An Easy Method for Plant Polysome Profiling.

PubMed

Lecampion, Cécile; Floris, Maïna; Fantino, Jean Raphaël; Robaglia, Christophe; Laloi, Christophe

2016-08-28

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

Posttranscriptional Control of Photosynthetic mRNA Decay under Stress Conditions Requires 3′ and 5′ Untranslated Regions and Correlates with Differential Polysome Association in Rice1[W][OA

PubMed Central

Park, Su-Hyun; Chung, Pil Joong; Juntawong, Piyada; Bailey-Serres, Julia; Kim, Youn Shic; Jung, Harin; Bang, Seung Woon; Kim, Yeon-Ki; Do Choi, Yang; Kim, Ju-Kon

2012-01-01

Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3′ and 5′ untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3′ and 5′ untranslated regions and correlates with differential polysome association. PMID:22566494

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

PubMed Central

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-01-01

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

PubMed

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-05-05

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

Diurnal Changes of Polysome Loading Track Sucrose Content in the Rosette of Wild-Type Arabidopsis and the Starchless pgm Mutant1[W][OA

PubMed Central

Pal, Sunil Kumar; Liput, Magdalena; Piques, Maria; Ishihara, Hirofumi; Obata, Toshihiro; Martins, Marina C.M.; Sulpice, Ronan; van Dongen, Joost T.; Fernie, Alisdair R.; Yadav, Umesh Prasad; Lunn, John E.; Usadel, Björn; Stitt, Mark

2013-01-01

Growth is driven by newly fixed carbon in the light, but at night it depends on reserves, like starch, that are laid down in the light. Unless plants coordinate their growth with diurnal changes in the carbon supply, they will experience acute carbon starvation during the night. Protein synthesis represents a major component of cellular growth. Polysome loading was investigated during the diurnal cycle, an extended night, and low CO2 in Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) and in the starchless phosphoglucomutase (pgm) mutant. In Col-0, polysome loading was 60% to 70% in the light, 40% to 45% for much of the night, and less than 20% in an extended night, while in pgm, it fell to less than 25% early in the night. Quantification of ribosomal RNA species using quantitative reverse transcription-polymerase chain reaction revealed that polysome loading remained high for much of the night in the cytosol, was strongly light dependent in the plastid, and was always high in mitochondria. The rosette sucrose content correlated with overall and with cytosolic polysome loading. Ribosome abundance did not show significant diurnal changes. However, compared with Col-0, pgm had decreased and increased abundance of plastidic and mitochondrial ribosomes, respectively. Incorporation of label from 13CO2 into protein confirmed that protein synthesis continues at a diminished rate in the dark. Modeling revealed that a decrease in polysome loading at night is required to balance protein synthesis with the availability of carbon from starch breakdown. Costs are also reduced by using amino acids that accumulated in the previous light period. These results uncover a tight coordination of protein synthesis with the momentary supply of carbon. PMID:23674104

Isolation of Polysomal RNA for Analyzing Stress-Responsive Genes Regulated at the Translational Level in Plants.

PubMed

Li, Yong-Fang; Mahalingam, Ramamurthy; Sunkar, Ramanjulu

2017-01-01

Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, indicating regulation at translational level is another major regulator for gene expression. Analysis of translatome, which refers to all mRNAs associated with ribosomes, thus has the potential to bridge the gap between transcriptome and proteome. Polysomal RNA profiling and recently developed ribosome profiling (Ribo-seq) are two main methods for translatome analysis at global level. Here, we describe the classical procedure for polysomal RNA isolation by sucrose gradient ultracentrifugation followed by highthroughput RNA-seq to identify genes regulated at translational level. Polysomal RNA can be further used for a variety of downstream applications including Northern blot analysis, qRT-PCR, RNase protection assay, and microarray-based gene expression profiling.

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h

PubMed Central

Schepetilnikov, Mikhail; Dimitrova, Maria; Mancera-Martínez, Eder; Geldreich, Angèle; Keller, Mario; Ryabova, Lyubov A

2013-01-01

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1—eIF3h—is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation. PMID:23524850

Toward a Molecular Understanding of Noise-Induced Hearing Loss

DTIC Science & Technology

2017-10-01

cell, SAHA, Heat shock, sex differences 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON...threshold shift, Temporary threshold shift, Noise induced hearing loss, Ribotag, RNA-seq, Hair cell, Supporting cell, SAHA, Heat shock, Sex ...also sex -specific. TTS-inducing noise exposure: crosses, calibration, validation cytocochleograms, noise exposure, tissue harvesting, polysome IP

EuPathDB: the eukaryotic pathogen genomics database resource

PubMed Central

Aurrecoechea, Cristina; Barreto, Ana; Basenko, Evelina Y.; Brestelli, John; Brunk, Brian P.; Cade, Shon; Crouch, Kathryn; Doherty, Ryan; Falke, Dave; Fischer, Steve; Gajria, Bindu; Harb, Omar S.; Heiges, Mark; Hertz-Fowler, Christiane; Hu, Sufen; Iodice, John; Kissinger, Jessica C.; Lawrence, Cris; Li, Wei; Pinney, Deborah F.; Pulman, Jane A.; Roos, David S.; Shanmugasundram, Achchuthan; Silva-Franco, Fatima; Steinbiss, Sascha; Stoeckert, Christian J.; Spruill, Drew; Wang, Haiming; Warrenfeltz, Susanne; Zheng, Jie

