age, sex, smoking history, anatomic location, histological type and grade, stage, molecular subtype and biological pathways. We found connections between CGI methylation and hypermutability, microsatellite instability, IDH1 mutation, 19p gain and polycomb features, and backbone demethylation with chromosomal instability, NSD1 and TP53 mutations, 5q and 19p loss and long repressive domains. These broad epigenetic patterns add a new dimension to our understanding of tumor biology and its clinical implications. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of a fluorescence-activated cell sorting method coupled with whole genome amplification to analyze minority and trace Dehalococcoides genomes in microbial communities.

PubMed

Lee, Patrick K H; Men, Yujie; Wang, Shanquan; He, Jianzhong; Alvarez-Cohen, Lisa

2015-02-03

Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 10(6) cells or ∼1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing ∼10% and ∼1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low.

Analysis of the Complete Mitochondrial Genome Sequence of the Diploid Cotton Gossypium raimondii by Comparative Genomics Approaches

PubMed Central

Paterson, Andrew H.; Wang, Xuelin; Xu, Yiqing; Wu, Dongyang; Qu, Yanshu; Jiang, Anna; Ye, Qiaolin

2016-01-01

Cotton is one of the most important economic crops and the primary source of natural fiber and is an important protein source for animal feed. The complete nuclear and chloroplast (cp) genome sequences of G. raimondii are already available but not mitochondria. Here, we assembled the complete mitochondrial (mt) DNA sequence of G. raimondii into a circular genome of length of 676,078 bp and performed comparative analyses with other higher plants. The genome contains 39 protein-coding genes, 6 rRNA genes, and 25 tRNA genes. We also identified four larger repeats (63.9 kb, 10.6 kb, 9.1 kb, and 2.5 kb) in this mt genome, which may be active in intramolecular recombination in the evolution of cotton. Strikingly, nearly all of the G. raimondii mt genome has been transferred to nucleus on Chr1, and the transfer event must be very recent. Phylogenetic analysis reveals that G. raimondii, as a member of Malvaceae, is much closer to another cotton (G. barbadense) than other rosids, and the clade formed by two Gossypium species is sister to Brassicales. The G. raimondii mt genome may provide a crucial foundation for evolutionary analysis, molecular biology, and cytoplasmic male sterility in cotton and other higher plants. PMID:27847816

Genome-wide study of correlations between genomic features and their relationship with the regulation of gene expression.

PubMed

Kravatsky, Yuri V; Chechetkin, Vladimir R; Tchurikov, Nikolai A; Kravatskaya, Galina I

2015-02-01

The broad class of tasks in genetics and epigenetics can be reduced to the study of various features that are distributed over the genome (genome tracks). The rapid and efficient processing of the huge amount of data stored in the genome-scale databases cannot be achieved without the software packages based on the analytical criteria. However, strong inhomogeneity of genome tracks hampers the development of relevant statistics. We developed the criteria for the assessment of genome track inhomogeneity and correlations between two genome tracks. We also developed a software package, Genome Track Analyzer, based on this theory. The theory and software were tested on simulated data and were applied to the study of correlations between CpG islands and transcription start sites in the Homo sapiens genome, between profiles of protein-binding sites in chromosomes of Drosophila melanogaster, and between DNA double-strand breaks and histone marks in the H. sapiens genome. Significant correlations between transcription start sites on the forward and the reverse strands were observed in genomes of D. melanogaster, Caenorhabditis elegans, Mus musculus, H. sapiens, and Danio rerio. The observed correlations may be related to the regulation of gene expression in eukaryotes. Genome Track Analyzer is freely available at http://ancorr.eimb.ru/. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

SL1 revisited: functional analysis of the structure and conformation of HIV-1 genome RNA.

PubMed

Sakuragi, Sayuri; Yokoyama, Masaru; Shioda, Tatsuo; Sato, Hironori; Sakuragi, Jun-Ichi

2016-11-11

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of HIV is located on the 5' end of the viral genome and suggested to form complex secondary/tertiary structures. Within this structure, stem-loop 1 (SL1) is believed to be most important and an essential key to dimerization, since the sequence and predicted secondary structure of SL1 are highly stable and conserved among various virus subtypes. In particular, a six-base palindromic sequence is always present at the hairpin loop of SL1 and the formation of kissing-loop structure at this position between the two strands of genomic RNA is suggested to trigger dimerization. Although the higher-order structure model of SL1 is well accepted and perhaps even undoubted lately, there could be stillroom for consideration to depict the functional SL1 structure while in vivo (in virion or cell). In this study, we performed several analyses to identify the nucleotides and/or basepairing within SL1 which are necessary for HIV-1 genome dimerization, encapsidation, recombination and infectivity. We unexpectedly found that some nucleotides that are believed to contribute the formation of the stem do not impact dimerization or infectivity. On the other hand, we found that one G-C basepair involved in stem formation may serve as an alternative dimer interactive site. We also report on our further investigation of the roles of the palindromic sequences on viral replication. Collectively, we aim to assemble a more-comprehensive functional map of SL1 on the HIV-1 viral life cycle. We discovered several possibilities for a novel structure of SL1 in HIV-1 DLS. The newly proposed structure model suggested that the hairpin loop of SL1 appeared larger, and genome dimerization process might consist of more complicated mechanism than previously understood. Further investigations would be still required to fully understand the genome packaging and dimerization of HIV.

The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

PubMed Central

de Solis, Christopher A.; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E.

2016-01-01

The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well. PMID:27587996

The Personal Genome Project Canada: findings from whole genome sequences of the inaugural 56 participants

PubMed Central

Reuter, Miriam S.; Walker, Susan; Thiruvahindrapuram, Bhooma; Whitney, Joe; Cohn, Iris; Sondheimer, Neal; Yuen, Ryan K.C.; Trost, Brett; Paton, Tara A.; Pereira, Sergio L.; Herbrick, Jo-Anne; Wintle, Richard F.; Merico, Daniele; Howe, Jennifer; MacDonald, Jeffrey R.; Lu, Chao; Nalpathamkalam, Thomas; Sung, Wilson W.L.; Wang, Zhuozhi; Patel, Rohan V.; Pellecchia, Giovanna; Wei, John; Strug, Lisa J.; Bell, Sherilyn; Kellam, Barbara; Mahtani, Melanie M.; Bassett, Anne S.; Bombard, Yvonne; Weksberg, Rosanna; Shuman, Cheryl; Cohn, Ronald D.; Stavropoulos, Dimitri J.; Bowdin, Sarah; Hildebrandt, Matthew R.; Wei, Wei; Romm, Asli; Pasceri, Peter; Ellis, James; Ray, Peter; Meyn, M. Stephen; Monfared, Nasim; Hosseini, S. Mohsen; Joseph-George, Ann M.; Keeley, Fred W.; Cook, Ryan A.; Fiume, Marc; Lee, Hin C.; Marshall, Christian R.; Davies, Jill; Hazell, Allison; Buchanan, Janet A.; Szego, Michael J.; Scherer, Stephen W.

2018-01-01

BACKGROUND: The Personal Genome Project Canada is a comprehensive public data resource that integrates whole genome sequencing data and health information. We describe genomic variation identified in the initial recruitment cohort of 56 volunteers. METHODS: Volunteers were screened for eligibility and provided informed consent for open data sharing. Using blood DNA, we performed whole genome sequencing and identified all possible classes of DNA variants. A genetic counsellor explained the implication of the results to each participant. RESULTS: Whole genome sequencing of the first 56 participants identified 207 662 805 sequence variants and 27 494 copy number variations. We analyzed a prioritized disease-associated data set (n = 1606 variants) according to standardized guidelines, and interpreted 19 variants in 14 participants (25%) as having obvious health implications. Six of these variants (e.g., in BRCA1 or mosaic loss of an X chromosome) were pathogenic or likely pathogenic. Seven were risk factors for cancer, cardiovascular or neurobehavioural conditions. Four other variants — associated with cancer, cardiac or neurodegenerative phenotypes — remained of uncertain significance because of discrepancies among databases. We also identified a large structural chromosome aberration and a likely pathogenic mitochondrial variant. There were 172 recessive disease alleles (e.g., 5 individuals carried mutations for cystic fibrosis). Pharmacogenomics analyses revealed another 3.9 potentially relevant genotypes per individual. INTERPRETATION: Our analyses identified a spectrum of genetic variants with potential health impact in 25% of participants. When also considering recessive alleles and variants with potential pharmacologic relevance, all 56 participants had medically relevant findings. Although access is mostly limited to research, whole genome sequencing can provide specific and novel information with the potential of major impact for health care. PMID:29431110

The Personal Genome Project Canada: findings from whole genome sequences of the inaugural 56 participants.

PubMed

Reuter, Miriam S; Walker, Susan; Thiruvahindrapuram, Bhooma; Whitney, Joe; Cohn, Iris; Sondheimer, Neal; Yuen, Ryan K C; Trost, Brett; Paton, Tara A; Pereira, Sergio L; Herbrick, Jo-Anne; Wintle, Richard F; Merico, Daniele; Howe, Jennifer; MacDonald, Jeffrey R; Lu, Chao; Nalpathamkalam, Thomas; Sung, Wilson W L; Wang, Zhuozhi; Patel, Rohan V; Pellecchia, Giovanna; Wei, John; Strug, Lisa J; Bell, Sherilyn; Kellam, Barbara; Mahtani, Melanie M; Bassett, Anne S; Bombard, Yvonne; Weksberg, Rosanna; Shuman, Cheryl; Cohn, Ronald D; Stavropoulos, Dimitri J; Bowdin, Sarah; Hildebrandt, Matthew R; Wei, Wei; Romm, Asli; Pasceri, Peter; Ellis, James; Ray, Peter; Meyn, M Stephen; Monfared, Nasim; Hosseini, S Mohsen; Joseph-George, Ann M; Keeley, Fred W; Cook, Ryan A; Fiume, Marc; Lee, Hin C; Marshall, Christian R; Davies, Jill; Hazell, Allison; Buchanan, Janet A; Szego, Michael J; Scherer, Stephen W

2018-02-05

The Personal Genome Project Canada is a comprehensive public data resource that integrates whole genome sequencing data and health information. We describe genomic variation identified in the initial recruitment cohort of 56 volunteers. Volunteers were screened for eligibility and provided informed consent for open data sharing. Using blood DNA, we performed whole genome sequencing and identified all possible classes of DNA variants. A genetic counsellor explained the implication of the results to each participant. Whole genome sequencing of the first 56 participants identified 207 662 805 sequence variants and 27 494 copy number variations. We analyzed a prioritized disease-associated data set ( n = 1606 variants) according to standardized guidelines, and interpreted 19 variants in 14 participants (25%) as having obvious health implications. Six of these variants (e.g., in BRCA1 or mosaic loss of an X chromosome) were pathogenic or likely pathogenic. Seven were risk factors for cancer, cardiovascular or neurobehavioural conditions. Four other variants - associated with cancer, cardiac or neurodegenerative phenotypes - remained of uncertain significance because of discrepancies among databases. We also identified a large structural chromosome aberration and a likely pathogenic mitochondrial variant. There were 172 recessive disease alleles (e.g., 5 individuals carried mutations for cystic fibrosis). Pharmacogenomics analyses revealed another 3.9 potentially relevant genotypes per individual. Our analyses identified a spectrum of genetic variants with potential health impact in 25% of participants. When also considering recessive alleles and variants with potential pharmacologic relevance, all 56 participants had medically relevant findings. Although access is mostly limited to research, whole genome sequencing can provide specific and novel information with the potential of major impact for health care. © 2018 Joule Inc. or its licensors.

Genomic and expression analysis of the vanG-like gene cluster of Clostridium difficile.

PubMed

Peltier, Johann; Courtin, Pascal; El Meouche, Imane; Catel-Ferreira, Manuella; Chapot-Chartier, Marie-Pierre; Lemée, Ludovic; Pons, Jean-Louis

2013-07-01

Primary antibiotic treatment of Clostridium difficile intestinal diseases requires metronidazole or vancomycin therapy. A cluster of genes homologous to enterococcal glycopeptides resistance vanG genes was found in the genome of C. difficile 630, although this strain remains sensitive to vancomycin. This vanG-like gene cluster was found to consist of five ORFs: the regulatory region consisting of vanR and vanS and the effector region consisting of vanG, vanXY and vanT. We found that 57 out of 83 C. difficile strains, representative of the main lineages of the species, harbour this vanG-like cluster. The cluster is expressed as an operon and, when present, is found at the same genomic location in all strains. The vanG, vanXY and vanT homologues in C. difficile 630 are co-transcribed and expressed to a low level throughout the growth phases in the absence of vancomycin. Conversely, the expression of these genes is strongly induced in the presence of subinhibitory concentrations of vancomycin, indicating that the vanG-like operon is functional at the transcriptional level in C. difficile. Hydrophilic interaction liquid chromatography (HILIC-HPLC) and MS analysis of cytoplasmic peptidoglycan precursors of C. difficile 630 grown without vancomycin revealed the exclusive presence of a UDP-MurNAc-pentapeptide with an alanine at the C terminus. UDP-MurNAc-pentapeptide [d-Ala] was also the only peptidoglycan precursor detected in C. difficile grown in the presence of vancomycin, corroborating the lack of vancomycin resistance. Peptidoglycan structures of a vanG-like mutant strain and of a strain lacking the vanG-like cluster did not differ from the C. difficile 630 strain, indicating that the vanG-like cluster also has no impact on cell-wall composition.

Genome-wide identification of significant aberrations in cancer genome.

PubMed

Yuan, Xiguo; Yu, Guoqiang; Hou, Xuchu; Shih, Ie-Ming; Clarke, Robert; Zhang, Junying; Hoffman, Eric P; Wang, Roger R; Zhang, Zhen; Wang, Yue

2012-07-27

Somatic Copy Number Alterations (CNAs) in human genomes are present in almost all human cancers. Systematic efforts to characterize such structural variants must effectively distinguish significant consensus events from random background aberrations. Here we introduce Significant Aberration in Cancer (SAIC), a new method for characterizing and assessing the statistical significance of recurrent CNA units. Three main features of SAIC include: (1) exploiting the intrinsic correlation among consecutive probes to assign a score to each CNA unit instead of single probes; (2) performing permutations on CNA units that preserve correlations inherent in the copy number data; and (3) iteratively detecting Significant Copy Number Aberrations (SCAs) and estimating an unbiased null distribution by applying an SCA-exclusive permutation scheme. We test and compare the performance of SAIC against four peer methods (GISTIC, STAC, KC-SMART, CMDS) on a large number of simulation datasets. Experimental results show that SAIC outperforms peer methods in terms of larger area under the Receiver Operating Characteristics curve and increased detection power. We then apply SAIC to analyze structural genomic aberrations acquired in four real cancer genome-wide copy number data sets (ovarian cancer, metastatic prostate cancer, lung adenocarcinoma, glioblastoma). When compared with previously reported results, SAIC successfully identifies most SCAs known to be of biological significance and associated with oncogenes (e.g., KRAS, CCNE1, and MYC) or tumor suppressor genes (e.g., CDKN2A/B). Furthermore, SAIC identifies a number of novel SCAs in these copy number data that encompass tumor related genes and may warrant further studies. Supported by a well-grounded theoretical framework, SAIC has been developed and used to identify SCAs in various cancer copy number data sets, providing useful information to study the landscape of cancer genomes. Open-source and platform-independent SAIC software is

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding*

PubMed Central

Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

2015-01-01

The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation. PMID:25627683

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

PubMed Central

Sorokin, Dmitry V; Stixová, Lenka; Sehnalová, Petra; Legartová, Soňa; Suchánková, Jana; Šimara, Pavel; Kozubek, Stanislav; Matula, Pavel; Skalníková, Magdalena; Raška, Ivan; Bártová, Eva

2015-01-01

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase. PMID:26208041

Genomic Distribution and Inter-Sample Variation of Non-CpG Methylation across Human Cell Types

PubMed Central

Liao, Jing; Zhang, Yingying; Gu, Hongcang; Bock, Christoph; Boyle, Patrick; Epstein, Charles B.; Bernstein, Bradley E.; Lengauer, Thomas; Gnirke, Andreas; Meissner, Alexander

2011-01-01

DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set. PMID:22174693

Compositional searching of CpG islands in the human genome

NASA Astrophysics Data System (ADS)

Luque-Escamilla, Pedro Luis; Martínez-Aroza, José; Oliver, José L.; Gómez-Lopera, Juan Francisco; Román-Roldán, Ramón

2005-06-01

We report on an entropic edge detector based on the local calculation of the Jensen-Shannon divergence with application to the search for CpG islands. CpG islands are pieces of the genome related to gene expression and cell differentiation, and thus to cancer formation. Searching for these CpG islands is a major task in genetics and bioinformatics. Some algorithms have been proposed in the literature, based on moving statistics in a sliding window, but its size may greatly influence the results. The local use of Jensen-Shannon divergence is a completely different strategy: the nucleotide composition inside the islands is different from that in their environment, so a statistical distance—the Jensen-Shannon divergence—between the composition of two adjacent windows may be used as a measure of their dissimilarity. Sliding this double window over the entire sequence allows us to segment it compositionally. The fusion of those segments into greater ones that satisfy certain identification criteria must be achieved in order to obtain the definitive results. We find that the local use of Jensen-Shannon divergence is very suitable in processing DNA sequences for searching for compositionally different structures such as CpG islands, as compared to other algorithms in literature.

Inference of Gorilla Demographic and Selective History from Whole-Genome Sequence Data

PubMed Central

McManus, Kimberly F.; Kelley, Joanna L.; Song, Shiya; Veeramah, Krishna R.; Woerner, August E.; Stevison, Laurie S.; Ryder, Oliver A.; Ape Genome Project, Great; Kidd, Jeffrey M.; Wall, Jeffrey D.; Bustamante, Carlos D.; Hammer, Michael F.

2015-01-01

Although population-level genomic sequence data have been gathered extensively for humans, similar data from our closest living relatives are just beginning to emerge. Examination of genomic variation within great apes offers many opportunities to increase our understanding of the forces that have differentially shaped the evolutionary history of hominid taxa. Here, we expand upon the work of the Great Ape Genome Project by analyzing medium to high coverage whole-genome sequences from 14 western lowland gorillas (Gorilla gorilla gorilla), 2 eastern lowland gorillas (G. beringei graueri), and a single Cross River individual (G. gorilla diehli). We infer that the ancestors of western and eastern lowland gorillas diverged from a common ancestor approximately 261 ka, and that the ancestors of the Cross River population diverged from the western lowland gorilla lineage approximately 68 ka. Using a diffusion approximation approach to model the genome-wide site frequency spectrum, we infer a history of western lowland gorillas that includes an ancestral population expansion of 1.4-fold around 970 ka and a recent 5.6-fold contraction in population size 23 ka. The latter may correspond to a major reduction in African equatorial forests around the Last Glacial Maximum. We also analyze patterns of variation among western lowland gorillas to identify several genomic regions with strong signatures of recent selective sweeps. We find that processes related to taste, pancreatic and saliva secretion, sodium ion transmembrane transport, and cardiac muscle function are overrepresented in genomic regions predicted to have experienced recent positive selection. PMID:25534031

G-Protein Genomic Association With Normal Variation in Gray Matter Density

PubMed Central

Chen, Jiayu; Calhoun, Vince D.; Arias-Vasquez, Alejandro; Zwiers, Marcel P.; van Hulzen, Kimm; Fernández, Guillén; Fisher, Simon E.; Franke, Barbara; Turner, Jessica A.; Liu, Jingyu

2017-01-01

While detecting genetic variations underlying brain structures helps reveal mechanisms of neural disorders, high data dimensionality poses a major challenge for imaging genomic association studies. In this work, we present the application of a recently proposed approach, parallel independent component analysis with reference (pICA-R), to investigate genomic factors potentially regulating gray matter variation in a healthy population. This approach simultaneously assesses many variables for an aggregate effect and helps to elicit particular features in the data. We applied pICA-R to analyze gray matter density (GMD) images (274,131 voxels) in conjunction with single nucleotide polymorphism (SNP) data (666,019 markers) collected from 1,256 healthy individuals of the Brain Imaging Genetics (BIG) study. Guided by a genetic reference derived from the gene GNA14, pICA-R identified a significant SNP-GMD association (r = −0.16, P = 2.34 × 10−8), implying that subjects with specific genotypes have lower localized GMD. The identified components were then projected to an independent dataset from the Mind Clinical Imaging Consortium (MCIC) including 89 healthy individuals, and the obtained loadings again yielded a significant SNP-GMD association (r = −0.25, P = 0.02). The imaging component reflected GMD variations in frontal, precuneus, and cingulate regions. The SNP component was enriched in genes with neuronal functions, including synaptic plasticity, axon guidance, molecular signal transduction via PKA and CREB, highlighting the GRM1, PRKCH, GNA12, and CAMK2B genes. Collectively, our findings suggest that GNA12 and GNA14 play a key role in the genetic architecture underlying normal GMD variation in frontal and parietal regions. PMID:26248772

Analyzing medical costs with time-dependent treatment: The nested g-formula.