2017-01-01

The Eukaryotic Pathogen Genomics Database Resource (EuPathDB, http://eupathdb.org) is a collection of databases covering 170+ eukaryotic pathogens (protists & fungi), along with relevant free-living and non-pathogenic species, and select pathogen hosts. To facilitate the discovery of meaningful biological relationships, the databases couple preconfigured searches with visualization and analysis tools for comprehensive data mining via intuitive graphical interfaces and APIs. All data are analyzed with the same workflows, including creation of gene orthology profiles, so data are easily compared across data sets, data types and organisms. EuPathDB is updated with numerous new analysis tools, features, data sets and data types. New tools include GO, metabolic pathway and word enrichment analyses plus an online workspace for analysis of personal, non-public, large-scale data. Expanded data content is mostly genomic and functional genomic data while new data types include protein microarray, metabolic pathways, compounds, quantitative proteomics, copy number variation, and polysomal transcriptomics. New features include consistent categorization of searches, data sets and genome browser tracks; redesigned gene pages; effective integration of alternative transcripts; and a EuPathDB Galaxy instance for private analyses of a user's data. Forthcoming upgrades include user workspaces for private integration of data with existing EuPathDB data and improved integration and presentation of host–pathogen interactions. PMID:27903906

Polysome Immunoselection Combined with cDNA Cloning to Obtain Specific Genes from Chlamydomonas reinhardtii

DTIC Science & Technology

1989-02-05

speclficlty of an avallable antitody (IgG) aginst the ribulose bisphosphate carboxylase sma"Ll subunit ( RUBISCO SSU). First, polysomal poly A+) RNA was...Lnlunopreclpitated by the antibody is the same size as the precursor of the RUBISCO S&U ,Fig. ,. When intact polysones were added to the wheat germ

Polysome Profiling in Leishmania, Human Cells and Mouse Testis.

PubMed

Karamysheva, Zemfira N; Tikhonova, Elena B; Grozdanov, Petar N; Huffman, James C; Baca, Kristen R; Karamyshev, Alexander; Denison, R Brian; MacDonald, Clinton C; Zhang, Kai; Karamyshev, Andrey L

2018-04-08

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Ribosome Patterns in Escherichia coli Growing at Various Rates

PubMed Central

Varricchio, Frederick; Monier, Roger

1971-01-01

The distribution of ribosomes, 30 and 50S subunits and polysomes, at three different growth rates of Escherichia coli strains B and K-12 has been studied. The usual percentage of subunits is about 20%. However, at the lowest growth rate (μ = generations/hour), μ = 0.45 at 30C, the proportion of subunits is about 30%. An exceptional situation exists in K-12 strains growing at maximum growth rate, μ = 1.35, where the percentage of subunits is 45%. Several points of control over ribosome production are thus indicated. It is suggested that “subunit pool” is essentially a reserve. Furthermore, the polysome content when related to deoxyribonucleic acid content varies directly with the growth rate, which indicates the average efficiency of polysomes in protein synthesis does not vary over the range of growth rates tested. PMID:5001192

Translatome analysis at the egg-to-embryo transition in sea urchin

PubMed Central

Chassé, Héloïse; Aubert, Julie; Boulben, Sandrine; Le Corguillé, Gildas; Corre, Erwan; Cormier, Patrick

2018-01-01

Abstract Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis. PMID:29660001

Translatome analysis at the egg-to-embryo transition in sea urchin.

PubMed

Chassé, Héloïse; Aubert, Julie; Boulben, Sandrine; Le Corguillé, Gildas; Corre, Erwan; Cormier, Patrick; Morales, Julia

2018-05-18

Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis.

Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis

PubMed Central

Liiv, Aivar; Karitkina, Diana; Maiväli, Ülo; Remme, Jaanus

2005-01-01

Background The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors. Results We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Ψ1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome. Conclusion Mutations A1912G, A1919G, and Ψ1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Ψ1911C, A1913G, C1914A, Ψ1915C, and A1918G lack clear phenotypes. PMID:16053518

Development of polysomic microsatellite markers for characterization of population structuring and phylogeography in the shortnose sturgeon (Acipenser brevirostrum)

USGS Publications Warehouse

Henderson, Anne P.; King, Tim L.

2012-01-01

Shortnose sturgeon Acipenser brevirostrum is an endangered polyploid fish species for which no nuclear DNA markers previously existed. To address this need, 86 polysomic loci were developed and characterized in 20 A. brevirostrum from five river systems and eight members (parents and six progeny) of a captive-bred family. All markers proved to be polymorphic, polysomic, and demonstrated direct inheritance when tested in a captive family. Eleven loci were included in a range-wide survey of 561 fish sampled from 17 geographic collections. Allelic diversity at these markers ranged from 7 to 24 alleles/locus and averaged 16.5 alleles/locus; sufficient diversity to produce unique multilocus genotypes. In the range-wide survey, a Mantel comparison of an ecological (1-Jaccard’s) and genetic (ΦPT; an analog to FST) distance metrics, identified a strong positive correlation (r = 0.98, P PT represents a viable metric for assessing genetic relatedness using this class of marker.

Vanillin Inhibits Translation and Induces Messenger Ribonucleoprotein (mRNP) Granule Formation in Saccharomyces cerevisiae: Application and Validation of High-Content, Image-Based Profiling

PubMed Central

Suga, Yohei; Izawa, Shingo; Ohya, Yoshikazu

2013-01-01

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells. PMID:23637899

Radiation-induced alternative transcripts as detected in total and polysome-bound mRNA.