PubMed

Spieker, Andrew; Roy, Jason; Mitra, Nandita

2018-04-16

As medical expenses continue to rise, methods to properly analyze cost outcomes are becoming of increasing relevance when seeking to compare average costs across treatments. Inverse probability weighted regression models have been developed to address the challenge of cost censoring in order to identify intent-to-treat effects (i.e., to compare mean costs between groups on the basis of their initial treatment assignment, irrespective of any subsequent changes to their treatment status). In this paper, we describe a nested g-computation procedure that can be used to compare mean costs between two or more time-varying treatment regimes. We highlight the relative advantages and limitations of this approach when compared with existing regression-based models. We illustrate the utility of this approach as a means to inform public policy by applying it to a simulated data example motivated by costs associated with cancer treatments. Simulations confirm that inference regarding intent-to-treat effects versus the joint causal effects estimated by the nested g-formula can lead to markedly different conclusions regarding differential costs. Therefore, it is essential to prespecify the desired target of inference when choosing between these two frameworks. The nested g-formula should be considered as a useful, complementary tool to existing methods when analyzing cost outcomes. Copyright © 2018 John Wiley & Sons, Ltd.

G protein-coupled estrogen receptor 1 agonist G-1 induces cell cycle arrest in the mitotic phase, leading to apoptosis in endometriosis.

PubMed

Mori, Taisuke; Ito, Fumitake; Matsushima, Hiroshi; Takaoka, Osamu; Tanaka, Yukiko; Koshiba, Akemi; Kusuki, Izumi; Kitawaki, Jo

2015-05-01

To demonstrate the effects of the selective G protein-coupled estrogen receptor 1 (GPER) agonist G-1 in human ovarian endometriotic stromal cells (ESCs). Experimental in vitro study. University hospital. A total of 33 patients with ovarian endometrioma. Endometriotic stromal cells from ovarian chocolate cysts were treated with the GPER agonist G-1. The primary outcomes were cell proliferation, measured using the WST-8 assay; cell cycle, as analyzed using flow cytometry, fluorescent immunocytochemistry, and cytotoxicity; caspase activity, as measured by fluorescent and luminescent enzyme assays; and protein expression levels, as determined by Western blot analysis. G-1 suppressed ESC proliferation in a concentration-dependent manner. The inhibitory effect was not blocked when GPER signaling pathways, including the GPER itself, were inhibited. G-1 induced cell cycle arrest and accumulation in the sub-G1 phase in ESCs. Immunofluorescence analysis demonstrated that G-1 interrupted microtubule assembly at the mitotic phase. G-1 also induced caspase-3-dependent apoptosis without significant cytotoxicity. G-1 suppressed proliferation and induced apoptosis in ESCs, suggesting the potential use of this compound as a therapeutic drug for the treatment of endometriosis. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

PubMed Central

Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

2015-01-01

Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

Echinococcus ortleppi (G5) and Echinococcus granulosus sensu stricto (G1) loads in cattle from Southern Brazil.

PubMed

Balbinotti, Helier; Santos, Guilherme B; Badaraco, Jeferson; Arend, Ana C; Graichen, Daniel Ângelo S; Haag, Karen L; Zaha, Arnaldo

2012-09-10

Echinococcus granulosus sensu stricto (G1) and Echinococcus ortleppi (G5) are haplotypes of the parasite formerly known as Echinococcus granulosus sensu lato, which in its larval stage causes cystic hydatid disease, endemic in Southern Brazil. Epidemiological and molecular knowledge about the haplotypes occurring in a region is essential to control the spread of the disease. The aim of this work was to analyze the haplotype frequency and fertility of hydatid cysts in cattle from the state of Rio Grande do Sul. Cysts were collected and classified according to their fertility status. DNA was extracted from protoscoleces and germinal layers and then used as template for the amplification of the cytochrome c oxidase subunit 1 gene by PCR. Amplicons were purified and sequenced, and the sequences were analyzed for haplotype identification. A total of 638 fertile cysts collected in the last ten years were genotyped. On average, G1 (56.6%) was more frequent than G5 (43.4%). In lungs, the G5 haplotype exhibited a higher parasite load (52.8%), whereas in the liver, G1 was more frequent (90.4%). The analysis revealed an increase in the frequency of G5 haplotype cysts during the period of sampling, and an increase in the abundance of fertile cysts has also been observed in the last several years. Most infertile cysts were genotyped as G1. The possible factors involved in the increase in the proportion of E. ortleppi (G5) and the consequences of this increase are discussed. This study suggests that the proportion of E. ortleppi (G5) loads in cattle may be increasing overtime. Copyright © 2012 Elsevier B.V. All rights reserved.

Analyzing JAVAD TR-G2 GPS Receiver's Sensitivities to SLS Trajectory

NASA Technical Reports Server (NTRS)

Schuler, Tristan

2017-01-01

Automated guidance and navigation systems are an integral part to successful space missions. Previous researchers created Python tools to receive and parse data from a JAVAD TR-G2 space-capable GPS receiver. I improved the tool by customizing the output for plotting and comparing several simulations. I analyzed position errors, data loss, and signal loss by comparing simulated receiver data from an IFEN GPS simulator to ‘truth data’ from a proposed trajectory. By adjusting the trajectory simulation’s gain, attitude, and start time, NASA can assess the best time to launch the SLS, where to position the antennas on the Block 1-B, and which filter to use. Some additional testing has begun with the Novatel SpaceQuestGPS receiver as well as a GNSS SDR receiver.

Prediction of CpG-island function: CpG clustering vs. sliding-window methods

PubMed Central

2010-01-01

Background Unmethylated stretches of CpG dinucleotides (CpG islands) are an outstanding property of mammal genomes. Conventionally, these regions are detected by sliding window approaches using %G + C, CpG observed/expected ratio and length thresholds as main parameters. Recently, clustering methods directly detect clusters of CpG dinucleotides as a statistical property of the genome sequence. Results We compare sliding-window to clustering (i.e. CpGcluster) predictions by applying new ways to detect putative functionality of CpG islands. Analyzing the co-localization with several genomic regions as a function of window size vs. statistical significance (p-value), CpGcluster shows a higher overlap with promoter regions and highly conserved elements, at the same time showing less overlap with Alu retrotransposons. The major difference in the prediction was found for short islands (CpG islets), often exclusively predicted by CpGcluster. Many of these islets seem to be functional, as they are unmethylated, highly conserved and/or located within the promoter region. Finally, we show that window-based islands can spuriously overlap several, differentially regulated promoters as well as different methylation domains, which might indicate a wrong merge of several CpG islands into a single, very long island. The shorter CpGcluster islands seem to be much more specific when concerning the overlap with alternative transcription start sites or the detection of homogenous methylation domains. Conclusions The main difference between sliding-window approaches and clustering methods is the length of the predicted islands. Short islands, often differentially methylated, are almost exclusively predicted by CpGcluster. This suggests that CpGcluster may be the algorithm of choice to explore the function of these short, but putatively functional CpG islands. PMID:20500903

PGSB/MIPS Plant Genome Information Resources and Concepts for the Analysis of Complex Grass Genomes.

PubMed

Spannagl, Manuel; Bader, Kai; Pfeifer, Matthias; Nussbaumer, Thomas; Mayer, Klaus F X

2016-01-01

PGSB (Plant Genome and Systems Biology; formerly MIPS-Munich Institute for Protein Sequences) has been involved in developing, implementing and maintaining plant genome databases for more than a decade. Genome databases and analysis resources have focused on individual genomes and aim to provide flexible and maintainable datasets for model plant genomes as a backbone against which experimental data, e.g., from high-throughput functional genomics, can be organized and analyzed. In addition, genomes from both model and crop plants form a scaffold for comparative genomics, assisted by specialized tools such as the CrowsNest viewer to explore conserved gene order (synteny) between related species on macro- and micro-levels.The genomes of many economically important Triticeae plants such as wheat, barley, and rye present a great challenge for sequence assembly and bioinformatic analysis due to their enormous complexity and large genome size. Novel concepts and strategies have been developed to deal with these difficulties and have been applied to the genomes of wheat, barley, rye, and other cereals. This includes the GenomeZipper concept, reference-guided exome assembly, and "chromosome genomics" based on flow cytometry sorted chromosomes.

Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G/sub 0//G/sub 1/

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

Freytag, S.O.

1988-04-01

A broad base of data has implicated a role for the c-myc proto-oncogene in the control of the cell cycle and cell differentiation. To further define the role of myc in these processes, the authors examined the effect of enforced myc expression on several events that are thought to be important steps leading to the terminally differentiated state: (i) the ability to arrest growth in G/sub 0//G/sub 1/, (ii) the ability to replicate the genome upon initiation of the differentiation program, and (iii) the ability to loose responsiveness to mitogens and withdraw from the cell cycle. 3T3-L1 preadipocyte cell linesmore » expressing various levels of myc mRNA were established by transfection with a recombinant myc gene under the transcriptional control of the Rous sarcoma virus (RSV) promoter. Cells that expressed high constitutive levels of pRSV myc mRNA arrested in G/sub 0//G/sub 1/ at densities similar to those of normal cells at confluence. Upon initiation of the differentiation program, such cells traversed the cell cycle with kinetics similar to those of normal cells and subsequently arrested in G/sub 0//G/sub 1/. Thus, enforced expression of myc had no effect on the ability of cells to arrest growth in G/sub 0//G/sub 1/ or to replicate the genome upon initiation of the differentiation program. Cells were then tested for their ability to reenter the cell cycle upon exposure to high concentrations of serum and for their capacity to differentiate. In contrast to normal cells, cells expressing high constitutive levels of myc RNA reentered the cell cycle when challenged with 30% serum and failed to terminally differentiate.« less

53BP1 promotes microhomology-mediated end-joining in G1-phase cells

PubMed Central

Xiong, Xiahui; Du, Zhanwen; Wang, Ying; Feng, Zhihui; Fan, Pan; Yan, Chunhong; Willers, Henning; Zhang, Junran

2015-01-01

Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair. PMID:25586219

The left end of rat L1 (L1Rn, long interspersed repeated) DNA which is a CpG island can function as a promoter.

PubMed Central

Nur, I; Pascale, E; Furano, A V

1988-01-01

Here we report that the 600 bp promoter-like region at the left end of a newly isolated and characterized rat L1 DNA element can activate the prokaryotic chloramphenicol acyltransferase gene in a rat cell line. Activation only occurs when the promoter region is oriented to the transferase gene as it is to the L1 protein encoding sequences and is 75% inhibited by methylation of just 5 of the 22 CpGs present in the promoter. The G + C rich promoter contains enough CpGs to qualify it as a CpG island, but in contrast to other CpG islands, genomic L1 promoters are fully methylated in both somatic cell and sperm DNA as judged by restriction enzyme analysis. Partial demethylation of the genomic promoters by treatment with 5-azacytidine failed to produce discrete L1 transcripts. The relationship of methylation to the evolutionary history and fate of the rat L1 promoter is discussed. Images PMID:2459662

Draft Genome Sequence of Thalassospira xiamenensis Strain MCCC 1A03042T

PubMed Central

Li, Meiqing; Yang, Shuo; Lai, Qiliang

2017-01-01

ABSTRACT Thalassospira xiamenensis strain MCCC 1A03042T was isolated from deep-sea sediment of the Indian Ocean, and it was characterized with heavy-metal arsenic tolerance. Here, we present the draft genome of strain MCCC 1A03042T, which contains 4,786,207 bp with a G+C content of 52.6% and 4,359 protein-coding genes. PMID:28254975

Draft Genome Sequence of Thalassospira xiamenensis Strain MCCC 1A03042T.

PubMed

Li, Meiqing; Yang, Shuo; Lai, Qiliang; Shao, Zongze

2017-03-02

Thalassospira xiamenensis strain MCCC 1A03042 T was isolated from deep-sea sediment of the Indian Ocean, and it was characterized with heavy-metal arsenic tolerance. Here, we present the draft genome of strain MCCC 1A03042 T , which contains 4,786,207 bp with a G+C content of 52.6% and 4,359 protein-coding genes. Copyright © 2017 Li et al.

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

PubMed Central

Barr, Alexis R.; Cooper, Samuel; Heldt, Frank S.; Butera, Francesca; Stoy, Henriette; Mansfeld, Jörg; Novák, Béla; Bakal, Chris

2017-01-01

Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability. PMID:28317845

Do Plants Contain G Protein-Coupled Receptors?1[C][W][OPEN

PubMed Central

Taddese, Bruck; Upton, Graham J.G.; Bailey, Gregory R.; Jordan, Siân R.D.; Abdulla, Nuradin Y.; Reeves, Philip J.; Reynolds, Christopher A.

2014-01-01

Whether G protein-coupled receptors (GPCRs) exist in plants is a fundamental biological question. Interest in deorphanizing new GPCRs arises because of their importance in signaling. Within plants, this is controversial, as genome analysis has identified 56 putative GPCRs, including G protein-coupled receptor1 (GCR1), which is reportedly a remote homolog to class A, B, and E GPCRs. Of these, GCR2 is not a GPCR; more recently, it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix-alignment method, which has been benchmarked against the class A-class B-class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologs to class A, class B, and class F GPCRs and shown that GCR1 is closer to class A and/or class B GPCRs than class A, class B, or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the six GPCR classes. Variability comparisons provide additional evidence that GCR1 homologs have the GPCR fold. From the alignments and a GCR1 comparative model, we have identified motifs that are common to GCR1, class A, B, and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fold. PMID:24246381

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

DOE PAGES

Alsøe, Lene; Sarno, Antonio; Carracedo, Sergio; ...

2017-08-03

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/ - mice. Here, we found that 5-hydroxymethyluracil accumulated in Smug1 -/ - tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1more » -/ - mice did not accumulate uracil in their genome and Ung -/ - mice showed slightly elevated uracil levels. Contrastingly, Ung -/ -Smug1 -/ - mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.« less

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

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

Alsøe, Lene; Sarno, Antonio; Carracedo, Sergio

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/ - mice. Here, we found that 5-hydroxymethyluracil accumulated in Smug1 -/ - tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1more » -/ - mice did not accumulate uracil in their genome and Ung -/ - mice showed slightly elevated uracil levels. Contrastingly, Ung -/ -Smug1 -/ - mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.« less

Genome-wide study of methotrexate clearance replicates SLCO1B1

PubMed Central

Ramsey, Laura B.; Panetta, John C.; Smith, Colton; Yang, Wenjian; Fan, Yiping; Winick, Naomi J.; Martin, Paul L.; Cheng, Cheng; Devidas, Meenakshi; Pui, Ching-Hon; Evans, William E.; Hunger, Stephen P.; Loh, Mignon

2013-01-01

Methotrexate clearance can influence the cure of and toxicity in children with acute lymphoblastic leukemia (ALL). We estimated methotrexate plasma clearance for 1279 patients with ALL treated with methotrexate (24-hour infusion of a 1 g/m2 dose or 4-hour infusion of a 2 g/m2 dose) on the Children's Oncology Group P9904 and P9905 protocols. Methotrexate clearance was lower in older children (P = 7 × 10−7), girls (P = 2.7 × 10−4), and those who received a delayed-intensification phase (P = .0022). A genome-wide analysis showed that methotrexate clearance was associated with polymorphisms in the organic anion transporter gene SLCO1B1 (P = 2.1 × 10−11). This replicates findings using different schedules of high-dose methotrexate in St Jude ALL treatment protocols; a combined meta-analysis yields a P value of 5.7 × 10−19 for the association of methotrexate clearance with SLCO1B1 SNP rs4149056. Validation of this variant with 5 different treatment regimens of methotrexate solidifies the robustness of this pharmacogenomic determinant of methotrexate clearance. This study is registered at http://www.clinicaltrials.gov as NCT00005585 and NCT00005596. PMID:23233662

Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins1[OPEN

PubMed Central

Hackenberg, Dieter; Quatrano, Ralph

2016-01-01

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution. PMID:27550997

On the Selective Packaging of Genomic RNA by HIV-1.

PubMed

Comas-Garcia, Mauricio; Davis, Sean R; Rein, Alan

2016-09-12

Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag-gRNA complex will nucleate particle assembly more efficiently than other Gag-RNA complexes. New data shows that among cellular mRNAs, those with long 3'-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3'-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.

Whole genome sequence analysis of BT-474 using complete Genomics' standard and long fragment read technologies.

PubMed

Ciotlos, Serban; Mao, Qing; Zhang, Rebecca Yu; Li, Zhenyu; Chin, Robert; Gulbahce, Natali; Liu, Sophie Jia; Drmanac, Radoje; Peters, Brock A

2016-01-01

The cell line BT-474 is a popular cell line for studying the biology of cancer and developing novel drugs. However, there is no complete, published genome sequence for this highly utilized scientific resource. In this study we sought to provide a comprehensive and useful data set for the scientific community by generating a whole genome sequence for BT-474. Five μg of genomic DNA, isolated from an early passage of the BT-474 cell line, was used to generate a whole genome sequence (114X coverage) using Complete Genomics' standard sequencing process. To provide additional variant phasing and structural variation data we also processed and analyzed two separate libraries of 5 and 6 individual cells to depths of 99X and 87X, respectively, using Complete Genomics' Long Fragment Read (LFR) technology. BT-474 is a highly aneuploid cell line with an extremely complex genome sequence. This ~300X total coverage genome sequence provides a more complete understanding of this highly utilized cell line at the genomic level.

Lack of a p21waf1/cip -dependent G1/S checkpoint in neural stem and progenitor cells after DNA damage in vivo.

PubMed

Roque, Telma; Haton, Céline; Etienne, Olivier; Chicheportiche, Alexandra; Rousseau, Laure; Martin, Ludovic; Mouthon, Marc-André; Boussin, François D

2012-03-01

The cyclin-dependent kinase inhibitor p21(waf1/cip) mediates the p53-dependent G1/S checkpoint, which is generally considered to be a critical requirement to maintain genomic stability after DNA damage. We used staggered 5-ethynyl-2'deoxyuridine/5-bromo-2'-deoxyuridine double-labeling in vivo to investigate the cell cycle progression and the role of p21(waf1/cip) in the DNA damage response of neural stem and progenitor cells (NSPCs) after exposure of the developing mouse cortex to ionizing radiation. We observed a radiation-induced p21-dependent apoptotic response in migrating postmitotic cortical cells. However, neural stem and progenitor cells (NSPCs) did not initiate a p21(waf1/cip1) -dependent G1/S block and continued to enter S-phase at a similar rate to the non-irradiated controls. The G1/S checkpoint is not involved in the mechanisms underlying the faithful transmission of the NSPC genome and/or the elimination of critically damaged cells. These processes typically involve intra-S and G2/M checkpoints that are rapidly activated after irradiation. p21 is normally repressed in neural cells during brain development except at the G1 to G0 transition. Lack of activation of a G1/S checkpoint and apoptosis of postmitotic migrating cells after DNA damage appear to depend on the expression of p21 in neural cells, since substantial cell-to-cell variations are found in the irradiated cortex. This suggests that repression of p21 during brain development prevents the induction of the G1/S checkpoint after DNA damage. Copyright © 2011 AlphaMed Press.

Revealing the Bacterial Butyrate Synthesis Pathways by Analyzing (Meta)genomic Data

PubMed Central

Vital, Marius; Howe, Adina Chuang

2014-01-01

ABSTRACT Butyrate-producing bacteria have recently gained attention, since they are important for a healthy colon and when altered contribute to emerging diseases, such as ulcerative colitis and type II diabetes. This guild is polyphyletic and cannot be accurately detected by 16S rRNA gene sequencing. Consequently, approaches targeting the terminal genes of the main butyrate-producing pathway have been developed. However, since additional pathways exist and alternative, newly recognized enzymes catalyzing the terminal reaction have been described, previous investigations are often incomplete. We undertook a broad analysis of butyrate-producing pathways and individual genes by screening 3,184 sequenced bacterial genomes from the Integrated Microbial Genome database. Genomes of 225 bacteria with a potential to produce butyrate were identified, including many previously unknown candidates. The majority of candidates belong to distinct families within the Firmicutes, but members of nine other phyla, especially from Actinobacteria, Bacteroidetes, Fusobacteria, Proteobacteria, Spirochaetes, and Thermotogae, were also identified as potential butyrate producers. The established gene catalogue (3,055 entries) was used to screen for butyrate synthesis pathways in 15 metagenomes derived from stool samples of healthy individuals provided by the HMP (Human Microbiome Project) consortium. A high percentage of total genomes exhibited a butyrate-producing pathway (mean, 19.1%; range, 3.2% to 39.4%), where the acetyl-coenzyme A (CoA) pathway was the most prevalent (mean, 79.7% of all pathways), followed by the lysine pathway (mean, 11.2%). Diversity analysis for the acetyl-CoA pathway showed that the same few firmicute groups associated with several Lachnospiraceae and Ruminococcaceae were dominating in most individuals, whereas the other pathways were associated primarily with Bacteroidetes. PMID:24757212

Permanent draft genome sequence of Comamonas testosteroni KF-1

PubMed Central

Weiss, Michael; Kesberg, Anna I.; LaButti, Kurt M.; Pitluck, Sam; Bruce, David; Hauser, Loren; Copeland, Alex; Woyke, Tanja; Lowry, Stephen; Lucas, Susan; Land, Miriam; Goodwin, Lynne; Kjelleberg, Staffan; Cook, Alasdair M.; Buhmann, Matthias; Thomas, Torsten; Schleheck, David

2013-01-01

Comamonas testosteroni KF-1 is a model organism for the elucidation of the novel biochemical degradation pathways for xenobiotic 4-sulfophenylcarboxylates (SPC) formed during biodegradation of synthetic 4-sulfophenylalkane surfactants (linear alkylbenzenesulfonates, LAS) by bacterial communities. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,026,527 bp long chromosome (one sequencing gap) exhibits an average G+C content of 61.79% and is predicted to encode 5,492 protein-coding genes and 114 RNA genes. PMID:23991256

Inference of gorilla demographic and selective history from whole-genome sequence data.