PubMed

Wahba, Amy; Ryan, Michael C; Shankavaram, Uma T; Camphausen, Kevin; Tofilon, Philip J

2018-01-02

Alternative splicing is a critical event in the posttranscriptional regulation of gene expression. To investigate whether this process influences radiation-induced gene expression we defined the effects of ionizing radiation on the generation of alternative transcripts in total cellular mRNA (the transcriptome) and polysome-bound mRNA (the translatome) of the human glioblastoma stem-like cell line NSC11. For these studies, RNA-Seq profiles from control and irradiated cells were compared using the program SpliceSeq to identify transcripts and splice variations induced by radiation. As compared to the transcriptome (total RNA) of untreated cells, the radiation-induced transcriptome contained 92 splice events suggesting that radiation induced alternative splicing. As compared to the translatome (polysome-bound RNA) of untreated cells, the radiation-induced translatome contained 280 splice events of which only 24 were overlapping with the radiation-induced transcriptome. These results suggest that radiation not only modifies alternative splicing of precursor mRNA, but also results in the selective association of existing mRNA isoforms with polysomes. Comparison of radiation-induced alternative transcripts to radiation-induced gene expression in total RNA revealed little overlap (about 3%). In contrast, in the radiation-induced translatome, about 38% of the induced alternative transcripts corresponded to genes whose expression level was affected in the translatome. This study suggests that whereas radiation induces alternate splicing, the alternative transcripts present at the time of irradiation may play a role in the radiation-induced translational control of gene expression and thus cellular radioresponse.

Water Deficit and Abscisic Acid Cause Differential Inhibition of Shoot versus Root Growth in Soybean Seedlings : Analysis of Growth, Sugar Accumulation, and Gene Expression.

PubMed

Creelman, R A; Mason, H S; Bensen, R J; Boyer, J S; Mullet, J E

1990-01-01

Roots often continue to elongate while shoot growth is inhibited in plants subjected to low-water potentials. The cause of this differential response to water deficit was investigated. We examined hypocotyl and root growth, polysome status and mRNA populations, and abscisic acid (ABA) content in etiolated soybean (Glycine max [L.] Merr. cv Williams) seedlings whose growth was inhibited by transfer to low-water potential vermiculite or exogenous ABA. Both treatments affected growth and dry weight in a similar fashion. Maximum inhibition of hypocotyl growth occurred when internal ABA levels (modulated by ABA application) reached the endogenous level found in the elongating zone of seedlings grown in water-deficient vermiculite. Conversely, root growth was affected to only a slight extent in low-water potential seedlings and by most ABA treatments (in some, growth was promoted). In every seedling section examined, transfer of seedlings into low-water potential vermiculite caused ABA levels to increase approximately 5- to 10-fold over that found in well-watered seedlings. Changes in soluble sugar content, polysome status, and polysome mRNA translation products seen in low-water potential seedlings did not occur with ABA treatments sufficient to cause significant inhibition of hypocotyl elongation. These data suggest that both variation in endogenous ABA levels, and differing sensitivity to ABA in hypocotyls and roots can modulate root/shoot growth ratios. However, exogenous ABA did not induce changes in sugar accumulation, polysome status, and mRNA populations seen after transfer into low-water potential vermiculite.

Water Deficit and Abscisic Acid Cause Differential Inhibition of Shoot versus Root Growth in Soybean Seedlings 1

PubMed Central

Creelman, Robert A.; Mason, Hugh S.; Bensen, Robert J.; Boyer, John S.; Mullet, John E.

1990-01-01

Roots often continue to elongate while shoot growth is inhibited in plants subjected to low-water potentials. The cause of this differential response to water deficit was investigated. We examined hypocotyl and root growth, polysome status and mRNA populations, and abscisic acid (ABA) content in etiolated soybean (Glycine max [L.] Merr. cv Williams) seedlings whose growth was inhibited by transfer to low-water potential vermiculite or exogenous ABA. Both treatments affected growth and dry weight in a similar fashion. Maximum inhibition of hypocotyl growth occurred when internal ABA levels (modulated by ABA application) reached the endogenous level found in the elongating zone of seedlings grown in water-deficient vermiculite. Conversely, root growth was affected to only a slight extent in low-water potential seedlings and by most ABA treatments (in some, growth was promoted). In every seedling section examined, transfer of seedlings into low-water potential vermiculite caused ABA levels to increase approximately 5- to 10-fold over that found in well-watered seedlings. Changes in soluble sugar content, polysome status, and polysome mRNA translation products seen in low-water potential seedlings did not occur with ABA treatments sufficient to cause significant inhibition of hypocotyl elongation. These data suggest that both variation in endogenous ABA levels, and differing sensitivity to ABA in hypocotyls and roots can modulate root/shoot growth ratios. However, exogenous ABA did not induce changes in sugar accumulation, polysome status, and mRNA populations seen after transfer into low-water potential vermiculite. Images Figure 6 Figure 7 PMID:16667248

Indirect genetic estimates of breeding population size in the polyploid green sturgeon (Acipenser medirostris).

PubMed

Israel, J A; May, B

2010-03-01

The utility of genetic measures for kinship reconstruction in polysomic species is not well evaluated. We developed a framework to test hypotheses about estimating breeding population size indirectly from collections of outmigrating green sturgeon juveniles. We evaluated a polysomic dataset, in allelic frequency and phenotypic formats, from green sturgeon to describe the relationship among known progeny from experimental families. The distributions of relatedness values for kin classes were used for reconstructing green sturgeon pedigrees from juveniles of unknown relationship. We compared three rarefaction functions that described the relationship between the number of kin groups and number of samples in a pedigree to estimate the annual abundance of spawners contributing to the threatened green sturgeon Southern Distinct Population Segment in the upper Sacramento River. Results suggested the estimated abundance of breeding green sturgeon remained roughly constant in the upper Sacramento River over a 5-year period, ranging from 10 to 28 individuals depending on the year and rarefaction method. These results demonstrate an empirical understanding for the distribution of relatedness values among individuals is a benefit for assessing pedigree reconstruction methods and identifying misclassification rates. Monitoring of rare species using these indirect methods is feasible and can provide insight into breeding and ontogenetic behaviour. While this framework was developed for specific application to studying fish populations in a riverscape, the framework could be advanced to improve genetic estimation of breeding population size and to identify important breeding habitats of rare species when combined with finer-scaled sampling of offspring.