PubMed

McManus, Kimberly F; Kelley, Joanna L; Song, Shiya; Veeramah, Krishna R; Woerner, August E; Stevison, Laurie S; Ryder, Oliver A; Ape Genome Project, Great; Kidd, Jeffrey M; Wall, Jeffrey D; Bustamante, Carlos D; Hammer, Michael F

2015-03-01

Although population-level genomic sequence data have been gathered extensively for humans, similar data from our closest living relatives are just beginning to emerge. Examination of genomic variation within great apes offers many opportunities to increase our understanding of the forces that have differentially shaped the evolutionary history of hominid taxa. Here, we expand upon the work of the Great Ape Genome Project by analyzing medium to high coverage whole-genome sequences from 14 western lowland gorillas (Gorilla gorilla gorilla), 2 eastern lowland gorillas (G. beringei graueri), and a single Cross River individual (G. gorilla diehli). We infer that the ancestors of western and eastern lowland gorillas diverged from a common ancestor approximately 261 ka, and that the ancestors of the Cross River population diverged from the western lowland gorilla lineage approximately 68 ka. Using a diffusion approximation approach to model the genome-wide site frequency spectrum, we infer a history of western lowland gorillas that includes an ancestral population expansion of 1.4-fold around 970 ka and a recent 5.6-fold contraction in population size 23 ka. The latter may correspond to a major reduction in African equatorial forests around the Last Glacial Maximum. We also analyze patterns of variation among western lowland gorillas to identify several genomic regions with strong signatures of recent selective sweeps. We find that processes related to taste, pancreatic and saliva secretion, sodium ion transmembrane transport, and cardiac muscle function are overrepresented in genomic regions predicted to have experienced recent positive selection. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Polymorphisms of endothelin 1 (G5665T and T-1370G) and endothelin receptor type A (C+70G and G-231A) in Graves' disease.

PubMed

Aydın, A Fatih; Develi-İş, Seval; Doğru-Abbasoğlu, Semra; Vural, Pervin; Ozderya, Ayşenur; Karadağ, Berrin; Uysal, Müjdat

2014-01-01

Endothelin 1 (EDN1) is a strong angiogenic and mitogenic factor, playing a key role in hypervascularization, thyroid follicle cell hyperplasia, and lymphocyte infiltration in the thyroid gland of patients with Graves' disease (GD). EDN1 induces angiogenesis and mitogenesis via endothelin receptor type A (EDNRA). This study examined the possible association of EDN1 (G5665T and T-1370G) and EDNRA (C+70G and G-231A) single nucleotide polymorphisms (SNPs) with the occurrence of GD, and evaluates the relationship between genotypes and clinical/laboratory manifestations of GD. We analyzed genotype and allele distributions of EDN1 and EDNRA polymorphisms in 165 patients with GD and 181 healthy controls by real-time PCR combined with melting curve analysis. No significant associations between GD and variant alleles of the studied polymorphisms were observed. However, the anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-TG) levels in EDN1 G5665T GG genotype were higher than those in T allele carriers (GT+TT) (p=0.001 and p=0.026, respectively). In addition, anti-TPO levels in EDN1 T-1370G wild-type homozygous patients were found to be higher than in mutant gene carrying patients (GT+GG) (p=0.006). The presence of EDNRA+70G allele was associated with 3.37-fold increased risk for development of ophthalmopathy in GD patients (p=0.009). Although there were no associations between EDN1 (G5665T and T-1370G) and EDNRA (C+70G and G-231A) SNPs and susceptibility to GD, EDN1 G5665T and T-1370G polymorphisms were related to alterations of autoantibody production and EDNRA C+70G polymorphism is related with increased risk for ophthalmopathy in GD patients. Copyright © 2013 Elsevier B.V. All rights reserved.

Draft Genome Sequence of Methylomicrobium buryatense Strain 5G, a Haloalkaline-Tolerant Methanotrophic Bacterium

PubMed Central

Khmelenina, Valentina N.; Beck, David A. C.; Munk, Christine; Davenport, Karen; Daligault, Hajnalka; Erkkila, Tracy; Goodwin, Lynne; Gu, Wei; Lo, Chien-Chi; Scholz, Matthew; Teshima, Hazuki; Xu, Yan; Chain, Patrick; Bringel, Francoise; Vuilleumier, Stéphane; DiSpirito, Alan; Dunfield, Peter; Jetten, Mike S. M.; Klotz, Martin G.; Knief, Claudia; Murrell, J. Colin; Op den Camp, Huub J. M.; Sakai, Yasuyoshi; Semrau, Jeremy; Svenning, Mette; Stein, Lisa Y.; Trotsenko, Yuri A.

2013-01-01

Robust growth of the gammaproteobacterium Methylomicrobium buryatense strain 5G on methane makes it an attractive system for CH4-based biocatalysis. Here we present a draft genome sequence of the strain that will provide a valuable framework for metabolic engineering of the core pathways for the production of valuable chemicals from methane. PMID:23814105

Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil.

PubMed

Melo, Ricardo Rodrigues de; Persinoti, Gabriela Felix; Paixão, Douglas Antonio Alvaredo; Squina, Fábio Márcio; Ruller, Roberto; Sato, Helia Harumi

Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296bp and G+C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Non-Canonical G-quadruplexes cause the hCEB1 minisatellite instability in Saccharomyces cerevisiae

PubMed Central

Piazza, Aurèle; Cui, Xiaojie; Adrian, Michael; Samazan, Frédéric; Heddi, Brahim; Phan, Anh-Tuan; Nicolas, Alain G

2017-01-01

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids in vitro, but the sequence and structural features dictating their formation and function in vivo remains uncertain. Here we report a structure-function analysis of the complex hCEB1 G4-forming sequence. We isolated four G4 conformations in vitro, all of which bear unusual structural features: Form 1 bears a V-shaped loop and a snapback guanine; Form 2 contains a terminal G-triad; Form 3 bears a zero-nucleotide loop; and Form 4 is a zero-nucleotide loop monomer or an interlocked dimer. In vivo, Form 1 and Form 2 differently account for 2/3rd of the genomic instability of hCEB1 in two G4-stabilizing conditions. Form 3 and an unidentified form contribute to the remaining instability, while Form 4 has no detectable effect. This work underscores the structural polymorphisms originated from a single highly G-rich sequence and demonstrates the existence of non-canonical G4s in cells, thus broadening the definition of G4-forming sequences. DOI: http://dx.doi.org/10.7554/eLife.26884.001 PMID:28661396

Bluejay 1.0: genome browsing and comparison with rich customization provision and dynamic resource linking

PubMed Central

Soh, Jung; Gordon, Paul MK; Taschuk, Morgan L; Dong, Anguo; Ah-Seng, Andrew C; Turinsky, Andrei L; Sensen, Christoph W

2008-01-01

Background The Bluejay genome browser has been developed over several years to address the challenges posed by the ever increasing number of data types as well as the increasing volume of data in genome research. Beginning with a browser capable of rendering views of XML-based genomic information and providing scalable vector graphics output, we have now completed version 1.0 of the system with many additional features. Our development efforts were guided by our observation that biologists who use both gene expression profiling and comparative genomics gain functional insights above and beyond those provided by traditional per-gene analyses. Results Bluejay 1.0 is a genome viewer integrating genome annotation with: (i) gene expression information; and (ii) comparative analysis with an unlimited number of other genomes in the same view. This allows the biologist to see a gene not just in the context of its genome, but also its regulation and its evolution. Bluejay now has rich provision for personalization by users: (i) numerous display customization features; (ii) the availability of waypoints for marking multiple points of interest on a genome and subsequently utilizing them; and (iii) the ability to take user relevance feedback of annotated genes or textual items to offer personalized recommendations. Bluejay 1.0 also embeds the Seahawk browser for the Moby protocol, enabling users to seamlessly invoke hundreds of Web Services on genomic data of interest without any hard-coding. Conclusion Bluejay offers a unique set of customizable genome-browsing features, with the goal of allowing biologists to quickly focus on, analyze, compare, and retrieve related information on the parts of the genomic data they are most interested in. We expect these capabilities of Bluejay to benefit the many biologists who want to answer complex questions using the information available from completely sequenced genomes. PMID:18940007

Clinical Significance of MLH1 Methylation and CpG Island Methylator Phenotype as Prognostic Markers in Patients with Gastric Cancer

PubMed Central

Shigeyasu, Kunitoshi; Nagasaka, Takeshi; Mori, Yoshiko; Yokomichi, Naosuke; Kawai, Takashi; Fuji, Tomokazu; Kimura, Keisuke; Umeda, Yuzo; Kagawa, Shunsuke; Goel, Ajay; Fujiwara, Toshiyoshi

2015-01-01

Background To improve the outcome of patients suffering from gastric cancer, a better understanding of underlying genetic and epigenetic events in this malignancy is required. Although CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) have been shown to play pivotal roles in gastric cancer pathogenesis, the clinical significance of these events on survival outcomes in patients with gastric cancer remains unknown. Methods This study included a patient cohort with pathologically confirmed gastric cancer who had surgical resections. A cohort of 68 gastric cancers was analyzed. CIMP and MSI statuses were determined by analyzing promoter CpG island methylation status of 28 genes/loci, and genomic instability at 10 microsatellite markers, respectively. A Cox’s proportional hazards model was performed for multivariate analysis including age, stage, tumor differentiation, KRAS mutation status, and combined CIMP/MLH1 methylation status in relation to overall survival (OS). Results By multivariate analysis, longer OS was significantly correlated with lower pathologic stage (P = 0.0088), better tumor differentiation (P = 0.0267) and CIMP-high and MLH1 3' methylated status (P = 0.0312). Stratification of CIMP status with regards to MLH1 methylation status further enabled prediction of gastric cancer prognosis. Conclusions CIMP and/or MLH1 methylation status may have a potential to be prognostic biomarkers for patients with gastric cancer. PMID:26121593

Proteus genomic island 1 (PGI1), a new resistance genomic island from two Proteus mirabilis French clinical isolates.

PubMed

Siebor, Eliane; Neuwirth, Catherine

2014-12-01

To analyse the genetic environment of the antibiotic resistance genes in two clinical Proteus mirabilis isolates resistant to multiple antibiotics. PCR, gene walking and whole-genome sequencing were used to determine the sequence of the resistance regions, the surrounding genetic structure and the flanking chromosomal regions. A genomic island of 81.1 kb named Proteus genomic island 1 (PGI1) located at the 3'-end of trmE (formerly known as thdF) was characterized. The large MDR region of PGI1 (55.4 kb) included a class 1 integron (aadB and aadA2) and regions deriving from several transposons: Tn2 (blaTEM-135), Tn21, Tn6020-like transposon (aphA1b), a hybrid Tn502/Tn5053 transposon, Tn501, a hybrid Tn1696/Tn1721 transposon [tetA(A)] carrying a class 1 integron (aadA1) and Tn5393 (strA and strB). Several ISs were also present (IS4321, IS1R and IS26). The PGI1 backbone (25.7 kb) was identical to that identified in Salmonella Heidelberg SL476 and shared some identity with the Salmonella genomic island 1 (SGI1) backbone. An IS26-mediated recombination event caused the division of the MDR region into two parts separated by a large chromosomal DNA fragment of 197 kb, the right end of PGI1 and this chromosomal sequence being in inverse orientation. PGI1 is a new resistance genomic island from P. mirabilis belonging to the same island family as SGI1. The role of PGI1 in the spread of antimicrobial resistance genes among Enterobacteriaceae of medical importance needs to be evaluated. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

HAL: a hierarchical format for storing and analyzing multiple genome alignments.

PubMed

Hickey, Glenn; Paten, Benedict; Earl, Dent; Zerbino, Daniel; Haussler, David

2013-05-15

Large multiple genome alignments and inferred ancestral genomes are ideal resources for comparative studies of molecular evolution, and advances in sequencing and computing technology are making them increasingly obtainable. These structures can provide a rich understanding of the genetic relationships between all subsets of species they contain. Current formats for storing genomic alignments, such as XMFA and MAF, are all indexed or ordered using a single reference genome, however, which limits the information that can be queried with respect to other species and clades. This loss of information grows with the number of species under comparison, as well as their phylogenetic distance. We present HAL, a compressed, graph-based hierarchical alignment format for storing multiple genome alignments and ancestral reconstructions. HAL graphs are indexed on all genomes they contain. Furthermore, they are organized phylogenetically, which allows for modular and parallel access to arbitrary subclades without fragmentation because of rearrangements that have occurred in other lineages. HAL graphs can be created or read with a comprehensive C++ API. A set of tools is also provided to perform basic operations, such as importing and exporting data, identifying mutations and coordinate mapping (liftover). All documentation and source code for the HAL API and tools are freely available at http://github.com/glennhickey/hal. hickey@soe.ucsc.edu or haussler@soe.ucsc.edu Supplementary data are available at Bioinformatics online.

The evolution of microbial species - a view through the genomic lens

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

Varghese, Neha; Mukherjee, Supratim; ivanova, Natalia

2014-03-17

For a long time prokaryotic species definition has been under debate and a constant source of turmoil in microbiology. This has recently prompted the ASM to call for a scalable and reproducible technique, which uses meaningful commonalities to cluster microorganisms into groups corresponding to prokaryotic species. Whole-genome Average Nucleotide Identity (gANI) was previously suggested as a measure of genetic distance that generally agrees with prokaryotic species assignments based on the accepted best practices (DNA-DNA hybridization and 16S rDNA similarity). In this work, we prove that gANI is indeed the meaningful commonality based on which microorganisms can be grouped into themore » aforementioned clusters. By analyzing 1.76 million pairs of genomes we find that identification of the closest relatives of an organism via gANI is precise, scalable, reproducible, and reflects the evolutionary dynamics of microbes. We model the previously unexplored statistical properties of gANI using 6,000 microbial genomes and apply species-specific gANI cutoffs to reveal anomalies in the current taxonomic species definitions for almost 50percent of the species with multiple genome sequences. We also provide evidence of speciation events and genetic continuums in 17.8percent of those species. We consider disagreements between gANI-based groupings and named species and demonstrate that the former have all the desired features to serve as the much-needed natural groups for moving forward with taxonomy. Further, the groupings identified are presented in detail at http://ani.jgi-psf.org to facilitate comprehensive downstream analysis for researchers across different disciplines« less

Draft Genome Sequence of Lutibaculum baratangense Strain AMV1T, Isolated from a Mud Volcano in Andamans, India.

PubMed

Singh, Aditya; Sreenivas, Ara; Sathyanarayana Reddy, Gundlapally; Pinnaka, Anil Kumar; Shivaji, Sisinthy

2014-07-24

The 4.3-Mb genome of Lutibaculum baratangense strain AMV1(T), isolated from a soil sample collected from a mud volcano in Andamans, India, is reported. The draft genome of strain Lutibaculum baratangense AMV1(T) consists of 4,300,776 bp with a G+C content of 66.93 mol% and 4,198 predicted coding regions, including 56 RNAs. Copyright © 2014 Singh et al.

Chrysler improved numerical differencing analyzer for third generation computers CINDA-3G

NASA Technical Reports Server (NTRS)

Gaski, J. D.; Lewis, D. R.; Thompson, L. R.

1972-01-01

New and versatile method has been developed to supplement or replace use of original CINDA thermal analyzer program in order to take advantage of improved systems software and machine speeds of third generation computers. CINDA-3G program options offer variety of methods for solution of thermal analog models presented in network format.

PAI-1 promoter 4G/5G polymorphism (rs1799768) contributes to tumor susceptibility: Evidence from meta-analysis.

PubMed

Xu, Xin; Xie, Yanqi; Lin, Yiwei; Xu, Xianglai; Zhu, Yi; Mao, Yeqing; Hu, Zhenghui; Wu, Jian; Chen, Hong; Zheng, Xiangyi; Qin, Jie; Xie, Liping

2012-12-01

Plasminogen activator inhibitor-1 (PAI-1), belonging to the urokinase plasminogen activation (uPA) system, is involved in cancer development and progression. The PAI-1 promoter 4G/5G polymorphism was shown to contribute to genetic susceptibility to cancer, although the results were inconsistent. To assess this relationship more precisely, a meta-analysis was performed. The electronic databases PubMed, Scopus, Web of Science and Chinese National Knowledge Infrastructure (CNKI) were searched; data were extracted and analyzed independently by two reviewers. Ultimately, 21 eligible case-control studies with a total of 8,415 cancer cases and 9,208 controls were included. The overall odds ratio (OR) with its 95% confidence interval (CI) showed a statistically significant association between the PAI-1 promoter 4G/5G polymorphism and cancer risk (4G/4G vs. 5G/5G: OR=1.25, 95% CI=1.07-1.47, P(heterogeneity)=0.001; 4G/4G vs. 4G/5G+5G/5G: OR=1.10, 95% CI=1.03-1.17, P(heterogeneity)=0.194; 4G/4G+4G/5G vs. 5G/5G: OR=1.17, 95% CI=1.01-1.35, P(heterogeneity)=0.041). In further subgroup analyses, the increased risk of cancer was observed in a subgroup of Caucasians with regards to endometrial cancer. Our meta-analysis suggests that the PAI-1 4G/5G polymorphism most likely contributes to susceptibility to cancer, particularly in Caucasians. Furthermore, the 4G allele may be associated with an increased risk of endometrial cancer.

PAI-1 promoter 4G/5G polymorphism (rs1799768) contributes to tumor susceptibility: Evidence from meta-analysis

PubMed Central

XU, XIN; XIE, YANQI; LIN, YIWEI; XU, XIANGLAI; ZHU, YI; MAO, YEQING; HU, ZHENGHUI; WU, JIAN; CHEN, HONG; ZHENG, XIANGYI; QIN, JIE; XIE, LIPING

2012-01-01

Plasminogen activator inhibitor-1 (PAI-1), belonging to the urokinase plasminogen activation (uPA) system, is involved in cancer development and progression. The PAI-1 promoter 4G/5G polymorphism was shown to contribute to genetic susceptibility to cancer, although the results were inconsistent. To assess this relationship more precisely, a meta-analysis was performed. The electronic databases PubMed, Scopus, Web of Science and Chinese National Knowledge Infrastructure (CNKI) were searched; data were extracted and analyzed independently by two reviewers. Ultimately, 21 eligible case-control studies with a total of 8,415 cancer cases and 9,208 controls were included. The overall odds ratio (OR) with its 95% confidence interval (CI) showed a statistically significant association between the PAI-1 promoter 4G/5G polymorphism and cancer risk (4G/4G vs. 5G/5G: OR=1.25, 95% CI=1.07–1.47, Pheterogeneity=0.001; 4G/4G vs. 4G/5G+5G/5G: OR=1.10, 95% CI=1.03–1.17, Pheterogeneity=0.194; 4G/4G+4G/5G vs. 5G/5G: OR=1.17, 95% CI=1.01–1.35, Pheterogeneity=0.041). In further subgroup analyses, the increased risk of cancer was observed in a subgroup of Caucasians with regards to endometrial cancer. Our meta-analysis suggests that the PAI-1 4G/5G polymorphism most likely contributes to susceptibility to cancer, particularly in Caucasians. Furthermore, the 4G allele may be associated with an increased risk of endometrial cancer. PMID:23226787

4G/5G and A-844G Polymorphisms of Plasminogen Activator Inhibitor-1 Associated with Glioblastoma in Iran--a Case-Control Study.