CCL3L1 copy number and susceptibility to malaria

PubMed Central

Carpenter, Danielle; Färnert, Anna; Rooth, Ingegerd; Armour, John A.L.; Shaw, Marie-Anne

2012-01-01

Copy number variation can contribute to the variation observed in susceptibility to complex diseases. Here we present the first study to investigate copy number variation of the chemokine gene CCL3L1 with susceptibility to malaria. We present a family-based genetic analysis of a Tanzanian population (n = 922), using parasite load, mean number of clinical infections of malaria and haemoglobin levels as phenotypes. Copy number of CCL3L1 was measured using the paralogue ratio test (PRT) and the dataset exhibited copy numbers ranging between 1 and 10 copies per diploid genome (pdg). Association between copy number and phenotypes was assessed. Furthermore, we were able to identify copy number haplotypes in some families, using microsatellites within the copy variable region, for transmission disequilibrium testing. We identified a high level of copy number haplotype diversity and find some evidence for an association of low CCL3L1 copy number with protection from anaemia. PMID:22484763

CCL3L1 copy number and susceptibility to malaria.

PubMed

Carpenter, Danielle; Färnert, Anna; Rooth, Ingegerd; Armour, John A L; Shaw, Marie-Anne

2012-07-01

Copy number variation can contribute to the variation observed in susceptibility to complex diseases. Here we present the first study to investigate copy number variation of the chemokine gene CCL3L1 with susceptibility to malaria. We present a family-based genetic analysis of a Tanzanian population (n=922), using parasite load, mean number of clinical infections of malaria and haemoglobin levels as phenotypes. Copy number of CCL3L1 was measured using the paralogue ratio test (PRT) and the dataset exhibited copy numbers ranging between 1 and 10 copies per diploid genome (pdg). Association between copy number and phenotypes was assessed. Furthermore, we were able to identify copy number haplotypes in some families, using microsatellites within the copy variable region, for transmission disequilibrium testing. We identified a high level of copy number haplotype diversity and find some evidence for an association of low CCL3L1 copy number with protection from anaemia. Copyright © 2012 Elsevier B.V. All rights reserved.

Mitochondrial genomic variation associated with higher mitochondrial copy number: the Cache County Study on Memory Health and Aging.

PubMed

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

2014-01-01

The mitochondria are essential organelles and are the location of cellular respiration, which is responsible for the majority of ATP production. Each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. The regulation of mitochondrial copy number by nuclear genes has been studied extensively. While mitochondrial variation has been associated with longevity and some of the diseases known to have reduced mitochondrial copy number, the role that the mitochondrial genome itself has in regulating mitochondrial copy number remains poorly understood. We analyzed the complete mitochondrial genomes from 1007 individuals randomly selected from the Cache County Study on Memory Health and Aging utilizing the inferred evolutionary history of the mitochondrial haplotypes present in our dataset to identify sequence variation and mitochondrial haplotypes associated with changes in mitochondrial copy number. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. We identified three variants associated with higher mitochondrial copy number and suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that causes changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes.

Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability. | Office of Cancer Genomics

Cancer.gov

Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent.

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

Hicks, S.E.; Wallwork, J.C.

There are conflicting reports in the literature concerning the role of zinc in protein synthesis. This study presents evidence for the direct involvement of zinc in the translation of polypeptide chains in rats. Cell-free systems for incorporation of amino acids into acid-insoluble proteins were prepared from livers of three populations of rats: (1) rats fed ad libitum a diet containing 25 ppm zinc; (2) rats fed a diet containing less than 1 ppm zinc and (3) rats pair-fed a diet containing 25 ppm zinc. The diets contained 20% egg white and were enriched with biotin. Distilled deionized drinking water wasmore » given. The animals were maintained on the regimen for 45 days with precautions to limit zinc contamination. Group 2 showed typical signs of zinc deficiency, including decreased bone zinc. In vitro systems containing liver polysomes and a pH5 precipitate enzyme fraction indicated that the synthetic ability of systems isolated from zinc-deficient rats was considerably depressed, resulting in incorporation of amino acids 15 to 30% less than systems from pair-fed rats and 30 to 50% less than ad libitum-fed control animals. The results of crossover experiments performed by mixing polysome and enzyme fractions from the different groups indicated that the defect is due primarily to the pH precipitate enzyme fraction and not the polysomes.« less

Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis

PubMed Central

Hofmann, Sarah; Elman, Tamar; Shenoy, Anjana; Geiger, Tamar; Elkon, Ran; Ehrlich, Marcelo

2017-01-01

Abstract Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC–MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase. PMID:28460002

Translation Regulation and RNA Granule Formation after Heat Shock of Procyclic Form Trypanosoma brucei: Many Heat-Induced mRNAs Are also Increased during Differentiation to Mammalian-Infective Forms.