PubMed

Pooyan, Honari; Ahmad, Ebrahimi; Azadeh, Rakhshan

2015-01-01

Glioblastoma is a highly aggressive and malignant brain tumor. Risk factors are largely unknown however, although several biomarkers have been identified which may support development, angiogenesis and invasion of tumor cells. One of these biomarkers is PAI-1. 4G/5G and A-844G are two common polymorphisms in the gene promotor of PAI 1 that may be related to high transcription and expression of this gene. Studies have shown that the prevalence of the 4G and 844G allele is significantly higher in patients with some cancers and genetic disorders. We here assessed the association of 4G/5G and A-844G polymorphisms with glioblastoma cancer risk in Iranians in a case-control study. All 71 patients with clinically confirmed and 140 volunteers with no history and symptoms of glioblastoma as control group were screened for 4G/5G and A-844G polymorphisms of PAI-1, using ARMS-PCR. Genotype and allele frequencies of case and control groups were analyzed using the DeFinetti program. Our results showed significant associations between 4G/5G (p=0.01824) and A-844G (p=0.02012) polymorphisms of the PAI-1 gene with glioblastoma cancer risk in our Iranian population. The results of this study supporting an association of the PAI-1 4G/5G (p=0.01824) and A-844G (p=0.02012) polymorphisms with increasing glioblastoma cancer risk in Iranian patients.

FORESEE: Fully Outsourced secuRe gEnome Study basEd on homomorphic Encryption

PubMed Central

2015-01-01

Background The increasing availability of genome data motivates massive research studies in personalized treatment and precision medicine. Public cloud services provide a flexible way to mitigate the storage and computation burden in conducting genome-wide association studies (GWAS). However, data privacy has been widely concerned when sharing the sensitive information in a cloud environment. Methods We presented a novel framework (FORESEE: Fully Outsourced secuRe gEnome Study basEd on homomorphic Encryption) to fully outsource GWAS (i.e., chi-square statistic computation) using homomorphic encryption. The proposed framework enables secure divisions over encrypted data. We introduced two division protocols (i.e., secure errorless division and secure approximation division) with a trade-off between complexity and accuracy in computing chi-square statistics. Results The proposed framework was evaluated for the task of chi-square statistic computation with two case-control datasets from the 2015 iDASH genome privacy protection challenge. Experimental results show that the performance of FORESEE can be significantly improved through algorithmic optimization and parallel computation. Remarkably, the secure approximation division provides significant performance gain, but without missing any significance SNPs in the chi-square association test using the aforementioned datasets. Conclusions Unlike many existing HME based studies, in which final results need to be computed by the data owner due to the lack of the secure division operation, the proposed FORESEE framework support complete outsourcing to the cloud and output the final encrypted chi-square statistics. PMID:26733391

FORESEE: Fully Outsourced secuRe gEnome Study basEd on homomorphic Encryption.

PubMed

Zhang, Yuchen; Dai, Wenrui; Jiang, Xiaoqian; Xiong, Hongkai; Wang, Shuang

2015-01-01

The increasing availability of genome data motivates massive research studies in personalized treatment and precision medicine. Public cloud services provide a flexible way to mitigate the storage and computation burden in conducting genome-wide association studies (GWAS). However, data privacy has been widely concerned when sharing the sensitive information in a cloud environment. We presented a novel framework (FORESEE: Fully Outsourced secuRe gEnome Study basEd on homomorphic Encryption) to fully outsource GWAS (i.e., chi-square statistic computation) using homomorphic encryption. The proposed framework enables secure divisions over encrypted data. We introduced two division protocols (i.e., secure errorless division and secure approximation division) with a trade-off between complexity and accuracy in computing chi-square statistics. The proposed framework was evaluated for the task of chi-square statistic computation with two case-control datasets from the 2015 iDASH genome privacy protection challenge. Experimental results show that the performance of FORESEE can be significantly improved through algorithmic optimization and parallel computation. Remarkably, the secure approximation division provides significant performance gain, but without missing any significance SNPs in the chi-square association test using the aforementioned datasets. Unlike many existing HME based studies, in which final results need to be computed by the data owner due to the lack of the secure division operation, the proposed FORESEE framework support complete outsourcing to the cloud and output the final encrypted chi-square statistics.

Predicting aberrant CpG island methylation

PubMed Central

Feltus, F. A.; Lee, E. K.; Costello, J. F.; Plass, C.; Vertino, P. M.

2003-01-01

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context. PMID:14519846

Predicting aberrant CpG island methylation.

PubMed

Feltus, F A; Lee, E K; Costello, J F; Plass, C; Vertino, P M

2003-10-14

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context.

BiQ Analyzer HT: locus-specific analysis of DNA methylation by high-throughput bisulfite sequencing

PubMed Central

Lutsik, Pavlo; Feuerbach, Lars; Arand, Julia; Lengauer, Thomas; Walter, Jörn; Bock, Christoph

2011-01-01

Bisulfite sequencing is a widely used method for measuring DNA methylation in eukaryotic genomes. The assay provides single-base pair resolution and, given sufficient sequencing depth, its quantitative accuracy is excellent. High-throughput sequencing of bisulfite-converted DNA can be applied either genome wide or targeted to a defined set of genomic loci (e.g. using locus-specific PCR primers or DNA capture probes). Here, we describe BiQ Analyzer HT (http://biq-analyzer-ht.bioinf.mpi-inf.mpg.de/), a user-friendly software tool that supports locus-specific analysis and visualization of high-throughput bisulfite sequencing data. The software facilitates the shift from time-consuming clonal bisulfite sequencing to the more quantitative and cost-efficient use of high-throughput sequencing for studying locus-specific DNA methylation patterns. In addition, it is useful for locus-specific visualization of genome-wide bisulfite sequencing data. PMID:21565797

Human RTEL1 stabilizes long G-overhangs allowing telomerase-dependent over-extension.

PubMed

Porreca, Rosa M; Glousker, Galina; Awad, Aya; Matilla Fernandez, Maria I; Gibaud, Anne; Naucke, Christian; Cohen, Scott B; Bryan, Tracy M; Tzfati, Yehuda; Draskovic, Irena; Londoño-Vallejo, Arturo

2018-05-18

Telomere maintenance protects the cell against genome instability and senescence. Accelerated telomere attrition is a characteristic of premature aging syndromes including Dyskeratosis congenita (DC). Mutations in hRTEL1 are associated with a severe form of DC called Hoyeraal-Hreidarsson syndrome (HHS). HHS patients carry short telomeres and HHS cells display telomere damage. Here we investigated how hRTEL1 contributes to telomere maintenance in human primary as well as tumor cells. Transient depletion of hRTEL1 resulted in rapid telomere shortening only in the context of telomerase-positive cells with very long telomeres and high levels of telomerase. The effect of hRTEL1 on telomere length is telomerase dependent without impacting telomerase biogenesis or targeting of the enzyme to telomeres. Instead, RTEL1 depletion led to a decrease in both G-overhang content and POT1 association with telomeres with limited telomere uncapping. Strikingly, overexpression of POT1 restored telomere length but not the overhang, demonstrating that G-overhang loss is the primary defect caused by RTEL1 depletion. We propose that hRTEL1 contributes to the maintenance of long telomeres by preserving long G-overhangs, thereby facilitating POT1 binding and elongation by telomerase.

Investigation of mutations in the HBB gene using the 1,000 genomes database.

PubMed

Carlice-Dos-Reis, Tânia; Viana, Jaime; Moreira, Fabiano Cordeiro; Cardoso, Greice de Lemos; Guerreiro, João; Santos, Sidney; Ribeiro-Dos-Santos, Ândrea

2017-01-01

Mutations in the HBB gene are responsible for several serious hemoglobinopathies, such as sickle cell anemia and β-thalassemia. Sickle cell anemia is one of the most common monogenic diseases worldwide. Due to its prevalence, diverse strategies have been developed for a better understanding of its molecular mechanisms. In silico analysis has been increasingly used to investigate the genotype-phenotype relationship of many diseases, and the sequences of healthy individuals deposited in the 1,000 Genomes database appear to be an excellent tool for such analysis. The objective of this study is to analyze the variations in the HBB gene in the 1,000 Genomes database, to describe the mutation frequencies in the different population groups, and to investigate the pattern of pathogenicity. The computational tool SNPEFF was used to align the data from 2,504 samples of the 1,000 Genomes database with the HG19 genome reference. The pathogenicity of each amino acid change was investigated using the databases CLINVAR, dbSNP and HbVar and five different predictors. Twenty different mutations were found in 209 healthy individuals. The African group had the highest number of individuals with mutations, and the European group had the lowest number. Thus, it is concluded that approximately 8.3% of phenotypically healthy individuals from the 1,000 Genomes database have some mutation in the HBB gene. The frequency of mutated genes was estimated at 0.042, so that the expected frequency of being homozygous or compound heterozygous for these variants in the next generation is approximately 0.002. In total, 193 subjects had a non-synonymous mutation, which 186 (7.4%) have a deleterious mutation. Considering that the 1,000 Genomes database is representative of the world's population, it can be estimated that fourteen out of every 10,000 individuals in the world will have a hemoglobinopathy in the next generation.

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells

PubMed Central

Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z.; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung’u, Thumbi; Walker, Bruce D.; Rosenberg, Eric S.; Yu, Xu G.

2017-01-01

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells. PMID:28628034

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

PubMed

Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-06-30

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

Emergence of Double- and Triple-Gene Reassortant G1P[8] Rotaviruses Possessing a DS-1-Like Backbone after Rotavirus Vaccine Introduction in Malawi.

PubMed

Jere, Khuzwayo C; Chaguza, Chrispin; Bar-Zeev, Naor; Lowe, Jenna; Peno, Chikondi; Kumwenda, Benjamin; Nakagomi, Osamu; Tate, Jacqueline E; Parashar, Umesh D; Heyderman, Robert S; French, Neil; Cunliffe, Nigel A; Iturriza-Gomara, Miren

2018-02-01

To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunization programs. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarix) into its immunization schedule in 2012. Utilizing a surveillance platform of hospitalized rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 to 2012) and after (2013 to 2014) vaccine introduction. Analysis of whole genomes obtained through next-generation sequencing revealed that all randomly selected prevaccine G1P[8] strains sequenced ( n = 32) possessed a Wa-like genetic constellation, whereas postvaccine G1P[8] strains ( n = 18) had a DS-1-like constellation. Phylodynamic analyses indicated that postvaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990s and hence were classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981 to 1994; their evolutionary rates ranged from 9.7 × 10 -4 to 4.1 × 10 -3 nucleotide substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation. IMPORTANCE The error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic mutation and

Emergence of Double- and Triple-Gene Reassortant G1P[8] Rotaviruses Possessing a DS-1-Like Backbone after Rotavirus Vaccine Introduction in Malawi

PubMed Central

2017-01-01

ABSTRACT To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunization programs. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarix) into its immunization schedule in 2012. Utilizing a surveillance platform of hospitalized rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 to 2012) and after (2013 to 2014) vaccine introduction. Analysis of whole genomes obtained through next-generation sequencing revealed that all randomly selected prevaccine G1P[8] strains sequenced (n = 32) possessed a Wa-like genetic constellation, whereas postvaccine G1P[8] strains (n = 18) had a DS-1-like constellation. Phylodynamic analyses indicated that postvaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990s and hence were classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981 to 1994; their evolutionary rates ranged from 9.7 × 10−4 to 4.1 × 10−3 nucleotide substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation. IMPORTANCE The error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic

Unanticipated error in HbA(1c) measurement on the HLC-723 G7 analyzer.

PubMed

van den Ouweland, Johannes M W; de Keijzer, Marinus H; van Daal, Henny

2010-04-01

Investigation of falsely elevated HbA(1c) measurements on the HLC-723 G7 analyser. Comparison of HbA(1c) in blood samples that were diluted either in hemolysis reagent or water. HbA(1c) results became falsely elevated when samples were diluted in hemolysis reagent, but not in water. QC-procedures failed to detect this error as calibrator and QC samples were manually diluted in water, according to manufacturer's instructions, whereas patient samples were automatically diluted using hemolysing reagent. After replacement of the instruments' sample-loop and rotor seal comparable HbA(1c) results were obtained, irrespective of dilution with hemolysing reagent or water. This case illustrates the importance of treating calibrator and QC materials similar to routine patient samples in order to prevent unnoticed drift in patient HbA(1c) results. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Ensembl Plants: Integrating Tools for Visualizing, Mining, and Analyzing Plant Genomics Data.

PubMed

Bolser, Dan; Staines, Daniel M; Pritchard, Emily; Kersey, Paul

2016-01-01

Ensembl Plants ( http://plants.ensembl.org ) is an integrative resource presenting genome-scale information for a growing number of sequenced plant species (currently 33). Data provided includes genome sequence, gene models, functional annotation, and polymorphic loci. Various additional information are provided for variation data, including population structure, individual genotypes, linkage, and phenotype data. In each release, comparative analyses are performed on whole genome and protein sequences, and genome alignments and gene trees are made available that show the implied evolutionary history of each gene family. Access to the data is provided through a genome browser incorporating many specialist interfaces for different data types, and through a variety of additional methods for programmatic access and data mining. These access routes are consistent with those offered through the Ensembl interface for the genomes of non-plant species, including those of plant pathogens, pests, and pollinators.Ensembl Plants is updated 4-5 times a year and is developed in collaboration with our international partners in the Gramene ( http://www.gramene.org ) and transPLANT projects ( http://www.transplantdb.org ).

Whole genome sequencing of a rare rotavirus from archived stool sample demonstrates independent zoonotic origin of human G8P[14] strains in Hungary.

PubMed

Marton, Szilvia; Dóró, Renáta; Fehér, Enikő; Forró, Barbara; Ihász, Katalin; Varga-Kugler, Renáta; Farkas, Szilvia L; Bányai, Krisztián

2017-01-02

Genotype P[14] rotaviruses in humans are thought to be zoonotic strains originating from bovine or ovine host species. Over the past 30 years only few genotype P[14] strains were identified in Hungary totaling<0.1% of all human rotaviruses whose genotype had been determined. In this study we report the genome sequence and phylogenetic analysis of a human genotype G8P[14] strain, RVA/Human-wt/HUN/182-02/2001/G8P[14]. The whole genome constellation (G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3) of this strain was shared with another Hungarian zoonotic G8P[14] strain, RVA/Human-wt/HUN/BP1062/2004/G8P[14], although phylogenetic analyses revealed the two rotaviruses likely had different progenitors. Overall, our findings indicate that human G8P[14] rotavirus detected in Hungary in the past originated from independent zoonotic events. Further studies are needed to assess the public health risk associated with infections by various animal rotavirus strains. Copyright © 2016. Published by Elsevier B.V.

G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

PubMed Central

Zhang, Bo; Wu, Wen-Qiang; Liu, Na-Nv; Duan, Xiao-Lei; Li, Ming; Dou, Shuo-Xing; Hou, Xi-Miao; Xi, Xu-Guang

2016-01-01

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells. PMID:27471032

An improved high-quality draft genome sequence of Carnobacterium inhibens subsp. inhibens strain K1 T

DOE PAGES

Nicholson, Wayne L.; Davis, Christina L.; Shapiro, Nicole; ...

2016-09-08

Despite their ubiquity and their involvement in food spoilage, the genus Carnobacterium remains rather sparsely characterized at the genome level. Carnobacterium inhibens K1 T is a member of the Carnobacteriaceae family within the class Bacilli. This strain is a Gram-positive, rod-shaped bacterium isolated from the intestine of an Atlantic salmon. The present study determined the genome sequence and annotation of Carnobacterium inhibens K1 T. The genome comprised 2,748,608 bp with a G+C content of 34.85 %, which included 2621 protein-coding genes and 116 RNA genes. The strain contained five contigs corresponding to presumptive plasmids of sizes: 19,036; 24,250; 26,581; 65,272;more » and 65,904 bp.« less

An improved high-quality draft genome sequence of Carnobacterium inhibens subsp. inhibens strain K1 T

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

Nicholson, Wayne L.; Davis, Christina L.; Shapiro, Nicole

Despite their ubiquity and their involvement in food spoilage, the genus Carnobacterium remains rather sparsely characterized at the genome level. Carnobacterium inhibens K1 T is a member of the Carnobacteriaceae family within the class Bacilli. This strain is a Gram-positive, rod-shaped bacterium isolated from the intestine of an Atlantic salmon. The present study determined the genome sequence and annotation of Carnobacterium inhibens K1 T. The genome comprised 2,748,608 bp with a G+C content of 34.85 %, which included 2621 protein-coding genes and 116 RNA genes. The strain contained five contigs corresponding to presumptive plasmids of sizes: 19,036; 24,250; 26,581; 65,272;more » and 65,904 bp.« less

Comparative analysis of the Rotarix™ vaccine strain and G1P[8] rotaviruses detected before and after vaccine introduction in Belgium.

PubMed

Zeller, Mark; Heylen, Elisabeth; Tamim, Sana; McAllen, John K; Kirkness, Ewen F; Akopov, Asmik; De Coster, Sarah; Van Ranst, Marc; Matthijnssens, Jelle

2017-01-01

G1P[8] rotaviruses are responsible for the majority of human rotavirus infections worldwide. The effect of universal mass vaccination with rotavirus vaccines on circulating G1P[8] rotaviruses is still poorly understood. Therefore we analyzed the complete genomes of the Rotarix™ vaccine strain, and 70 G1P[8] rotaviruses, detected between 1999 and 2010 in Belgium (36 before and 34 after vaccine introduction) to investigate the impact of rotavirus vaccine introduction on circulating G1P[8] strains. All rotaviruses possessed a complete Wa-like genotype constellation, but frequent intra-genogroup reassortments were observed as well as multiple different cluster constellations circulating in a single season. In addition, identical cluster constellations were found to circulate persistently over multiple seasons. The Rotarix™ vaccine strain possessed a unique cluster constellation that was not present in currently circulating G1P[8] strains. At the nucleotide level, the VP6, VP2 and NSP2 gene segments of Rotarix™ were relatively distantly related to any Belgian G1P[8] strain, but other gene segments of Rotarix™ were found in clusters also containing circulating Belgian strains. At the amino acid level, the genetic distance between Rotarix™ and circulating Belgian strains was considerably lower, except for NSP1. When we compared the Belgian G1P[8] strains collected before and after vaccine introduction a reduction in the proportion of strains that were found in the same cluster as the Rotarix™ vaccine strain was observed for most gene segments. The reduction in the proportion of strains belonging to the same cluster may be the result of the vaccine introduction, although natural fluctuations cannot be ruled out.

Concise classification of the genomic porcine endogenous retroviral gamma1 load to defined lineages.

PubMed

Klymiuk, Nikolai; Wolf, Eckhard; Aigner, Bernhard

2008-02-05

We investigated the infection history of porcine endogenous retroviruses (PERV) gamma1 by analyzing published env and LTR sequences. PERV sequences from various breeds, porcine cell lines and infected human primary cells were included in the study. We identified a considerable number of retroviral lineages indicating multiple independent colonization events of the porcine genome. A recent boost of the proviral load in an isolated pig herd and exclusive occurrence of distinct lineages in single studies indicated the ongoing colonization of the porcine genome with endogenous retroviruses. Retroviral recombination between co-packaged genomes was a general factor for PERV gamma1 diversity which indicated the simultaneous expression of different proviral loci over a period of time. In total, our detailed description of endogenous retroviral lineages is the prerequisite for breeding approaches to minimize the infectious potential of porcine tissues for the subsequent use in xenotransplantation.

Genome-wide divergence, haplotype distribution and population demographic histories for Gossypium hirsutum and Gossypium barbadense as revealed by genome-anchored SNPs

PubMed Central

Reddy, Umesh K.; Nimmakayala, Padma; Abburi, Venkata Lakshmi; Reddy, C. V. C. M.; Saminathan, Thangasamy; Percy, Richard G.; Yu, John Z.; Frelichowski, James; Udall, Joshua A.; Page, Justin T.; Zhang, Dong; Shehzad, Tariq; Paterson, Andrew H.

2017-01-01

Use of 10,129 singleton SNPs of known genomic location in tetraploid cotton provided unique opportunities to characterize genome-wide diversity among 440 Gossypium hirsutum and 219 G. barbadense cultivars and landrace accessions of widespread origin. Using the SNPs distributed genome-wide, we examined genetic diversity, haplotype distribution and linkage disequilibrium patterns in the G. hirsutum and G. barbadense genomes to clarify population demographic history. Diversity and identity-by-state analyses have revealed little sharing of alleles between the two cultivated allotetraploid genomes, with a few exceptions that indicated sporadic gene flow. We found a high number of new alleles, representing increased nucleotide diversity, on chromosomes 1 and 2 in cultivated G. hirsutum as compared with low nucleotide diversity on these chromosomes in landrace G. hirsutum. In contrast, G. barbadense chromosomes showed negative Tajima’s D on several chromosomes for both cultivated and landrace types, which indicate that speciation of G. barbadense itself, might have occurred with relatively narrow genetic diversity. The presence of conserved linkage disequilibrium (LD) blocks and haplotypes between G. hirsutum and G. barbadense provides strong evidence for comparable patterns of evolution in their domestication processes. Our study illustrates the potential use of population genetic techniques to identify genomic regions for domestication. PMID:28128280

Selection, periodicity and potential function for Highly Iterative Palindrome-1 (HIP1) in cyanobacterial genomes.