PubMed

Minia, Igor; Merce, Clementine; Terrao, Monica; Clayton, Christine

2016-09-01

African trypanosome procyclic forms multiply in the midgut of tsetse flies, and are routinely cultured at 27°C. Heat shocks of 37°C and above result in general inhibition of translation, and severe heat shock (41°C) results in sequestration of mRNA in granules. The mRNAs that are bound by the zinc-finger protein ZC3H11, including those encoding refolding chaperones, escape heat-induced translation inhibition. At 27°C, ZC3H11 mRNA is predominantly present as an untranslated cytosolic messenger ribonucleoprotein particle, but after heat shocks of 37°C-41°C, the ZC3H11 mRNA moves into the polysomal fraction. To investigate the scope and specificities of heat-shock translational regulation and granule formation, we analysed the distributions of mRNAs on polysomes at 27°C and after 1 hour at 39°C, and the mRNA content of 41°C heat shock granules. We found that mRNAs that bind to ZC3H11 remained in polysomes at 39°C and were protected from sequestration in granules at 41°C. As previously seen for starvation stress granules, the mRNAs that encode ribosomal proteins were excluded from heat-shock granules. 70 mRNAs moved towards the polysomal fraction after the 39°C heat shock, and 260 increased in relative abundance. Surprisingly, many of these mRNAs are also increased when trypanosomes migrate to the tsetse salivary glands. It therefore seems possible that in the wild, temperature changes due to diurnal variations and periodic intake of warm blood might influence the efficiency with which procyclic forms develop into mammalian-infective forms.

Development of genome- and transcriptome-derived microsatellites in related species of snapping shrimps with highly duplicated genomes.

PubMed

Gaynor, Kaitlyn M; Solomon, Joseph W; Siller, Stefanie; Jessell, Linnet; Duffy, J Emmett; Rubenstein, Dustin R

2017-11-01

Molecular markers are powerful tools for studying patterns of relatedness and parentage within populations and for making inferences about social evolution. However, the development of molecular markers for simultaneous study of multiple species presents challenges, particularly when species exhibit genome duplication or polyploidy. We developed microsatellite markers for Synalpheus shrimp, a genus in which species exhibit not only great variation in social organization, but also interspecific variation in genome size and partial genome duplication. From the four primary clades within Synalpheus, we identified microsatellites in the genomes of four species and in the consensus transcriptome of two species. Ultimately, we designed and tested primers for 143 microsatellite markers across 25 species. Although the majority of markers were disomic, many markers were polysomic for certain species. Surprisingly, we found no relationship between genome size and the number of polysomic markers. As expected, markers developed for a given species amplified better for closely related species than for more distant relatives. Finally, the markers developed from the transcriptome were more likely to work successfully and to be disomic than those developed from the genome, suggesting that consensus transcriptomes are likely to be conserved across species. Our findings suggest that the transcriptome, particularly consensus sequences from multiple species, can be a valuable source of molecular markers for taxa with complex, duplicated genomes. © 2017 John Wiley & Sons Ltd.

Autopolyploidy genome duplication preserves other ancient genome duplications in Atlantic salmon (Salmo salar).

PubMed

Christensen, Kris A; Davidson, William S

2017-01-01

Salmonids (e.g. Atlantic salmon, Pacific salmon, and trouts) have a long legacy of genome duplication. In addition to three ancient genome duplications that all teleosts are thought to share, salmonids have had one additional genome duplication. We explored a methodology for untangling these duplications from each other to better understand them in Atlantic salmon. In this methodology, homeologous regions (paralogous/duplicated genomic regions originating from a whole genome duplication) from the most recent genome duplication were assumed to have duplicated genes at greater density and have greater sequence similarity. This assumption was used to differentiate duplicated gene pairs in Atlantic salmon that are either from the most recent genome duplication or from earlier duplications. From a comparison with multiple vertebrate species, it is clear that Atlantic salmon have retained more duplicated genes from ancient genome duplications than other vertebrates--often at higher density in the genome and containing fewer synonymous mutations. It may be that polysomic inheritance is the mechanism responsible for maintaining ancient gene duplicates in salmonids. Polysomic inheritance (when multiple chromosomes pair during meiosis) is thought to be relatively common in salmonids compared to other vertebrate species. These findings illuminate how genome duplications may not only increase the number of duplicated genes, but may also be involved in the maintenance of them from previous genome duplications as well.

Silibinin inhibits translation initiation: implications for anticancer therapy.

PubMed

Lin, Chen-Ju; Sukarieh, Rami; Pelletier, Jerry

2009-06-01

Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane.

PubMed

Sun, Yue; Joyce, Priya Aiyar

2017-11-01

Droplet digital PCR combined with the low copy ACT allele as endogenous reference gene, makes accurate and rapid estimation of gene copy number in Q208 A and Q240 A attainable. Sugarcane is an important cultivated crop with both high polyploidy and aneuploidy in its 10 Gb genome. Without a known copy number reference gene, it is difficult to accurately estimate the copy number of any gene of interest by PCR-based methods in sugarcane. Recently, a new technology, known as droplet digital PCR (ddPCR) has been developed which can measure the absolute amount of the target DNA in a given sample. In this study, we deduced the true copy number of three endogenous genes, actin depolymerizing factor (ADF), adenine phosphoribosyltransferase (APRT) and actin (ACT) in three Australian sugarcane varieties, using ddPCR by comparing the absolute amounts of the above genes with a transgene of known copy number. A single copy of the ACT allele was detected in Q208 A , two copies in Q240 A , but was absent in Q117. Copy number variation was also observed for both APRT and ADF, and ranged from 9 to 11 in the three tested varieties. Using this newly developed ddPCR method, transgene copy number was successfully determined in 19 transgenic Q208 A and Q240 A events using ACT as the reference endogenous gene. Our study demonstrates that ddPCR can be used for high-throughput genetic analysis and is a quick, accurate and reliable alternative method for gene copy number determination in sugarcane. This discovered ACT allele would be a suitable endogenous reference gene for future gene copy number variation and dosage studies of functional genes in Q208 A and Q240 A .

Functional effects of CCL3L1 copy number

PubMed Central

Carpenter, Danielle; McIntosh, Richard S; Pleass, Richard J; Armour, John AL

2012-01-01

Copy number variation (CNV) is becoming increasingly important as a feature of human variation in disease susceptibility studies. However, the consequences of copy number variation are not so well understood. Here we present data exploring the functional consequences of copy number variation of CCL3L1 in 55 independent UK samples with no known clinical phenotypes. Copy number of CCL3L1 was determined by the paralogue ratio test (PRT), and expression levels of MIP-1α and mRNA from stimulated monocytes were measured and analysed. The data show no statistically significant association of MIP-1α protein levels with copy number. However, there was a significant correlation between copy number and CCL3L1:CCL3 mRNA ratio. The data also provide evidence that expression of CCL3 predominates in both protein and mRNA, and therefore the observed variation of CCL3 is potentially more important biologically than that of copy number variation of CCL3L1. PMID:22476153

Beneficial effect of a high number of copies of salivary amylase AMY1 gene on obesity risk in Mexican children.

PubMed

Mejía-Benítez, María A; Bonnefond, Amélie; Yengo, Loïc; Huyvaert, Marlène; Dechaume, Aurélie; Peralta-Romero, Jesús; Klünder-Klünder, Miguel; García Mena, Jaime; El-Sayed Moustafa, Julia S; Falchi, Mario; Cruz, Miguel; Froguel, Philippe

2015-02-01

Childhood obesity is a major public health problem in Mexico, affecting one in every three children. Genome-wide association studies identified genetic variants associated with childhood obesity, but a large missing heritability remains to be elucidated. We have recently shown a strong association between a highly polymorphic copy number variant encompassing the salivary amylase gene (AMY1 also known as AMY1A) and obesity in European and Asian adults. In the present study, we aimed to evaluate the association between AMY1 copy number and obesity in Mexican children. We evaluated the number of AMY1 copies in 597 Mexican children (293 obese children and 304 normal weight controls) through highly sensitive digital PCR. The effect of AMY1 copy number on obesity status was assessed using a logistic regression model adjusted for age and sex. We identified a marked effect of AMY1 copy number on reduced risk of obesity (OR per estimated copy 0.84, with the number of copies ranging from one to 16 in this population; p = 4.25 × 10(-6)). The global association between AMY1 copy number and reduced risk of obesity seemed to be mostly driven by the contribution of the highest AMY1 copy number. Strikingly, all children with >10 AMY1 copies were normal weight controls. Salivary amylase initiates the digestion of dietary starch, which is highly consumed in Mexico. Our current study suggests putative benefits of high number of AMY1 copies (and related production of salivary amylase) on energy metabolism in Mexican children.

VIRUS-SPECIFIC POLYSOMES IN CELLS INFECTED WITH THE VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS,

DTIC Science & Technology

VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS, *RIBOSOMES, *TISSUE CULTURE CELLS, RIBOSOMES, GROWTH(PHYSIOLOGY), INFECTIOUS DISEASES, ARBOVIRUSES, VIRUSES, NUCLEIC ACIDS, BIOSYNTHESIS, USSR, MOLECULAR STRUCTURE.

The cost of copy number in a selfish genetic element: the 2-μm plasmid of Saccharomyces cerevisiae.

PubMed

Harrison, Ellie; Koufopanou, V; Burt, A; MacLean, R C

2012-11-01

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade-off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes

PubMed Central

Drummond, Sheona P.; Hildyard, John; Firczuk, Helena; Reamtong, Onrapak; Li, Ning; Kannambath, Shichina; Claydon, Amy J.; Beynon, Robert J.; Eyers, Claire E.; McCarthy, John E. G.

2011-01-01

Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shift-dependent modulation of the intracellular locations of Dhh1 and Pat1. Like the formation of P bodies, this phenomenon changes the spatial relationship between components involved in translation and mRNA degradation. We report significant spatial separation of Dhh1 and Pat1 from ribosomes in exponentially growing cells. Moreover, biochemical analyses reveal that these proteins are excluded from polysomal complexes in exponentially growing cells, indicating that they may not be associated with active states of the translation machinery. In contrast, under diauxic growth shift conditions, Dhh1 and Pat1 are found to co-localize with polysomal complexes. This work suggests that Dhh1 and Pat1 functions are modulated by a re-localization mechanism that involves eIF4A. Pull-down experiments reveal that the intracellular binding partners of Dhh1 and Pat1 change as cells undergo the diauxic growth shift. This reveals a new dimension to the relationship between translation activity and interactions between mRNA, the translation machinery and decapping activator proteins. PMID:21712243

Trypanosoma cruzi transcriptome during axenic epimastigote growth curve

PubMed Central

dos Santos, Cyndia Mara Bezerra; Ludwig, Adriana; Kessler, Rafael Luis; Rampazzo, Rita de Cássia Pontello; Inoue, Alexandre Haruo; Krieger, Marco Aurélio; Pavoni, Daniela Parada; Probst, Christian Macagnan

2018-01-01

BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins. PMID:29668769

Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

PubMed

Aviner, Ranen; Hofmann, Sarah; Elman, Tamar; Shenoy, Anjana; Geiger, Tamar; Elkon, Ran; Ehrlich, Marcelo; Elroy-Stein, Orna

2017-06-02

Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

40 CFR 262.22 - Number of copies.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 26 2011-07-01 2011-07-01 false Number of copies. 262.22 Section 262...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE The Manifest § 262.22 Number of copies. The manifest consists of at least the number of copies which will provide the generator, each transporter, and the owner...