PubMed

Xu, Minli; Lawrence, Jeffrey G; Durand, Dannie

2018-03-16

Highly Iterated Palindrome 1 (HIP1, GCGATCGC) is hyper-abundant in most cyanobacterial genomes. In some cyanobacteria, average HIP1 abundance exceeds one motif per gene. Such high abundance suggests a significant role in cyanobacterial biology. However, 20 years of study have not revealed whether HIP1 has a function, much less what that function might be. We show that HIP1 is 15- to 300-fold over-represented in genomes analyzed. More importantly, HIP1 sites are conserved both within and between open reading frames, suggesting that their overabundance is maintained by selection rather than by continual replenishment by neutral processes, such as biased DNA repair. This evidence for selection suggests a functional role for HIP1. No evidence was found to support a functional role as a peptide or RNA motif or a role in the regulation of gene expression. Rather, we demonstrate that the distribution of HIP1 along cyanobacterial chromosomes is significantly periodic, with periods ranging from 10 to 90 kb, consistent in scale with periodicities reported for co-regulated, co-expressed and evolutionarily correlated genes. The periodicity we observe is also comparable in scale to chromosomal interaction domains previously described in other bacteria. In this context, our findings imply HIP1 functions associated with chromosome and nucleoid structure.

The Plasminogen Activator Inhibitor 1 4G/5G Polymorphism and the Risk of Alzheimer's Disease.

PubMed

Fekih-Mrissa, Najiba; Mansour, Malek; Sayeh, Aicha; Bedoui, Ines; Mrad, Meriem; Riahi, Anis; Mrissa, Ridha; Nsiri, Brahim

2017-09-01

The aim of this study was to determine whether plasminogen activator inhibitor 1 (PAI-1) is associated with the risk of Alzheimer's disease (AD) in Tunisian patients. We analyzed the genotype and allele frequency distribution of the PAI-1 polymorphism in 60 Tunisian patients with AD and 120 healthy controls. The results show a significantly increased risk of AD in carriers of the 4G/4G and 4G/5G genotypes versus the wild-type 5G/5G genotype (4G/4G: 28.33% in patients vs 10.0% in controls; P < 10 -3 ; OR = 8.78; 4G/5G: 55.0% in patients vs 38.33% in controls; OR = 4.45; P < 10 -3 ). The 4G allele was also more frequently found in patients compared with controls; P < 10 -3 ; OR = 3.07. For all participants and by gender, homozygotic carriers (4G/4G) were at an increased risk of AD over heterozygotes and women were at an increased risk over their male genotype counterparts. The odds ratio for AD among 4G/4G carriers for any group was approximately twice that of heterozygotes in the same group. Women homozygotes ranked highest for AD risk (OR = 20.8) and, in fact, women heterozygotes (OR = 9.03) ranked higher for risk than male homozygotes (OR = 6.12). These preliminary exploratory results should be confirmed in a larger study.

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

PubMed

Bridger, J M; Kill, I R; Lichter, P

1998-01-01

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

Comparative Genomic Analyses of the Human NPHP1 Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates

PubMed Central

Yuan, Bo; Liu, Pengfei; Gupta, Aditya; Beck, Christine R.; Tejomurtula, Anusha; Campbell, Ian M.; Gambin, Tomasz; Simmons, Alexandra D.; Withers, Marjorie A.; Harris, R. Alan; Rogers, Jeffrey; Schwartz, David C.; Lupski, James R.

2015-01-01

Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100) is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs) are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases—about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR) between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV) haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual’s susceptibility to acquiring disease-associated alleles. PMID:26641089

Plasminogen activator inhibitor-1 5G/5G genotype is a protecting factor preventing posttransplant diabetes mellitus.

PubMed

Chang, Horng-Rong; Yang, Shun-Fa; Tsai, Jen-Pi; Hsieh, Ming-Chia; Wu, Sheng-Wen; Tsai, Hui-Ching; Hung, Tung-Wei; Huang, Jun-Huang; Lian, Jong-Da

2011-01-30

Plasminogen activator inhibitor 1 (PAI-1) is thought to play a role in the pathogenesis of obesity and insulin resistance. A connection between gestational diabetes mellitus and the functional -675 PAI-1 genotype has been reported. Therefore, we examined the role of the PAI-1 gene polymorphism in kidney transplant recipients. A total of 376 kidney transplant recipients were prospectively screened for posttransplant diabetes mellitus (PTDM). Eighty-one (21.5%) patients were diagnosed with PTDM and the other 295 patients were non-diabetic following kidney transplantation. DNA samples were isolated from the sera and analyzed for the functional -675 4G/5G promoter polymorphisms of the PAI-1 gene. Kidney transplant recipients with PTDM were significantly associated with tacrolimus use (p=0.03), older age (p=0.036), and higher body mass index (p=0.001). The genotype distribution was significantly different between the patients with PTDM (genotype 4G/4G:4G/5G:5G/5G=33.3%:60.5%:6.2%) and those without PTDM (genotype 4G/4G:4G/5G:5G/5G=36.9%:44.1%:19.0%) (p=0.018). Patients with homozygosity for 5G had a significantly lower rate of PTDM (aOR, 0.286, p=0.022) and higher cumulative event-free probability of time to PTDM (log rank test, p=0.0058). Homozygosity for the 5G allele of the PAI-1 gene constitutes a protecting factor for the development of PTDM. Our findings are similar to a previous study on gestational diabetes mellitus, and strongly support a possible genetic role of PAI-1 in the development of PTDM. Copyright © 2010 Elsevier B.V. All rights reserved.

Novel Gbeta Mimic Kelch Proteins (Gpb1 and Gpb2 Connect G-Protein Signaling to Ras via Yeast Neurofibromin Homologs Ira1 and Ira2: A Model for Human NF1

DTIC Science & Technology

2008-03-01

tractable fungal model system, Cryptococcus neoformans, and identified two kelch repeat homologs that are involved in mating (Kem1 and Kem2). To...find kelch-repeat proteins involved in G protein signaling, Cryptococcus homologues of Gpb1/2, which interacts with and negatively regulates the G...protein alpha subunit, Gpa2, in S. cerevisiae, were searched by BLAST (tblastn) in Cryptococcus genome database of serotype A (Duke University Medical

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.

Draft Genome Sequence of Oil-Degrading Bacterium Gallaecimonas pentaromativorans Strain YA_1 from the Southwest Indian Ocean

PubMed Central

Xu, Yiyuan; Ren, Chong; Chen, Ruixuan

2016-01-01

Gallaecimonas pentaromativorans has been previously reported to be capable of degrading crude oil and diesel oil. G. pentaromativorans strain YA_1 was isolated from the southwest Indian Ocean and can degrade crude oil. This study reports the draft genome sequence of G. pentaromativorans, which can provide insights into the mechanisms of microbial oil biodegradation. PMID:27491993

Project 1: Microbial Genomes: A Genomic Approach to Understanding the Evolution of Virulence. Project 2: From Genomes to Life: Drosophilia Development in Space and Time

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

Robert DeSalle

2004-09-10

This project seeks to use the genomes of two close relatives, A. actinomycetemcomitans and H. aphrophilus, to understand the evolutionary changes that take place in a genome to make it more or less virulent. Our primary specific aim of this project was to sequence, annotate, and analyze the genomes of Actinobacillus actinomycetemcomitans (CU1000, serotype f) and Haemophilus aphrophilus. With these genome sequences we have then compared the whole genome sequences to each other and to the current Aa (HK1651 www.genome.ou.edu) genome project sequence along with other fully sequenced Pasteurellaceae to determine inter and intra species differences that may account formore » the differences and similarities in disease. We also propose to create and curate a comprehensive database where sequence information and analysis for the Pasteurellaceae (family that includes the genera Actinobacillus and Haemophilus) are readily accessible. And finally we have proposed to develop phylogenetic techniques that can be used to efficiently and accurately examine the evolution of genomes. Below we report on progress we have made on these major specific aims. Progress on the specific aims is reported below under two major headings--experimental approaches and bioinformatics and systematic biology approaches.« less

Comparative Chloroplast Genomics of Gossypium Species: Insights Into Repeat Sequence Variations and Phylogeny

PubMed Central

Wu, Ying; Liu, Fang; Yang, Dai-Gang; Li, Wei; Zhou, Xiao-Jian; Pei, Xiao-Yu; Liu, Yan-Gai; He, Kun-Lun; Zhang, Wen-Sheng; Ren, Zhong-Ying; Zhou, Ke-Hai; Ma, Xiong-Feng; Li, Zhong-Hu

2018-01-01

Cotton is one of the most economically important fiber crop plants worldwide. The genus Gossypium contains a single allotetraploid group (AD) and eight diploid genome groups (A–G and K). However, the evolution of repeat sequences in the chloroplast genomes and the phylogenetic relationships of Gossypium species are unclear. Thus, we determined the variations in the repeat sequences and the evolutionary relationships of 40 cotton chloroplast genomes, which represented the most diverse in the genus, including five newly sequenced diploid species, i.e., G. nandewarense (C1-n), G. armourianum (D2-1), G. lobatum (D7), G. trilobum (D8), and G. schwendimanii (D11), and an important semi-wild race of upland cotton, G. hirsutum race latifolium (AD1). The genome structure, gene order, and GC content of cotton species were similar to those of other higher plant plastid genomes. In total, 2860 long sequence repeats (>10 bp in length) were identified, where the F-genome species had the largest number of repeats (G. longicalyx F1: 108) and E-genome species had the lowest (G. stocksii E1: 53). Large-scale repeat sequences possibly enrich the genetic information and maintain genome stability in cotton species. We also identified 10 divergence hotspot regions, i.e., rpl33-rps18, psbZ-trnG (GCC), rps4-trnT (UGU), trnL (UAG)-rpl32, trnE (UUC)-trnT (GGU), atpE, ndhI, rps2, ycf1, and ndhF, which could be useful molecular genetic markers for future population genetics and phylogenetic studies. Site-specific selection analysis showed that some of the coding sites of 10 chloroplast genes (atpB, atpE, rps2, rps3, petB, petD, ccsA, cemA, ycf1, and rbcL) were under protein sequence evolution. Phylogenetic analysis based on the whole plastomes suggested that the Gossypium species grouped into six previously identified genetic clades. Interestingly, all 13 D-genome species clustered into a strong monophyletic clade. Unexpectedly, the cotton species with C, G, and K-genomes were admixed and

Human RTEL1 stabilizes long G-overhangs allowing telomerase-dependent over-extension

PubMed Central

Porreca, Rosa M; Glousker, Galina; Awad, Aya; Matilla Fernandez, Maria I; Gibaud, Anne; Naucke, Christian; Cohen, Scott B; Bryan, Tracy M; Tzfati, Yehuda; Draskovic, Irena; Londoño-Vallejo, Arturo

2018-01-01

Abstract Telomere maintenance protects the cell against genome instability and senescence. Accelerated telomere attrition is a characteristic of premature aging syndromes including Dyskeratosis congenita (DC). Mutations in hRTEL1 are associated with a severe form of DC called Hoyeraal-Hreidarsson syndrome (HHS). HHS patients carry short telomeres and HHS cells display telomere damage. Here we investigated how hRTEL1 contributes to telomere maintenance in human primary as well as tumor cells. Transient depletion of hRTEL1 resulted in rapid telomere shortening only in the context of telomerase-positive cells with very long telomeres and high levels of telomerase. The effect of hRTEL1 on telomere length is telomerase dependent without impacting telomerase biogenesis or targeting of the enzyme to telomeres. Instead, RTEL1 depletion led to a decrease in both G-overhang content and POT1 association with telomeres with limited telomere uncapping. Strikingly, overexpression of POT1 restored telomere length but not the overhang, demonstrating that G-overhang loss is the primary defect caused by RTEL1 depletion. We propose that hRTEL1 contributes to the maintenance of long telomeres by preserving long G-overhangs, thereby facilitating POT1 binding and elongation by telomerase. PMID:29522136

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

PubMed

Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M; Michor, Franziska; Fan, Rong; Pan, Xinghua

2017-06-02

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

PubMed Central

Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L.; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M.

2017-01-01

Abstract Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. PMID:28126923

Ensembl Plants: Integrating Tools for Visualizing, Mining, and Analyzing Plant Genomic Data.

PubMed

Bolser, Dan M; Staines, Daniel M; Perry, Emily; Kersey, Paul J

2017-01-01

Ensembl Plants ( http://plants.ensembl.org ) is an integrative resource presenting genome-scale information for 39 sequenced plant species. Available data includes genome sequence, gene models, functional annotation, and polymorphic loci; for the latter, additional information including population structure, individual genotypes, linkage, and phenotype data is available for some species. Comparative data is also available, including genomic alignments and "gene trees," which show the inferred evolutionary history of each gene family represented in the resource. Access to the data is provided through a genome browser, which incorporates many specialist interfaces for different data types, through a variety of programmatic interfaces, and via a specialist data mining tool supporting rapid filtering and retrieval of bulk data. Genomic data from many non-plant species, including those of plant pathogens, pests, and pollinators, is also available via the same interfaces through other divisions of Ensembl.Ensembl Plants is updated 4-6 times a year and is developed in collaboration with our international partners in the Gramene ( http://www.gramene.org ) and transPLANT projects ( http://www.transplantdb.eu ).

Quadruplexes in 'Dicty': crystal structure of a four-quartet G-quadruplex formed by G-rich motif found in the Dictyostelium discoideum genome.

PubMed

Guédin, Aurore; Lin, Linda Yingqi; Armane, Samir; Lacroix, Laurent; Mergny, Jean-Louis; Thore, Stéphane; Yatsunyk, Liliya A

2018-06-01

Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249-1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson-Crick base pair and a A-T-A triad and displays high thermal stability (Tm > 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools.

Impacts, Effectiveness and Regional Inequalities of the GeoMIP G1 to G4 Solar Radiation Management Scenarios

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

Yu, Xiaoyong; Moore, John; Cui, Xuefeng

We evaluate the regional effectiveness of solar radiation management (SRM) to compensate for simultaneous changes in temperature and precipitation induced by increased greenhouse gas concentrations. We analyze results from multiple earth system models under four Geoengineering Model Intercomparison Project(GeoMIP) experiments with a modified form of the Residual Climate Response approach. Under the solar dimming geoengineering experiments G1(4xCO2) and G2(increasing CO2 by 1% per year), global average temperature is successfully restored to pre-industrial level over 50 years simulations. However, these two SRM experiments also produce a robust global precipitation decrease. The stratospheric aerosol GeoMIP geoengineering experiment, G4 has significantly greater regionalmore » inequality and lower effectiveness for compensating temperature change than G1 and G2. G4 also has significantly larger regional inequality for compensating precipitation change than G1and G2. However, there is no significant difference between precipitation change compensation effectiveness of G4 and G2, though there is much larger across model variability in G4 results. G3 has significant greater regional inequality for compensating temperature change than G1 and G2, and has significant lower effectiveness than G1. The effectiveness of four SRMs to compensate for temperature change is much higher than for precipitation. The large cross-model variation in adjustment percentage of compensated SAT and precipitation change by SRM to achieve optimal compensation effectiveness shed a light on the uncertainty accumulation effect in optimizing compensation effectiveness of SRM.« less

Search for the genome of bovine herpesvirus types 1, 4 and 5 in bovine semen

PubMed Central

Morán, P.E.; Favier, P.A.; Lomónaco, M.; Catena, M.C.; Chiapparrone, M.L.; Odeón, A.C.; Verna, A.E.; Pérez, S.E.

2013-01-01

Bovine herpesvirus type 1 (BoHV-1) causes respiratory and reproductive disorders in cattle. Recently, bovine herpesvirus type 5 (BoHV-5) and bovine herpesvirus type 4 (BoHV-4) have been identified to be associated with genital disease. In this study, the presence of the genome of BoHV-1, BoHV-4 and BoHV-5 in bovine semen of Argentinean and international origin was analyzed by PCR assays. The most important finding of this study is the detection of the genome of BoHV-1 and BoHV-4 in semen of bulls maintained at artificial insemination centers. It is particularly relevant that BoHV-1 DNA was also identified in one sample of international origin suggesting the need for extensive quality control measures on international transport of bovine semen. PMID:26623325

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method.

PubMed

Guo, Bingfu; Guo, Yong; Hong, Huilong; Qiu, Li-Juan

2016-01-01

Molecular characterization of sequence flanking exogenous fragment insertion is essential for safety assessment and labeling of genetically modified organism (GMO). In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS) method. More than 22.4 Gb sequence data (∼21 × coverage) for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767-50543792 and Chr17: 7980527-7980541 in these two transgenic lines. Identification of genomic insertion sites of G2-EPSPS and GAT transgenes will facilitate the utilization of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS was a cost-effective and rapid method for identifying sites of T-DNA insertions and flanking sequences in soybean.

Analyzing Somatic Genome Rearrangements in Human Cancers by Using Whole-Exome Sequencing

PubMed Central

Yang, Lixing; Lee, Mi-Sook; Lu, Hengyu; Oh, Doo-Yi; Kim, Yeon Jeong; Park, Donghyun; Park, Gahee; Ren, Xiaojia; Bristow, Christopher A.; Haseley, Psalm S.; Lee, Soohyun; Pantazi, Angeliki; Kucherlapati, Raju; Park, Woong-Yang; Scott, Kenneth L.; Choi, Yoon-La; Park, Peter J.

2016-01-01

Although exome sequencing data are generated primarily to detect single-nucleotide variants and indels, they can also be used to identify a subset of genomic rearrangements whose breakpoints are located in or near exons. Using >4,600 tumor and normal pairs across 15 cancer types, we identified over 9,000 high confidence somatic rearrangements, including a large number of gene fusions. We find that the 5′ fusion partners of functional fusions are often housekeeping genes, whereas the 3′ fusion partners are enriched in tyrosine kinases. We establish the oncogenic potential of ROR1-DNAJC6 and CEP85L-ROS1 fusions by showing that they can promote cell proliferation in vitro and tumor formation in vivo. Furthermore, we found that ∼4% of the samples have massively rearranged chromosomes, many of which are associated with upregulation of oncogenes such as ERBB2 and TERT. Although the sensitivity of detecting structural alterations from exomes is considerably lower than that from whole genomes, this approach will be fruitful for the multitude of exomes that have been and will be generated, both in cancer and in other diseases. PMID:27153396

Selection, periodicity and potential function for Highly Iterative Palindrome-1 (HIP1) in cyanobacterial genomes

PubMed Central

Xu, Minli; Lawrence, Jeffrey G; Durand, Dannie

2018-01-01

Abstract Highly Iterated Palindrome 1 (HIP1, GCGATCGC) is hyper-abundant in most cyanobacterial genomes. In some cyanobacteria, average HIP1 abundance exceeds one motif per gene. Such high abundance suggests a significant role in cyanobacterial biology. However, 20 years of study have not revealed whether HIP1 has a function, much less what that function might be. We show that HIP1 is 15- to 300-fold over-represented in genomes analyzed. More importantly, HIP1 sites are conserved both within and between open reading frames, suggesting that their overabundance is maintained by selection rather than by continual replenishment by neutral processes, such as biased DNA repair. This evidence for selection suggests a functional role for HIP1. No evidence was found to support a functional role as a peptide or RNA motif or a role in the regulation of gene expression. Rather, we demonstrate that the distribution of HIP1 along cyanobacterial chromosomes is significantly periodic, with periods ranging from 10 to 90 kb, consistent in scale with periodicities reported for co-regulated, co-expressed and evolutionarily correlated genes. The periodicity we observe is also comparable in scale to chromosomal interaction domains previously described in other bacteria. In this context, our findings imply HIP1 functions associated with chromosome and nucleoid structure. PMID:29432573

Whole genome analysis provides evidence for porcine-to-simian interspecies transmission of rotavirus-A.

PubMed

Navarro, Ryan; Aung, Meiji Soe; Cruz, Katalina; Ketzis, Jennifer; Gallagher, Christa Ann; Beierschmitt, Amy; Malik, Yashpal Singh; Kobayashi, Nobumichi; Ghosh, Souvik

2017-04-01

We report here whole genome analysis of a porcine rotavirus-A (RVA) strain RVA/Pig-wt/KNA/ET8B/2015/G5P[13] detected in a diarrheic piglet, and nearly whole genome (except for VP4 gene) analysis of a simian RVA strain RVA/Simian-wt/KNA/08979/2015/G5P[X] detected in a non-diarrheic African green monkey (AGM) on the island of St. Kitts, Caribbean region. Strain ET8B exhibited a G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 genotype constellation that was identical to those of Brazilian porcine RVA G5P[13] strains RVA/Pig-wt/BRA/ROTA01/2013/G5P[13] and RVA/Pig-wt/BRA/ROTA07/2013/G5P[13], the only porcine G5P[13] RVAs that have been analyzed for the whole genome so far. Phylogenetically, all the 11 gene segments of ET8B were closely related to those of porcine and porcine-like human RVAs within the respective genotypes. Although the porcine G5P[13] RVAs exhibited identical genotype constellations, ET8B did not appear to share common evolutionary pathways with the Brazilian porcine G5P[13] RVAs. Interestingly, the VP2, VP3, VP6, VP7, and NSP1-NSP5 genes of simian RVA strain 08979 were closely related to those of porcine and porcine-like human RVA strains, exhibiting 99%-100% nucleotide sequence identities to cognate genes of co-circulating porcine RVA strain ET8B. On the other hand, the VP1 of 08979 appeared to be genetically divergent from porcine and human RVAs within the R1 genotype, and its exact origin could not be ascertained. Taken together, these observations suggested that simian strain 08979 might have been derived from interspecies transmission events involving transmission of ET8B-like RVAs from pigs to AGMs. In St. Kitts, AGMs often stray from the wild into livestock farms. Therefore, it may be possible that the AGM acquired the infection from a pig farm on the island. To our knowledge, this is the first report on detection of porcine-like RVAs in monkeys. Also, the present study is the first to report whole genomic analysis of a porcine RVA strain from the Caribbean

The first genome sequence of a metatherian herpesvirus: Macropodid herpesvirus 1.

PubMed

Vaz, Paola K; Mahony, Timothy J; Hartley, Carol A; Fowler, Elizabeth V; Ficorilli, Nino; Lee, Sang W; Gilkerson, James R; Browning, Glenn F; Devlin, Joanne M

2016-01-22

While many placental herpesvirus genomes have been fully sequenced, the complete genome of a marsupial herpesvirus has not been described. Here we present the first genome sequence of a metatherian herpesvirus, Macropodid herpesvirus 1 (MaHV-1). The MaHV-1 viral genome was sequenced using an Illumina MiSeq sequencer, de novo assembly was performed and the genome was annotated. The MaHV-1 genome was 140 kbp in length and clustered phylogenetically with the primate simplexviruses, sharing 67% nucleotide sequence identity with Human herpesviruses 1 and 2. The MaHV-1 genome contained 66 predicted open reading frames (ORFs) homologous to those in other herpesvirus genomes, but lacked homologues of UL3, UL4, UL56 and glycoprotein J. This is the first alphaherpesvirus genome that has been found to lack the UL3 and UL4 homologues. We identified six novel ORFs and confirmed their transcription by RT-PCR. This is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade. Members of the Simplexvirus genus are remarkably conserved, so the absence of ORFs otherwise retained in eutherian and avian alphaherpesviruses contributes to our understanding of the Alphaherpesvirinae. Further study of metatherian herpesvirus genetics and pathogenesis provides a unique approach to understanding herpesvirus-mammalian interactions.

The analysis of APOL1 genetic variation and haplotype diversity provided by 1000 Genomes project.

PubMed

Peng, Ting; Wang, Li; Li, Guisen

2017-08-11

The APOL1 gene variants has been shown to be associated with an increased risk of multiple kinds of diseases, particularly in African Americans, but not in Caucasians and Asians. In this study, we explored the single nucleotide polymorphism (SNP) and haplotype diversity of APOL1 gene in different races provided by 1000 Genomes project. Variants of APOL1 gene in 1000 Genome Project were obtained and SNPs located in the regulatory region or coding region were selected for genetic variation analysis. Total 2504 individuals from 26 populations were classified as four groups that included Africa, Europe, Asia and Admixed populations. Tag SNPs were selected to evaluate the haplotype diversities in the four populations by HaploStats software. APOL1 gene was surrounded by some of the most polymorphic genes in the human genome, variation of APOL1 gene was common, with up to 613 SNP (1000 Genome Project reported) and 99 of them (16.2%) with MAF ≥ 1%. There were 79 SNPs in the URR and 92 SNPs in 3'UTR. Total 12 SNPs in URR and 24 SNPs in 3'UTR were considered as common variants with MAF ≥ 1%. It is worth noting that URR-1 was presents lower frequencies in European populations, while other three haplotypes taken an opposite pattern; 3'UTR presents several high-frequency variation sites in a short segment, and the differences of its haplotypes among different population were significant (P < 0.01), UTR-1 and UTR-5 presented much higher frequency in African population, while UTR-2, UTR-3 and UTR-4 were much lower. APOL1 coding region showed that two SNP of G1 with higher frequency are actually pull down the haplotype H-1 frequency when considering all populations pooled together, and the diversity among the four populations be widen by the G1 two mutation (P 1  = 3.33E-4 vs P 2  = 3.61E-30). The distributions of APOL1 gene variants and haplotypes were significantly different among the different populations, in either regulatory or coding regions. It could provide

Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.

PubMed

Yohda, Masafumi; Yagi, Osami; Takechi, Ayane; Kitajima, Mizuki; Matsuda, Hisashi; Miyamura, Naoaki; Aizawa, Tomoko; Nakajima, Mutsuyasu; Sunairi, Michio; Daiba, Akito; Miyajima, Takashi; Teruya, Morimi; Teruya, Kuniko; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Juan, Ayaka; Nakano, Kazuma; Aoyama, Misako; Terabayashi, Yasunobu; Satou, Kazuhito; Hirano, Takashi

2015-07-01

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Complete genome sequence of Peptoniphilus sp. strain ING2-D1G isolated from a mesophilic lab-scale completely stirred tank reactor utilizing maize silage in co-digestion with pig and cattle manure for biomethanation.

PubMed

Tomazetto, Geizecler; Hahnke, Sarah; Maus, Irena; Wibberg, Daniel; Pühler, Alfred; Schlüter, Andreas; Klocke, Michael

2014-12-20

The bacterium Peptoniphilus sp. strain ING2-D1G (DSM 28672), a mesophilic and obligate anaerobic bacterium belonging to the order Clostridiales was isolated from a biogas-producing lab-scale completely stirred tank reactor (CSTR) optimized for anaerobic digestion of maize silage in co-fermentation with pig and cattle manure. In this study, the whole genome sequence of Peptoniphilus sp. strain ING2-D1G, a new isolate potentially involved in protein breakdown and acidogenesis during biomass degradation, is reported. The chromosome of this strain is 1.6Mb in size and encodes genes predicted to be involved in the production of acetate, lactate and butyrate specifying the acidogenic metabolism of the isolate. Copyright © 2014 Elsevier B.V. All rights reserved.

PLK1 Activation in Late G2 Sets Up Commitment to Mitosis.

PubMed

Gheghiani, Lilia; Loew, Damarys; Lombard, Bérangère; Mansfeld, Jörg; Gavet, Olivier

2017-06-06

Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Roles of SLX1–SLX4, MUS81–EME1, and GEN1 in avoiding genome instability and mitotic catastrophe

PubMed Central

Sarbajna, Shriparna; Davies, Derek; West, Stephen C.

2014-01-01

The resolution of recombination intermediates containing Holliday junctions (HJs) is critical for genome maintenance and proper chromosome segregation. Three pathways for HJ processing exist in human cells and involve the following enzymes/complexes: BLM–TopoIIIα–RMI1–RMI2 (BTR complex), SLX1–SLX4–MUS81–EME1 (SLX–MUS complex), and GEN1. Cycling cells preferentially use the BTR complex for the removal of double HJs in S phase, with SLX–MUS and GEN1 acting at temporally distinct phases of the cell cycle. Cells lacking SLX–MUS and GEN1 exhibit chromosome missegregation, micronucleus formation, and elevated levels of 53BP1-positive G1 nuclear bodies, suggesting that defects in chromosome segregation lead to the transmission of extensive DNA damage to daughter cells. In addition, however, we found that the effects of SLX4, MUS81, and GEN1 depletion extend beyond mitosis, since genome instability is observed throughout all phases of the cell cycle. This is exemplified in the form of impaired replication fork movement and S-phase progression, endogenous checkpoint activation, chromosome segmentation, and multinucleation. In contrast to SLX4, SLX1, the nuclease subunit of the SLX1–SLX4 structure-selective nuclease, plays no role in the replication-related phenotypes associated with SLX4/MUS81 and GEN1 depletion. These observations demonstrate that the SLX1–SLX4 nuclease and the SLX4 scaffold play divergent roles in the maintenance of genome integrity in human cells. PMID:24831703

PCR Amplification Strategies towards full-length HIV-1 Genome sequencing.

PubMed

Liu, Chao Chun; Ji, Hezhao

2018-06-26

The advent of next generation sequencing has enabled greater resolution of viral diversity and improved feasibility of full viral genome sequencing allowing routine HIV-1 full genome sequencing in both research and diagnostic settings. Regardless of the sequencing platform selected, successful PCR amplification of the HIV-1 genome is essential for sequencing template preparation. As such, full HIV-1 genome amplification is a crucial step in dictating the successful and reliable sequencing downstream. Here we reviewed existing PCR protocols leading to HIV-1 full genome sequencing. In addition to the discussion on basic considerations on relevant PCR design, the advantages as well as the pitfalls of published protocols were reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense

PubMed Central

Zhu, Yingjie; Xu, Jiang; Sun, Chao; Zhou, Shiguo; Xu, Haibin; Nelson, David R.; Qian, Jun; Song, Jingyuan; Luo, Hongmei; Xiang, Li; Li, Ying; Xu, Zhichao; Ji, Aijia; Wang, Lizhi; Lu, Shanfa; Hayward, Alice; Sun, Wei; Li, Xiwen; Schwartz, David C.; Wang, Yitao; Chen, Shilin

2015-01-01

Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms. PMID:26046933

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells

NASA Technical Reports Server (NTRS)

Wang, Xiang; Khadpe, Jay; Hu, Baocheng; Iliakis, George; Wang, Ya

2003-01-01

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.

Full-length and defective enterovirus G genomes with distinct torovirus protease insertions are highly prevalent on a Chinese pig farm.

PubMed

Wang, Yan; Zhang, Wen; Liu, Zhijian; Fu, Xingli; Yuan, Jiaqi; Zhao, Jieji; Lin, Yuan; Shen, Quan; Wang, Xiaochun; Deng, Xutao; Delwart, Eric; Shan, Tongling; Yang, Shixing

2018-05-21

Recombination occurs frequently between enteroviruses (EVs) which are classified within the same species of the Picornaviridae family. Here, using viral metagenomics, the genomes of two recombinant EV-Gs (strains EVG 01/NC_CHI/2014 and EVG 02/NC_CHI/2014) found in the feces of pigs from a swine farm in China are described. The two strains are characterized by distinct insertion of a papain-like protease gene from toroviruses classified within the Coronaviridae family. According to recent reports the site of the torovirus protease insertion was located at the 2C/3A junction region in EVG 02/NC_CHI/2014. For the other variant EVG 01/NC_CHI/2014, the inserted protease sequence replaced the entire viral capsid protein region up to the VP1/2A junction. These two EV-G strains were highly prevalent in the same pig farm with all animals shedding the full-length genome (EVG 02/NC_CHI/2014) while 65% also shed the capsid deletion mutant (EVG 01/NC_CHI/2014). A helper-defective virus relationship between the two co-circulating EV-G recombinants is hypothesized.

Alternative polyadenylation drives genome-to-phenome information detours in the AMPKα1 and AMPKα2 knockout mice.

PubMed

Zhang, Shuwen; Zhang, Yangzi; Zhou, Xiang; Fu, Xing; Michal, Jennifer J; Ji, Guoli; Du, Min; Davis, Jon F; Jiang, Zhihua

2018-04-24

Currently available mouse knockout (KO) lines remain largely uncharacterized for genome-to-phenome (G2P) information flows. Here we test our hypothesis that altered myogenesis seen in AMPKα1- and AMPKα2-KO mice is caused by use of alternative polyadenylation sites (APSs). AMPKα1 and AMPKα2 are two α subunits of adenosine monophosphate-activated protein kinase (AMPK), which serves as a cellular sensor in regulation of many biological events. A total of 56,483 APSs were derived from gastrocnemius muscles. The differentially expressed APSs (DE-APSs) that were down-regulated tended to be distal. The DE-APSs that were related to reduced and increased muscle mass were down-regulated in AMPKα1-KO mice, but up-regulated in AMPKα2-KO mice, respectively. Five genes: Car3 (carbonic anhydrase 3), Mylk4 (myosin light chain kinase family, member 4), Neb (nebulin), Obscn (obscurin) and Pfkm (phosphofructokinase, muscle) utilized different APSs with potentially antagonistic effects on muscle function. Overall, gene knockout triggers genome plasticity via use of APSs, completing the G2P processes. However, gene-based analysis failed to reach such a resolution. Therefore, we propose that alternative transcripts are minimal functional units in genomes and the traditional central dogma concept should be now examined under a systems biology approach.

Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress.

PubMed

Macheret, Morgane; Halazonetis, Thanos D

2018-03-01

Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

Thromboelastography platelet mapping in healthy dogs using 1 analyzer versus 2 analyzers.

PubMed

Blois, Shauna L; Banerjee, Amrita; Wood, R Darren; Park, Fiona M

2013-07-01

The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer's guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy.

Thromboelastography platelet mapping in healthy dogs using 1 analyzer versus 2 analyzers

PubMed Central

Blois, Shauna L.; Banerjee, Amrita; Wood, R. Darren; Park, Fiona M.

2013-01-01

The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer’s guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy. PMID:24101802

Plasminogen activator inhibitor-1 4G/5G polymorphism and retinopathy risk in type 2 diabetes: a meta-analysis.

PubMed

Zhang, Tengyue; Pang, Chong; Li, Ningdong; Zhou, Elaine; Zhao, Kanxing

2013-01-02

Mounting evidence has suggested that plasminogen activator inhibitor-1 (PAI-1) is a candidate for increased risk of diabetic retinopathy. Studies have reported that insertion/deletion polymorphism in the PAI-1 gene may influence the risk of this disease. To comprehensively address this issue, we performed a meta-analysis to evaluate the association of PAI-1 4G/5G polymorphism with diabetic retinopathy in type 2 diabetes. Data were retrieved in a systematic manner and analyzed using Review Manager and STATA Statistical Software. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. Nine studies with 1, 217 cases and 1, 459 controls were included. Allelic and genotypic comparisons between cases and controls were evaluated. Overall analysis suggests a marginal association of the 4G/5G polymorphism with diabetic retinopathy (for 4G versus 5G: OR 1.13, 95%CI 1.01 to 1.26; for 4G/4G versus 5G/5G: OR 1.30, 95%CI 1.04 to 1.64; for 4G/4G versus 5G/5G + 4G/5G: OR 1.26, 95%CI 1.05 to 1.52). In subgroup analysis by ethnicity, we found an association among the Caucasian population (for 4G versus 5G: OR 1.14, 95% CI 1.00 to 1.30; for 4G/4G versus 5G/5G: OR 1.33, 95%CI 1.02 to 1.74; for 4G/4G versus 5G/5G + 4G/5G: OR 1.41, 95%CI 1.13 to 1.77). When stratified by the average duration of diabetes, patients with diabetes histories longer than 10 years have an elevated susceptibility to diabetic retinopathy than those with shorter histories (for 4G/4G versus 5G/5G: OR 1.47, 95%CI 1.08 to 2.00). We also detected a higher risk in hospital-based studies (for 4G/4G versus 5G/5G+4G/5G: OR 1.27, 95%CI 1.02 to 1.57). The present meta-analysis suggested that 4G/5G polymorphism in the PAI-1 gene potentially increased the risk of diabetic retinopathy in type 2 diabetes and showed a discrepancy in different ethnicities. A higher susceptibility in patients with longer duration of diabetes (more than 10 years) indicated a gene

Plasminogen activator inhibitor-1 4G/5G polymorphism and retinopathy risk in type 2 diabetes: a meta-analysis

PubMed Central

2013-01-01

Background Mounting evidence has suggested that plasminogen activator inhibitor-1 (PAI-1) is a candidate for increased risk of diabetic retinopathy. Studies have reported that insertion/deletion polymorphism in the PAI-1 gene may influence the risk of this disease. To comprehensively address this issue, we performed a meta-analysis to evaluate the association of PAI-1 4G/5G polymorphism with diabetic retinopathy in type 2 diabetes. Methods Data were retrieved in a systematic manner and analyzed using Review Manager and STATA Statistical Software. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. Results Nine studies with 1, 217 cases and 1, 459 controls were included. Allelic and genotypic comparisons between cases and controls were evaluated. Overall analysis suggests a marginal association of the 4G/5G polymorphism with diabetic retinopathy (for 4G versus 5G: OR 1.13, 95%CI 1.01 to 1.26; for 4G/4G versus 5G/5G: OR 1.30, 95%CI 1.04 to 1.64; for 4G/4G versus 5G/5G + 4G/5G: OR 1.26, 95%CI 1.05 to 1.52). In subgroup analysis by ethnicity, we found an association among the Caucasian population (for 4G versus 5G: OR 1.14, 95% CI 1.00 to 1.30; for 4G/4G versus 5G/5G: OR 1.33, 95%CI 1.02 to 1.74; for 4G/4G versus 5G/5G + 4G/5G: OR 1.41, 95%CI 1.13 to 1.77). When stratified by the average duration of diabetes, patients with diabetes histories longer than 10 years have an elevated susceptibility to diabetic retinopathy than those with shorter histories (for 4G/4G versus 5G/5G: OR 1.47, 95%CI 1.08 to 2.00). We also detected a higher risk in hospital-based studies (for 4G/4G versus 5G/5G+4G/5G: OR 1.27, 95%CI 1.02 to 1.57). Conclusions The present meta-analysis suggested that 4G/5G polymorphism in the PAI-1 gene potentially increased the risk of diabetic retinopathy in type 2 diabetes and showed a discrepancy in different ethnicities. A higher susceptibility in patients with longer duration of diabetes (more than 10

RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.

PubMed

Vannier, Jean-Baptiste; Pavicic-Kaltenbrunner, Visnja; Petalcorin, Mark I R; Ding, Hao; Boulton, Simon J

2012-05-11

T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(-/-) cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere. Copyright © 2012 Elsevier Inc. All rights reserved.

26 CFR 1.475(g)-1 - Effective dates.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 6 2012-04-01 2012-04-01 false Effective dates. 1.475(g)-1 Section 1.475(g)-1...) INCOME TAXES (CONTINUED) Inventories § 1.475(g)-1 Effective dates. (a)-(b) [Reserved] (c) Section 1.475(a...) applies on and after August 13, 1996 (the effective date of § 1.1275-6). (g) [Reserved] (h) Section 1.475...

26 CFR 1.475(g)-1 - Effective dates.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 6 2014-04-01 2014-04-01 false Effective dates. 1.475(g)-1 Section 1.475(g)-1...) INCOME TAXES (CONTINUED) Inventories § 1.475(g)-1 Effective dates. (a)-(b) [Reserved] (c) Section 1.475(a...) applies on and after August 13, 1996 (the effective date of § 1.1275-6). (g) [Reserved] (h) Section 1.475...

26 CFR 1.475(g)-1 - Effective dates.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 6 2013-04-01 2013-04-01 false Effective dates. 1.475(g)-1 Section 1.475(g)-1...) INCOME TAXES (CONTINUED) Inventories § 1.475(g)-1 Effective dates. (a)-(b) [Reserved] (c) Section 1.475(a...) applies on and after August 13, 1996 (the effective date of § 1.1275-6). (g) [Reserved] (h) Section 1.475...

26 CFR 1.475(g)-1 - Effective dates.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 6 2011-04-01 2011-04-01 false Effective dates. 1.475(g)-1 Section 1.475(g)-1...) INCOME TAXES (CONTINUED) Inventories § 1.475(g)-1 Effective dates. (a)-(b) [Reserved] (c) Section 1.475(a...) applies on and after August 13, 1996 (the effective date of § 1.1275-6). (g) [Reserved] (h) Section 1.475...

Body Weight Gain During a Discrete Nursing Episode in Suckling Rats Reared at 1.5-g or 1.5-g Exceeds that of 1.0-g Controls and is Independent of Material Hypergravity Exposure

NASA Technical Reports Server (NTRS)

Ronca, April E.; Baer, Lisa A.; Plaut, Karen; Wade, Charles E.; Sun, Sid (Technical Monitor)

2001-01-01

We recently reported that body weights of suckling rats reared during 1.5-g centrifugation are approximately 10% lower than those of 1.0-g controls. This finding raises the possibility that hypergravity exposed pups ingest less milk than controls due to either impairments in their ability to acquire milk from the nipple, or to decreased availability or palatability of their mother's milk. In the present study, we analyzed body weight gain in suckling rats reared during a discrete nursing episode following rearing at either 1.75-g, 1.5-g or 1.0-g. On Gestational day (G) 10 of the rats' 22-day pregnancy, time-bred SD rat dams were 1:1 matched based on body weight and assigned to either Hypergravity (HG) or Stationary Yoked Control (SYC) conditions and to either 1.75-g or 1.5-g conditions. Beginning on G11, HG dams and litters were exposed to 26 days of continuous centrifugation with brief daily stops for veterinary inspection and animal maintenance. On the day following birth (Postnatal day), litters were pooled within each condition then randomly re-assigned in equivalent proportions to HG and SYC dams. On P15, HG litters were removed from their mother's and placed in an incubator (33 C). Following a 4hr deprivation period, four neonates were tested from each litter, with two pups placed with either their own dam or the SYC dam; two pups from the yoked mother were paired with the HG pups. Pups were individually weighed, permitted to suckle for 75 min, then re-weighed. At the start of the test, the body weights of HG pups were significantly less than those of SYC pups (p less than 0.05). Relative to SYC pups, BG pups showed significantly greater proportional body weight gain (p less than 0.05), possibly due to augmented post-centrifugation feeding. Pup weight gain was independent of maternal hypergravity exposure. Neither impairments in milk acquisition nor milk availability or palatibility of hypergravity-exposed dams cannot account for reduced body mass of

Molecular characterization of two rare human G8P[14] rotavirus strains, detected in Italy in 2012.