40 CFR 262.22 - Number of copies.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 27 2012-07-01 2012-07-01 false Number of copies. 262.22 Section 262...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE The Manifest § 262.22 Number of copies. The manifest consists of at least the number of copies which will provide the generator, each transporter, and the owner...

40 CFR 262.22 - Number of copies.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Number of copies. 262.22 Section 262...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE The Manifest § 262.22 Number of copies. The manifest consists of at least the number of copies which will provide the generator, each transporter, and the owner...

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing.

PubMed

Castle, John C; Biery, Matthew; Bouzek, Heather; Xie, Tao; Chen, Ronghua; Misura, Kira; Jackson, Stuart; Armour, Christopher D; Johnson, Jason M; Rohl, Carol A; Raymond, Christopher K

2010-04-16

DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing

PubMed Central

2010-01-01

Background DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. Results We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. Conclusion The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads. PMID:20398377

Dietary starch intake modifies the relation between copy number variation in the salivary amylase gene and BMI.

PubMed

Rukh, Gull; Ericson, Ulrika; Andersson-Assarsson, Johanna; Orho-Melander, Marju; Sonestedt, Emily

2017-07-01

Background: Studies have shown conflicting associations between the salivary amylase gene ( AMY1 ) copy number and obesity. Salivary amylase initiates starch digestion in the oral cavity; starch is a major source of energy in the diet. Objective: We investigated the association between AMY1 copy number and obesity traits, and the effect of the interaction between AMY1 copy number and starch intake on these obesity traits. Design: We first assessed the association between AMY1 copy number (genotyped by digital droplet polymerase chain reaction) and obesity traits in 4800 individuals without diabetes (mean age: 57 y; 60% female) from the Malmö Diet and Cancer Cohort. Then we analyzed interactions between AMY1 copy number and energy-adjusted starch intake (obtained by a modified diet history method) on body mass index (BMI) and body fat percentage. Results: AMY1 copy number was not associated with BMI ( P = 0.80) or body fat percentage ( P = 0.38). We observed a significant effect of the interaction between AMY1 copy number and starch intake on BMI ( P -interaction = 0.007) and body fat percentage ( P -interaction = 0.03). Upon stratification by dietary starch intake, BMI tended to decrease with increasing AMY1 copy numbers in the low-starch intake group ( P = 0.07) and tended to increase with increasing AMY1 copy numbers in the high-starch intake group ( P = 0.08). The lowest mean BMI was observed in the group of participants with a low AMY1 copy number and a high dietary intake of starch. Conclusions: Our findings suggest an effect of the interaction between starch intake and AMY1 copy number on obesity. Individuals with high starch intake but low genetic capacity to digest starch had the lowest BMI, potentially because larger amounts of undigested starch are transported through the gastrointestinal tract, contributing to fewer calories extracted from ingested starch. © 2017 American Society for Nutrition.

Epidermal growth factor receptor and AKT1 gene copy numbers by multi-gene fluorescence in situ hybridization impact on prognosis in breast cancer.

PubMed

Li, Jiao; Su, Wei; Zhang, Sheng; Hu, Yunhui; Liu, Jingjing; Zhang, Xiaobei; Bai, Jingchao; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lei, Zhenmin; Zhang, Jin

2015-05-01

The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

PubMed

van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

2018-03-23

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L. lactis at near-zero growth rates, characteristic for cheese ripening. Moreover, we analysed the effect of pH, nutrient limitation and presence of citrate. This showed that plasmid copy numbers were stable giving insight into plasmid copy number dynamics in dairy fermentations. Copyright © 2018 American Society for Microbiology.

Tools for Genetic Studies in Experimental Populations of Polyploids.

PubMed

Bourke, Peter M; Voorrips, Roeland E; Visser, Richard G F; Maliepaard, Chris

2018-01-01

Polyploid organisms carry more than two copies of each chromosome, a condition rarely tolerated in animals but which occurs relatively frequently in the plant kingdom. One of the principal challenges faced by polyploid organisms is to evolve stable meiotic mechanisms to faithfully transmit genetic information to the next generation upon which the study of inheritance is based. In this review we look at the tools available to the research community to better understand polyploid inheritance, many of which have only recently been developed. Most of these tools are intended for experimental populations (rather than natural populations), facilitating genomics-assisted crop improvement and plant breeding. This is hardly surprising given that a large proportion of domesticated plant species are polyploid. We focus on three main areas: (1) polyploid genotyping; (2) genetic and physical mapping; and (3) quantitative trait analysis and genomic selection. We also briefly review some miscellaneous topics such as the mode of inheritance and the availability of polyploid simulation software. The current polyploid analytic toolbox includes software for assigning marker genotypes (and in particular, estimating the dosage of marker alleles in the heterozygous condition), establishing chromosome-scale linkage phase among marker alleles, constructing (short-range) haplotypes, generating linkage maps, performing genome-wide association studies (GWAS) and quantitative trait locus (QTL) analyses, and simulating polyploid populations. These tools can also help elucidate the mode of inheritance (disomic, polysomic or a mixture of both as in segmental allopolyploids) or reveal whether double reduction and multivalent chromosomal pairing occur. An increasing number of polyploids (or associated diploids) are being sequenced, leading to publicly available reference genome assemblies. Much work remains in order to keep pace with developments in genomic technologies. However, such technologies also offer the promise of understanding polyploid genomes at a level which hitherto has remained elusive.