PubMed

Delogu, Roberto; Ianiro, Giovanni; Morea, Anna; Chironna, Maria; Fiore, Lucia; Ruggeri, Franco M

2016-10-01

Since 2007, the Italian Rotavirus Surveillance Program (RotaNet-Italy) has monitored the diversity and distribution of genotypes identified in children hospitalized with rotavirus acute gastroenteritis. We report the genomic characterization of two rare human G8P[14] rotavirus strains, identified in two children hospitalized with acute gastroenteritis in the southern Italian region of Apulia during rotavirus strain surveillance in 2012. Both strains showed a G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genomic constellation (DS-1-like genomic background). Phylogenetic analysis of each genome segment revealed a mixed configuration of genes of animal and zoonotic human origin, indicating that genetic reassortment events generated these unusual human strains. Eight out of 11 genes (VP1, VP2, VP3, VP6, VP7, NSP3, NSP4 and NSP5) of the Italian G8P[14] strains exhibited close identity with a Spanish sheep strain, whereas the remaining genes (VP4, NSP1 and NSP2) were more closely related to human strains. The amino acid sequences of the antigenic regions of outer capsid proteins VP4 and VP7 were compared with vaccine and field strains, showing high conservation between the amino acid sequences of Apulia G8P[14] strains and human and animal strains bearing G8 and/or P[14] proteins, and revealing many substitutions with respect to the RotaTeq™ and Rotarix™ vaccine strains. Conversely, the amino acid analysis of the four antigenic sites of VP6 revealed a high degree of conservation between the two Apulia strains and the human and animal strains analyzed. These results reinforce the potential role of interspecies transmission and reassortment in generating novel rotavirus strains that might not be fully contrasted by current vaccines. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-wide analysis of a Wnt1-regulated transcriptional network implicates neurodegenerative pathways.

PubMed

Wexler, Eric M; Rosen, Ezra; Lu, Daning; Osborn, Gregory E; Martin, Elizabeth; Raybould, Helen; Geschwind, Daniel H

2011-10-04

Wnt proteins are critical to mammalian brain development and function. The canonical Wnt signaling pathway involves the stabilization and nuclear translocation of β-catenin; however, Wnt also signals through alternative, noncanonical pathways. To gain a systems-level, genome-wide view of Wnt signaling, we analyzed Wnt1-stimulated changes in gene expression by transcriptional microarray analysis in cultured human neural progenitor (hNP) cells at multiple time points over a 72-hour time course. We observed a widespread oscillatory-like pattern of changes in gene expression, involving components of both the canonical and the noncanonical Wnt signaling pathways. A higher-order, systems-level analysis that combined independent component analysis, waveform analysis, and mutual information-based network construction revealed effects on pathways related to cell death and neurodegenerative disease. Wnt effectors were tightly clustered with presenilin1 (PSEN1) and granulin (GRN), which cause dominantly inherited forms of Alzheimer's disease and frontotemporal dementia (FTD), respectively. We further explored a potential link between Wnt1 and GRN and found that Wnt1 decreased GRN expression by hNPs. Conversely, GRN knockdown increased WNT1 expression, demonstrating that Wnt and GRN reciprocally regulate each other. Finally, we provided in vivo validation of the in vitro findings by analyzing gene expression data from individuals with FTD. These unbiased and genome-wide analyses provide evidence for a connection between Wnt signaling and the transcriptional regulation of neurodegenerative disease genes.

Insulinotropic and antidiabetic effects of 17β-estradiol and the GPR30 agonist G-1 on human pancreatic islets.

PubMed

Kumar, Rajesh; Balhuizen, Alexander; Amisten, Stefan; Lundquist, Ingmar; Salehi, Albert

2011-07-01

We have recently shown that 17β-estradiol (E2) and the synthetic G protein-coupled receptor 30 (GPR30) ligand G-1 have antiapoptotic actions in mouse pancreatic islets, raising the prospect that they might exert beneficial effects also in human islets. The objective of the present study was to identify the expression of GPR30 in human islets and clarify the role of GPR30 in islet hormone secretion and β-cell survival. GPR30 expression was analyzed by confocal microscopy, Western blot, and quantitative PCR in islets from female and male donors. Hormone secretion, phosphatidylinositol hydrolysis, cAMP content, and caspase-3 activity in female islets were determined with conventional methods and apoptosis with the annexin-V method. Confocal microscopy revealed GPR30 expression in islet insulin, glucagon, and somatostatin cells. GPR30 mRNA and protein expression was markedly higher in female vs. male islets. An amplifying effect of G-1 or E2 on cAMP content and insulin secretion from isolated female islets was not influenced by the E2 genomic receptor (ERα and ERβ) antagonists ICI 182,780 and EM-652. Cytokine-induced (IL-1β plus TNFα plus interferon-γ) apoptosis in islets cultured for 24 h at 5 mmol/liter glucose was almost abolished by G-1 or E2 treatment and was not affected by the nuclear estrogen receptor antagonists. Concentration-response studies on female islets from healthy controls and type 2 diabetic subjects showed that both E2 and G-1 displayed important antidiabetic actions by improving glucose-stimulated insulin release while suppressing glucagon and somatostatin secretion. In view of these findings, we propose that small molecules activating GPR30 could be promising in the therapy of diabetes mellitus.

A New Sythetic Hybrid (A1D5) between Gossypium herbaceum and G. raimondii and Its Morphological, Cytogenetic, Molecular Characterization

PubMed Central

Zhu, Shuijin; Zhang, Lufei; Li, Lingjiao

2017-01-01

The diploid species G. herbaceum (A1) and G. raimondii (D5) are the progenitors of allotetraploid cotton, respectively. However, hybrids between G. herbaceum and G. raimondii haven’t been reported. In the present study, hybridization between G. herbaceum and G. raimondii was explored. Morphological, cytogenetic and molecular analyses were used to assess the hybridity. The interspecific hybrid plants were successfully obtained. Most of the morphological characteristics of the hybrids were intermediate between G. herbaceum and G. raimondii. However, the color of glands, anther cases, pollen and corolla, and the state of bracteoles in hybrids were associated with the G. herbaceum. The color of staminal columns and filaments in hybrids were associated with G. raimondii. Cytogenetic analysis confirmed abnormal meiotic behavior existed in hybrids. The hybrids couldn’t produce boll-set. Simple sequence repeat results found that besides the fragments inherited from the two parents, some novel bands were amplified in hybrids, indicating that potential mutations and chromosomal recombination occurred between parental genomes during hybridization. These results may provide some novel insights in speciation, genome interaction, and evolution of the tetraploid cotton species. PMID:28187145

The temperate marine phage PhiHAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome.

PubMed

Mobberley, Jennifer M; Authement, R Nathan; Segall, Anca M; Paul, John H

2008-07-01

A myovirus-like temperate phage, PhiHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 x 10(10) VLP ml(-1)) than control cultures (3.83 x 10(7) VLP ml(-1)) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The PhiHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10(-3)) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the PhiHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and PhiHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of PhiHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The PhiHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies

PubMed Central

Karamitros, Timokratis; Piorkowska, Renata; Katzourakis, Aris; Magiorkinis, Gkikas; Mbisa, Jean Lutamyo

2016-01-01

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal. PMID:27309375

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies.

PubMed

Karamitros, Timokratis; Harrison, Ian; Piorkowska, Renata; Katzourakis, Aris; Magiorkinis, Gkikas; Mbisa, Jean Lutamyo

2016-01-01

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal.

26 CFR 1.149(g)-1 - Hedge bonds.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 2 2012-04-01 2012-04-01 false Hedge bonds. 1.149(g)-1 Section 1.149(g)-1...) INCOME TAXES (CONTINUED) Tax Exemption Requirements for State and Local Bonds § 1.149(g)-1 Hedge bonds... for purposes of section 149(g) and this section. In addition, the following terms have the following...

26 CFR 1.149(g)-1 - Hedge bonds.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 2 2014-04-01 2014-04-01 false Hedge bonds. 1.149(g)-1 Section 1.149(g)-1...) INCOME TAXES (CONTINUED) Tax Exemption Requirements for State and Local Bonds § 1.149(g)-1 Hedge bonds... for purposes of section 149(g) and this section. In addition, the following terms have the following...

26 CFR 1.149(g)-1 - Hedge bonds.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 2 2011-04-01 2011-04-01 false Hedge bonds. 1.149(g)-1 Section 1.149(g)-1...) INCOME TAXES (CONTINUED) Tax Exemption Requirements for State and Local Bonds § 1.149(g)-1 Hedge bonds... for purposes of section 149(g) and this section. In addition, the following terms have the following...

26 CFR 1.149(g)-1 - Hedge bonds.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 2 2013-04-01 2013-04-01 false Hedge bonds. 1.149(g)-1 Section 1.149(g)-1...) INCOME TAXES (CONTINUED) Tax Exemption Requirements for State and Local Bonds § 1.149(g)-1 Hedge bonds... for purposes of section 149(g) and this section. In addition, the following terms have the following...

26 CFR 1.149(g)-1 - Hedge bonds.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 2 2010-04-01 2010-04-01 false Hedge bonds. 1.149(g)-1 Section 1.149(g)-1...) INCOME TAXES (CONTINUED) Tax Exemption Requirements for State and Local Bonds § 1.149(g)-1 Hedge bonds... for purposes of section 149(g) and this section. In addition, the following terms have the following...

Genome-wide association study for circulating levels of PAI-1 provides novel insights into its regulation

PubMed Central

Huang, Jie; Sabater-Lleal, Maria; Asselbergs, Folkert W.; Tregouet, David; Shin, So-Youn; Ding, Jingzhong; Baumert, Jens; Oudot-Mellakh, Tiphaine; Folkersen, Lasse; Johnson, Andrew D.; Smith, Nicholas L.; Williams, Scott M.; Ikram, Mohammad A.; Kleber, Marcus E.; Becker, Diane M.; Truong, Vinh; Mychaleckyj, Josyf C.; Tang, Weihong; Yang, Qiong; Sennblad, Bengt; Moore, Jason H.; Williams, Frances M. K.; Dehghan, Abbas; Silbernagel, Günther; Schrijvers, Elisabeth M. C.; Smith, Shelly; Karakas, Mahir; Tofler, Geoffrey H.; Silveira, Angela; Navis, Gerjan J.; Lohman, Kurt; Chen, Ming-Huei; Peters, Annette; Goel, Anuj; Hopewell, Jemma C.; Chambers, John C.; Saleheen, Danish; Lundmark, Per; Psaty, Bruce M.; Strawbridge, Rona J.; Boehm, Bernhard O.; Carter, Angela M.; Meisinger, Christa; Peden, John F.; Bis, Joshua C.; McKnight, Barbara; Öhrvik, John; Taylor, Kent; Franzosi, Maria Grazia; Seedorf, Udo; Collins, Rory; Franco-Cereceda, Anders; Syvänen, Ann-Christine; Goodall, Alison H.; Yanek, Lisa R.; Cushman, Mary; Müller-Nurasyid, Martina; Folsom, Aaron R.; Basu, Saonli; Matijevic, Nena; van Gilst, Wiek H.; Kooner, Jaspal S.; Hofman, Albert; Danesh, John; Clarke, Robert; Meigs, James B.; Kathiresan, Sekar; Reilly, Muredach P.; Klopp, Norman; Harris, Tamara B.; Winkelmann, Bernhard R.; Grant, Peter J.; Hillege, Hans L.; Watkins, Hugh; Spector, Timothy D.; Becker, Lewis C.; Tracy, Russell P.; März, Winfried; Uitterlinden, Andre G.; Eriksson, Per; Cambien, Francois; Morange, Pierre-Emmanuel; Koenig, Wolfgang; Soranzo, Nicole; van der Harst, Pim; Liu, Yongmei

2012-01-01

We conducted a genome-wide association study to identify novel associations between genetic variants and circulating plasminogen activator inhibitor-1 (PAI-1) concentration, and examined functional implications of variants and genes that were discovered. A discovery meta-analysis was performed in 19 599 subjects, followed by replication analysis of genome-wide significant (P < 5 × 10−8) single nucleotide polymorphisms (SNPs) in 10 796 independent samples. We further examined associations with type 2 diabetes and coronary artery disease, assessed the functional significance of the SNPs for gene expression in human tissues, and conducted RNA-silencing experiments for one novel association. We confirmed the association of the 4G/5G proxy SNP rs2227631 in the promoter region of SERPINE1 (7q22.1) and discovered genome-wide significant associations at 3 additional loci: chromosome 7q22.1 close to SERPINE1 (rs6976053, discovery P = 3.4 × 10−10); chromosome 11p15.2 within ARNTL (rs6486122, discovery P = 3.0 × 10−8); and chromosome 3p25.2 within PPARG (rs11128603, discovery P = 2.9 × 10−8). Replication was achieved for the 7q22.1 and 11p15.2 loci. There was nominal association with type 2 diabetes and coronary artery disease at ARNTL (P < .05). Functional studies identified MUC3 as a candidate gene for the second association signal on 7q22.1. In summary, SNPs in SERPINE1 and ARNTL and an SNP associated with the expression of MUC3 were robustly associated with circulating levels of PAI-1. PMID:22990020

Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System.

PubMed

Wang, Shaohua; Dong, Sheng; Wang, Pixiang; Tao, Yong; Wang, Yi

2017-05-15

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk , two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations. IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent

Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome.

PubMed

Halmillawewa, Anupama P; Restrepo-Córdoba, Marcela; Perry, Benjamin J; Yost, Christopher K; Hynes, Michael F

2016-02-01

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available.

Plasminogen activator inhibitor type 1 serum levels and 4G/5G gene polymorphism in morbidly obese Hispanic patients with non-alcoholic fatty liver disease.

PubMed

Espino, Alberto; Villagrán, Andrea; Vollrath, Valeska; Hanckes, Paulina; Salas, Roberto; Farah, Andrea; Solís, Nancy; Pizarro, Margarita; Escalona, Alex; Boza, Camilo; Pérez, Gustavo; Carrasco, Gonzalo; Padilla, Oslando; Miquel, Juan Francisco; Nervi, Flavio; Chavez-Tapia, Norberto C; Arab, Juan Pablo; Alvarez-Lobos, Manuel; Arrese, Marco; Riquelme, Arnoldo

2011-01-01

The plasminogen activator inhibitor type-1 (PAI-1) has been implicated in the regulation of fibrinolysis and extracellular matrix components. The single base pair guanine insertion/deletion polymorphism (4G/5G) within the promoter region of the PAI-1 gene influences PAI-1 synthesis and may modulate hepatic fibrogenesis. To evaluate the influence of PAI-1 serum levels and 4G/5G polymorphism on the risk of liver fibrosis associated to non-alcoholic fatty liver disease (NAFLD) in morbidly obese patients. Case-control study of 50 obese patients undergoing bariatric surgery and 71 non-obese subjects matched by age and sex. Anthropometric and biochemical measurements were performed, including PAI-1 serum levels. Genomic DNA was obtained to assess the presence of 4G/5G polymorphism. BMI, insulinemia, triglycerides, HOMA-IR, hypertension and diabetes were significantly higher in obese patients compared to control subjects. PAI-1 serum levels observed in obese patients were significantly lower (10.63 ± 4.82) compared to controls (14.26 ± 11.4; p < 0.05). No differences were observed in the PAI-1 4G/5G promoter genotypes frequencies (p = 0.12). No differences were observed in PAI-1 plasma levels among obese patients with liver fibrosis (10.64 ± 4.35) compared to patients without liver fibrosis (10.61 ± 5.2; p = 0.985). PAI-1 4G/5G promoter genotypes frequencies were similar in patients with or without liver fibrosis associated to NASH (p = 0.6). Morbidly obese patients had significantly lower PAI-1 serum levels with similar PAI-1 4G/5G genotypes frequencies compared to non-obese subjects. The frequency of 4G/5G genotypes in Chilean Hispanic healthy subjects was similar to that described in other populations. No association was found between PAI-1 serum levels or 4G/5G genotype with liver fibrosis in obese patients.

Genome analysis of Diploscapter coronatus: insights into molecular peculiarities of a nematode with parthenogenetic reproduction.

PubMed

Hiraki, Hideaki; Kagoshima, Hiroshi; Kraus, Christopher; Schiffer, Philipp H; Ueta, Yumiko; Kroiher, Michael; Schierenberg, Einhard; Kohara, Yuji

2017-06-24

Sexual reproduction involving the fusion of egg and sperm is prevailing among eukaryotes. In contrast, the nematode Diploscapter coronatus, a close relative of the model Caenorhabditis elegans, reproduces parthenogenetically. Neither males nor sperm have been observed and some steps of meiosis are apparently skipped in this species. To uncover the genomic changes associated with the evolution of parthenogenesis in this nematode, we carried out a genome analysis. We obtained a 170 Mbp draft genome in only 511 scaffolds with a N 50 length of 1 Mbp. Nearly 90% of these scaffolds constitute homologous pairs with a 5.7% heterozygosity on average and inversions and translocations, meaning that the 170 Mbp sequences correspond to the diploid genome. Fluorescent staining shows that the D. coronatus genome consists of two chromosomes (2n = 2). In our genome annotation, we found orthologs of 59% of the C. elegans genes. However, a number of genes were missing or very divergent. These include genes involved in sex determination (e.g. xol-1, tra-2) and meiosis (e.g. the kleisins rec-8 and coh-3/4) giving a possible explanation for the absence of males and the second meiotic division. The high degree of heterozygosity allowed us to analyze the expression level of individual alleles. Most of the homologous pairs show very similar expression levels but others exhibit a 2-5-fold difference. Our high-quality draft genome of D. coronatus reveals the peculiarities of the genome of parthenogenesis and provides some clues to the genetic basis for parthenogenetic reproduction. This draft genome should be the basis to elucidate fundamental questions related to parthenogenesis such as its origin and mechanisms through comparative analyses with other nematodes. Furthermore, being the closest outgroup to the genus Caenorhabditis, the draft genome will help to disclose many idiosyncrasies of the model C. elegans and its congeners in future studies.

Population genomics of intrapatient HIV-1 evolution

PubMed Central

Zanini, Fabio; Brodin, Johanna; Thebo, Lina; Lanz, Christa; Bratt, Göran; Albert, Jan; Neher, Richard A

2015-01-01

Many microbial populations rapidly adapt to changing environments with multiple variants competing for survival. To quantify such complex evolutionary dynamics in vivo, time resolved and genome wide data including rare variants are essential. We performed whole-genome deep sequencing of HIV-1 populations in 9 untreated patients, with 6-12 longitudinal samples per patient spanning 5-8 years of infection. The data can be accessed and explored via an interactive web application. We show that patterns of minor diversity are reproducible between patients and mirror global HIV-1 diversity, suggesting a universal landscape of fitness costs that control diversity. Reversions towards the ancestral HIV-1 sequence are observed throughout infection and account for almost one third of all sequence changes. Reversion rates depend strongly on conservation. Frequent recombination limits linkage disequilibrium to about 100bp in most of the genome, but strong hitch-hiking due to short range linkage limits diversity. DOI: http://dx.doi.org/10.7554/eLife.11282.001 PMID:26652000

Impact of the 4G/5G polymorphism in the plasminogen activator inhibitor-1 gene on primary nephrotic syndrome.

PubMed

Luo, Yuezhong; Wang, Chao; Tu, Haitao

2014-03-01

The aim of the present study was to investigate whether the four guanosines (4G)/five guanosines (5G) polymorphism in the gene coding for plasminogen activator inhibitor-1 (PAI-1) affects the clinical features of primary nephrotic syndrome (PNS). A cohort of 200 biopsy-diagnosed PNS patients was studied, with 40 healthy subjects as controls. The PAI-1 gene polymorphism was detected by polymerase chain reaction and DNA sequencing. Associations between the PAI-1 4G/5G polymorphism and clinical features and pathological types of PNS were analyzed. The results indicated that the PAI-1 genotype distribution is significantly different between patients with PNS and healthy controls, with significantly higher numbers of the 4G/4G genotype and lower numbers of the 5G5G genotype detected in PNS patients compared to controls (both P<0.05). The frequency of the 4G allele was also significantly higher in PNS patients compared to healthy controls (P<0.01). Among the different pathological types of PNS, IgA nephropathy (IgAN) and membranous nephropathy (MN) were associated with significantly increased frequencies of the 4G/4G and 4G/5G genotypes, as well as of the 4G allele. The increased 4G frequency was also detected in patients with minimal change disease (MCD). Significantly increased international normalized ratio (INR) and prolonged activated partial thromboplastin time (APTT) were observed in 4G/4G compared to 5G/5G PNS subjects. The response to steroids was not significantly different among the three genotypes. In conclusion, the 4G allele of the PAI-1 gene appears to be associated with PNS, especially in MN and IgAN patients. These findings suggest that specific targeting may be required for the treatment of PNS patients with the 4G/4G genotype.

CpG Distribution and Methylation Pattern in Porcine Parvovirus

PubMed Central

Tóth, Renáta; Mészáros, István; Stefancsik, Rajmund; Bartha, Dániel; Bálint, Ádám; Zádori, Zoltán

2013-01-01

Based on GC content and the observed/expected CpG ratio (oCpGr), we found three major groups among the members of subfamily Parvovirinae: Group I parvoviruses with low GC content and low oCpGr values, Group II with low GC content and high oCpGr values and Group III with high GC content and high oCpGr values. Porcine parvovirus belongs to Group I and it features an ascendant CpG distribution by position in its coding regions similarly to the majority of the parvoviruses. The entire PPV genome remains hypomethylated during the viral lifecycle independently from the tissue of origin. In vitro CpG methylation of the genome has a modest inhibitory effect on PPV replication. The in vitro hypermethylation disappears from the replicating PPV genome suggesting that beside the maintenance DNMT1 the de novo DNMT3a and DNMT3b DNA methyltransferases can’t methylate replicating PPV DNA effectively either, despite that the PPV infection does not seem to influence the expression, translation or localization of the DNA methylases. SNP analysis revealed high mutability of the CpG sites in the PPV genome, while introduction of 29 extra CpG sites into the genome has no significant biological effects on PPV replication in vitro. These experiments raise the possibility that beyond natural selection mutational pressure may also significantly contribute to the low level of the CpG sites in the PPV genome. PMID:24392033

Complete Mitochondrial Genome of the Medicinal Mushroom Ganoderma lucidum

PubMed Central

Chen, Haimei; Chen, Xiangdong; Lan, Jin; Liu, Chang

2013-01-01

Ganoderma lucidum is one of the well-known medicinal basidiomycetes worldwide. The mitochondrion, referred to as the second genome, is an organelle found in most eukaryotic cells and participates in critical cellular functions. Elucidating the structure and function of this genome is important to understand completely the genetic contents of G. lucidum. In this study, we assembled the mitochondrial genome of G. lucidum and analyzed the differential expressions of its encoded genes across three developmental stages. The mitochondrial genome is a typical circular DNA molecule of 60,630 bp with a GC content of 26.67%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNAs, four homing endonucleases, and two hypothetical proteins. Except for genes encoding trnW and two hypothetical proteins, all genes were located on the positive strand. For the repeat structure analysis, eight forward, two inverted, and three tandem repeats were detected. A pair of fragments with a total length around 5.5 kb was found in both the nuclear and mitochondrial genomes, which suggests the possible transfer of DNA sequences between two genomes. RNA-Seq data for samples derived from three stages, namely, mycelia, primordia, and fruiting bodies, were mapped to the mitochondrial genome and qualified. The protein-coding genes were expressed higher in mycelia or primordial stages compared with those in the fruiting bodies. The rRNA abundances were significantly higher in all three stages. Two regions were transcribed but did not contain any identified protein or tRNA genes. Furthermore, three RNA-editing sites were detected. Genome synteny analysis showed that significant genome rearrangements occurred in the mitochondrial genomes. This study provides valuable information on the gene contents of the mitochondrial genome and their differential expressions at various developmental stages of G. lucidum. The results contribute to the understanding of the

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.

Identification of Tf1 integration events in S. pombe under nonselective conditions

PubMed Central

Cherry, Kristina E.; Hearn, Willis E.; Seshie, Osborne Y.; Singleton, Teresa L.; Singleton, Teresa L.

2014-01-01

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the S. pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418S/neo+ Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418S/neo+ clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1’s ability to insert within silent regions of S. pombe’s genome. PMID:24680781

Identification of Tf1 integration events in S. pombe under nonselective conditions.

PubMed

Cherry, Kristina E; Hearn, Willis E; Seshie, Osborne Y K; Singleton, Teresa L

2014-06-01

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning the integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the Schizosaccharomyces pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418(S)/neo(+) Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418(S)/neo(+) clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1's ability to insert within silent regions of S. pombe's genome. Copyright © 2014 Elsevier B.V. All rights reserved.

Draft Genome Sequence of Sphingopyxis sp. Strain MWB1, a Crude-Oil-Degrading Marine Bacterium

PubMed Central

Kim, Jonghyun; Kim, Soo Jung; Kim, Seon Hee; Kim, Seung Il; Moon, Yoon-Jung; Park, Sung-Joon

2014-01-01

Sphingopyxis sp. strain MWB1, which is capable of degrading crude oil, diesel, and kerosene, was isolated from crude oil–contaminated seashore in Tae-an, South Korea. Here, we report the draft genome sequence of this strain, which comprises 3,118,428 bp with a G+C content of 62.85 mol%. PMID:25477411

Methylation-sensitive amplified polymorphism-based genome-wide analysis of cytosine methylation profiles in Nicotiana tabacum cultivars.

PubMed

Jiao, J; Wu, J; Lv, Z; Sun, C; Gao, L; Yan, X; Cui, L; Tang, Z; Yan, B; Jia, Y

2015-11-26

This study aimed to investigate cytosine methylation profiles in different tobacco (Nicotiana tabacum) cultivars grown in China. Methylation-sensitive amplified polymorphism was used to analyze genome-wide global methylation profiles in four tobacco cultivars (Yunyan 85, NC89, K326, and Yunyan 87). Amplicons with methylated C motifs were cloned by reamplified polymerase chain reaction, sequenced, and analyzed. The results show that geographical location had a greater effect on methylation patterns in the tobacco genome than did sampling time. Analysis of the CG dinucleotide distribution in methylation-sensitive polymorphic restriction fragments suggested that a CpG dinucleotide cluster-enriched area is a possible site of cytosine methylation in the tobacco genome. The sequence alignments of the Nia1 gene (that encodes nitrate reductase) in Yunyan 87 in different regions indicate that a C-T transition might be responsible for the tobacco phenotype. T-C nucleotide replacement might also be responsible for the tobacco phenotype and may be influenced by geographical location.

Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag.

PubMed

Grewe, Bastian; Hoffmann, Bianca; Ohs, Inga; Blissenbach, Maik; Brandt, Sabine; Tippler, Bettina; Grunwald, Thomas; Uberla, Klaus

2012-03-01

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

Insights into Conifer Giga-Genomes1

PubMed Central

De La Torre, Amanda R.; Birol, Inanc; Bousquet, Jean; Ingvarsson, Pär K.; Jansson, Stefan; Jones, Steven J.M.; Keeling, Christopher I.; MacKay, John; Nilsson, Ove; Ritland, Kermit; Street, Nathaniel; Yanchuk, Alvin; Zerbe, Philipp; Bohlmann, Jörg

2014-01-01

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world’s forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20–30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes. PMID:25349325

Human T-cell leukemia virus type 1 Tax interacts with Chk1 and attenuates DNA-damage induced G2 arrest mediated by Chk1.

PubMed

Park, Hyeon Ung; Jeong, Jae-Hoon; Chung, Jay H; Brady, John N

2004-06-24

Checkpoint kinase 1 (Chk1) mediates diverse cellular responses to genotoxic stress, regulating the network of genome-surveillance pathways that coordinate cell cycle progression with DNA repair. Chk1 is essential for mammalian development and viability, and has been shown to be important for both S and G(2) checkpoints. We now present evidence that the HTLV-1 Tax protein interacts directly with Chk1 and impairs its kinase activities in vitro and in vivo. The direct and physical interaction of Chk1 and Tax was observed in HTLV-1-infected T cells (C81, HuT 102 and MT-2) and transfected fibroblasts (293 T) by coimmunoprecipitation and by in vitro GST pull-down assays. Interestingly, Tax inhibited the kinase activity of Chk1 protein in in vitro and in vivo kinase assays. Consistent with these results, Tax inhibited the phosphorylation-dependent degradation of Cdc25A and G(2) arrest in response to gamma-irradiation (IR) in a dose-dependent manner in vivo. The G(2) arrest did not require Chk2 or p53. These studies provide the first example of a viral transforming protein targeting Chk1 and provide important insights into checkpoint pathway regulation.

Genomic deletions created upon LINE-1 retrotransposition.

PubMed

Gilbert, Nicolas; Lutz-Prigge, Sheila; Moran, John V

2002-08-09

LINE-1 (L1) retrotransposition continues to impact the human genome, yet little is known about how L1 integrates into DNA. Here, we developed a plasmid-based rescue system and have used it to recover 37 new L1 retrotransposition events from cultured human cells. Sequencing of the insertions revealed the usual L1 structural hallmarks; however, in four instances, retrotransposition generated large target site deletions. Remarkably, three of those resulted in the formation of chimeric L1s, containing the 5' end of an endogenous L1 fused precisely to our engineered L1. Thus, our data demonstrate multiple pathways for L1 integration in cultured cells, and show that L1 is not simply an insertional mutagen, but that its retrotransposition can result in significant deletions of genomic sequence.

The A, C, G, and T of Genome Assembly

PubMed Central

Wajid, Bilal; Sohail, Muhammad U.; Ekti, Ali R.; Serpedin, Erchin

2016-01-01

Genome assembly in its two decades of history has produced significant research, in terms of both biotechnology and computational biology. This contribution delineates sequencing platforms and their characteristics, examines key steps involved in filtering and processing raw data, explains assembly frameworks, and discusses quality statistics for the assessment of the assembled sequence. Furthermore, the paper explores recent Ubuntu-based software environments oriented towards genome assembly as well as some avenues for future research. PMID:27247941

The A, C, G, and T of Genome Assembly.

PubMed

Wajid, Bilal; Sohail, Muhammad U; Ekti, Ali R; Serpedin, Erchin

2016-01-01

Genome assembly in its two decades of history has produced significant research, in terms of both biotechnology and computational biology. This contribution delineates sequencing platforms and their characteristics, examines key steps involved in filtering and processing raw data, explains assembly frameworks, and discusses quality statistics for the assessment of the assembled sequence. Furthermore, the paper explores recent Ubuntu-based software environments oriented towards genome assembly as well as some avenues for future research.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

PubMed

Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A; Alperovich, Nina; MacMath, Derek; Najera, Claudia D; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N; Montague, Michael G; Friedman, Robert M; Desai, Prashant J; Vashee, Sanjay

2017-10-17

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS YA , replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods

PubMed Central

Grzesik, Peter; Voorhies, Alexander A.; Alperovich, Nina; MacMath, Derek; Najera, Claudia D.; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N.; Montague, Michael G.; Friedman, Robert M.; Desai, Prashant J.

2017-01-01

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats. PMID:28928148

Comparison of the genomic sequence of the microminipig, a novel breed of swine, with the genomic database for conventional pig.

PubMed

Miura, Naoki; Kucho, Ken-Ichi; Noguchi, Michiko; Miyoshi, Noriaki; Uchiumi, Toshiki; Kawaguchi, Hiroaki; Tanimoto, Akihide

2014-01-01

The microminipig, which weighs less than 10 kg at an early stage of maturity, has been reported as a potential experimental model animal. Its extremely small size and other distinct characteristics suggest the possibility of a number of differences between the genome of the microminipig and that of conventional pigs. In this study, we analyzed the genomes of two healthy microminipigs using a next-generation sequencer SOLiD™ system. We then compared the obtained genomic sequences with a genomic database for the domestic pig (Sus scrofa). The mapping coverage of sequenced tag from the microminipig to conventional pig genomic sequences was greater than 96% and we detected no clear, substantial genomic variance from these data. The results may indicate that the distinct characteristics of the microminipig derive from small-scale alterations in the genome, such as Single Nucleotide Polymorphisms or translational modifications, rather than large-scale deletion or insertion polymorphisms. Further investigation of the entire genomic sequence of the microminipig with methods enabling deeper coverage is required to elucidate the genetic basis of its distinct phenotypic traits. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

PubMed Central

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1

PubMed Central

de Bruin, R. A. M.; Kalashnikova, T. I.; Aslanian, A.; Wohlschlegel, J.; Chahwan, C.; Yates, J. R.; Russell, P.; Wittenberg, C.

2008-01-01

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication. PMID:18682565

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae

PubMed Central

Mukherjee, Munmun; Kakarla, Prathusha; Kumar, Sanath; Gonzalez, Esmeralda; Floyd, Jared T.; Inupakutika, Madhuri; Devireddy, Amith Reddy; Tirrell, Selena R.; Bruns, Merissa; He, Guixin; Lindquist, Ingrid E.; Sundararajan, Anitha; Schilkey, Faye D.; Mudge, Joann; Varela, Manuel F.

2015-01-01

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence–related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations. PMID:25722857

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae.

PubMed

Mukherjee, Munmun; Kakarla, Prathusha; Kumar, Sanath; Gonzalez, Esmeralda; Floyd, Jared T; Inupakutika, Madhuri; Devireddy, Amith Reddy; Tirrell, Selena R; Bruns, Merissa; He, Guixin; Lindquist, Ingrid E; Sundararajan, Anitha; Schilkey, Faye D; Mudge, Joann; Varela, Manuel F

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae . Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence-related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.

Genome Modification Leads to Phenotype Reversal in Human Myotonic Dystrophy type 1 iPS-cell Derived Neural Stem Cells

PubMed Central

Xia, Guangbin; Gao, Yuanzheng; Jin, Shouguang; Subramony, SH.; Terada, Naohiro; Ranum, Laura P.W.; Swanson, Maurice S.; Ashizawa, Tetsuo

2015-01-01

Objective Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats in the 3'-untranslated region (3’ UTR) of the DMPK gene. Correcting the mutation in DM1 stem cells would be an important step towards autologous stem cell therapy. The objective of this study is to demonstrate in vitro genome editing to prevent production of toxic mutant transcripts and reverse phenotypes in DM1 stem cells. Methods Genome editing was performed in DM1 neural stem cells (NSCs) derived from human DM1 iPS cells. An editing cassette containing SV40/bGH polyA signals was integrated upstream of the CTG repeats by TALEN-mediated homologous recombination (HR). The expression of mutant CUG repeats transcript was monitored by nuclear RNA foci, the molecular hallmarks of DM1, using RNA fluorescence in situ hybridization (RNA-FISH). Alternative splicing of microtubule-associated protein tau (MAPT) and muscleblind-like (MBNL) proteins were analyzed to further monitor the phenotype reversal after genome modification. Results The cassette was successfully inserted into DMPK intron 9 and this genomic modification led to complete disappearance of nuclear RNA foci. MAPT and MBNL 1, 2 aberrant splicing in DM1 NSCs was reversed to normal pattern in genome-modified NSCs. Interpretation Genome modification by integration of exogenous polyA signals upstream of the DMPK CTG repeat expansion prevents the production of toxic RNA and leads to phenotype reversal in human DM1 iPS-cells derived stem cells. Our data provide proof-of-principle evidence that genome modification may be used to generate genetically modified progenitor cells as a first step toward autologous cell transfer therapy for DM1. PMID:25702800

Use of genomic recursions and algorithm for proven and young animals for single-step genomic BLUP analyses--a simulation study.

PubMed

Fragomeni, B O; Lourenco, D A L; Tsuruta, S; Masuda, Y; Aguilar, I; Misztal, I

2015-10-01

The purpose of this study was to examine accuracy of genomic selection via single-step genomic BLUP (ssGBLUP) when the direct inverse of the genomic relationship matrix (G) is replaced by an approximation of G(-1) based on recursions for young genotyped animals conditioned on a subset of proven animals, termed algorithm for proven and young animals (APY). With the efficient implementation, this algorithm has a cubic cost with proven animals and linear with young animals. Ten duplicate data sets mimicking a dairy cattle population were simulated. In a first scenario, genomic information for 20k genotyped bulls, divided in 7k proven and 13k young bulls, was generated for each replicate. In a second scenario, 5k genotyped cows with phenotypes were included in the analysis as young animals. Accuracies (average for the 10 replicates) in regular EBV were 0.72 and 0.34 for proven and young animals, respectively. When genomic information was included, they increased to 0.75 and 0.50. No differences between genomic EBV (GEBV) obtained with the regular G(-1) and the approximated G(-1) via the recursive method were observed. In the second scenario, accuracies in GEBV (0.76, 0.51 and 0.59 for proven bulls, young males and young females, respectively) were also higher than those in EBV (0.72, 0.35 and 0.49). Again, no differences between GEBV with regular G(-1) and with recursions were observed. With the recursive algorithm, the number of iterations to achieve convergence was reduced from 227 to 206 in the first scenario and from 232 to 209 in the second scenario. Cows can be treated as young animals in APY without reducing the accuracy. The proposed algorithm can be implemented to reduce computing costs and to overcome current limitations on the number of genotyped animals in the ssGBLUP method. © 2015 Blackwell Verlag GmbH.

Predicting gene expression levels from codon biases in alpha-proteobacterial genomes.

PubMed

Karlin, Samuel; Barnett, Melanie J; Campbell, Allan M; Fisher, Robert F; Mrazek, Jan

2003-06-10

Predicted highly expressed (PHX) genes in five currently available high G+C complete alpha-proteobacterial genomes are analyzed. These include: the nitrogen-fixing plant symbionts Sinorhizobium meliloti (SINME) and Mesorhizobium loti (MESLO), the nonpathogenic aquatic bacterium Caulobacter crescentus (CAUCR), the plant pathogen Agrobacterium tumefaciens (AGRTU), and the mammalian pathogen Brucella melitensis (BRUME). Three of these genomes, SINME, AGRTU, and BRUME, contain multiple chromosomes or megaplasmids (>1 Mb length). PHX genes in these genomes are concentrated mainly in the major (largest) chromosome with few PHX genes found in the secondary chromosomes and megaplasmids. Tricarboxylic acid cycle and aerobic respiration genes are strongly PHX in all five genomes, whereas anaerobic pathways of glycolysis and fermentation are mostly not PHX. Only in MESLO (but not SINME) and BRUME are most glycolysis genes PHX. Many flagellar genes are PHX in MESLO and CAUCR, but mostly are not PHX in SINME and AGRTU. The nonmotile BRUME also carries many flagellar genes but these are generally not PHX and all but one are located in the second chromosome. CAUCR stands out among available prokaryotic genomes with 25 PHX TonB-dependent receptors. These are putatively involved in uptake of iron ions and other nonsoluble compounds.

Joint genomic evaluation of French dairy cattle breeds using multiple-trait models.

PubMed

Karoui, Sofiene; Carabaño, María Jesús; Díaz, Clara; Legarra, Andrés

2012-12-07

Using a multi-breed reference population might be a way of increasing the accuracy of genomic breeding values in small breeds. Models involving mixed-breed data do not take into account the fact that marker effects may differ among breeds. This study was aimed at investigating the impact on accuracy of increasing the number of genotyped candidates in the training set by using a multi-breed reference population, in contrast to single-breed genomic evaluations. Three traits (milk production, fat content and female fertility) were analyzed by genomic mixed linear models and Bayesian methodology. Three breeds of French dairy cattle were used: Holstein, Montbéliarde and Normande with 2976, 950 and 970 bulls in the training population, respectively and 964, 222 and 248 bulls in the validation population, respectively. All animals were genotyped with the Illumina Bovine SNP50 array. Accuracy of genomic breeding values was evaluated under three scenarios for the correlation of genomic breeding values between breeds (r(g)): uncorrelated (1), r(g) = 0; estimated r(g) (2); high, r(g) = 0.95 (3). Accuracy and bias of predictions obtained in the validation population with the multi-breed training set were assessed by the coefficient of determination (R(2)) and by the regression coefficient of daughter yield deviations of validation bulls on their predicted genomic breeding values, respectively. The genetic variation captured by the markers for each trait was similar to that estimated for routine pedigree-based genetic evaluation. Posterior means for rg ranged from -0.01 for fertility between Montbéliarde and Normande to 0.79 for milk yield between Montbéliarde and Holstein. Differences in R(2) between the three scenarios were notable only for fat content in the Montbéliarde breed: from 0.27 in scenario (1) to 0.33 in scenarios (2) and (3). Accuracies for fertility were lower than for other traits. Using a multi-breed reference population resulted in small or no increases in