Tools for Genetic Studies in Experimental Populations of Polyploids

PubMed Central

Bourke, Peter M.; Voorrips, Roeland E.; Visser, Richard G. F.; Maliepaard, Chris

2018-01-01

Polyploid organisms carry more than two copies of each chromosome, a condition rarely tolerated in animals but which occurs relatively frequently in the plant kingdom. One of the principal challenges faced by polyploid organisms is to evolve stable meiotic mechanisms to faithfully transmit genetic information to the next generation upon which the study of inheritance is based. In this review we look at the tools available to the research community to better understand polyploid inheritance, many of which have only recently been developed. Most of these tools are intended for experimental populations (rather than natural populations), facilitating genomics-assisted crop improvement and plant breeding. This is hardly surprising given that a large proportion of domesticated plant species are polyploid. We focus on three main areas: (1) polyploid genotyping; (2) genetic and physical mapping; and (3) quantitative trait analysis and genomic selection. We also briefly review some miscellaneous topics such as the mode of inheritance and the availability of polyploid simulation software. The current polyploid analytic toolbox includes software for assigning marker genotypes (and in particular, estimating the dosage of marker alleles in the heterozygous condition), establishing chromosome-scale linkage phase among marker alleles, constructing (short-range) haplotypes, generating linkage maps, performing genome-wide association studies (GWAS) and quantitative trait locus (QTL) analyses, and simulating polyploid populations. These tools can also help elucidate the mode of inheritance (disomic, polysomic or a mixture of both as in segmental allopolyploids) or reveal whether double reduction and multivalent chromosomal pairing occur. An increasing number of polyploids (or associated diploids) are being sequenced, leading to publicly available reference genome assemblies. Much work remains in order to keep pace with developments in genomic technologies. However, such technologies also offer the promise of understanding polyploid genomes at a level which hitherto has remained elusive. PMID:29720992

The relationship between mitochondrial DNA copy number and stallion sperm function.

PubMed

Darr, Christa R; Moraes, Luis E; Connon, Richard E; Love, Charles C; Teague, Sheila; Varner, Dickson D; Meyers, Stuart A

2017-05-01

Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, and humans, and has been suggested as a potential biomarker of fertility in stallion sperm. The results of the present study extend this recent discovery using sperm samples from American Quarter Horse stallions of varying age. By determining copy number of three mitochondrial genes, cytochrome b (CYTB), NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 4 (ND4), instead of a single gene, we demonstrate an improved understanding of mtDNA fate in stallion sperm mitochondria following spermatogenesis. Sperm samples from 37 stallions ranging from 3 to 24 years old were collected at four breeding ranches in north and central Texas during the 2015 breeding season. Samples were analyzed for sperm motion characteristics, nuclear DNA denaturability and mtDNA copy number. Mitochondrial DNA content in individual sperm was determined by real-time qPCR and normalized with a single copy nuclear gene, Beta actin. Exploratory correlation analysis revealed that total motility was negatively correlated with CYTB copy number and sperm chromatin structure. Stallion age did not have a significant effect on copy number for any of the genes. Copy number differences existed between the three genes with CYTB having the greatest number of copies (20.6 ± 1.2 copies, range: 6.0 to 41.1) followed by ND4 (15.5 ± 0.8 copies, range: 6.7 to 27.8) and finally ND1 (12.0 ± 1.0 copies, range: 0.4 to 26.6) (P < 0.05). Varying copy number across mitochondrial genes is likely to be a result of mtDNA fragmentation and degradation since downregulation of sperm mtDNA occurs during spermatogenesis and may be important for normal sperm function. Beta regression analysis suggested that for every unit increase in mtDNA copy number of CYTB, there was a 4% decrease in the odds of sperm movement (P = 0.001). Influential analysis suggested that results are robust and not highly influenced by data from individual stallions despite the low number of stallions sampled with low sperm motility. Further genome sequencing is necessary to investigate if mutations or deletions are the underlying causes of inconsistent copy numbers across mitochondrial genes. In conclusion, we show, for the first time, that increased mtDNA copy number is associated with decreased total sperm motility in stallions. We therefore suggest that mtDNA copy number may be an indicator of defective spermatogenesis in stallions. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparing CNV detection methods for SNP arrays.

PubMed

Winchester, Laura; Yau, Christopher; Ragoussis, Jiannis

2009-09-01

Data from whole genome association studies can now be used for dual purposes, genotyping and copy number detection. In this review we discuss some of the methods for using SNP data to detect copy number events. We examine a number of algorithms designed to detect copy number changes through the use of signal-intensity data and consider methods to evaluate the changes found. We describe the use of several statistical models in copy number detection in germline samples. We also present a comparison of data using these methods to assess accuracy of prediction and detection of changes in copy number.

NEDDylation promotes stress granule assembly.

PubMed

Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum-Yong; Baek, Je-Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

2016-07-06

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

The transcription factor titration effect dictates level of gene expression.

PubMed

Brewster, Robert C; Weinert, Franz M; Garcia, Hernan G; Song, Dan; Rydenfelt, Mattias; Phillips, Rob

2014-03-13

Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number-in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA Copy Number Signature to Predict Recurrence in Early Stage Ovarian Cancer

DTIC Science & Technology

2016-08-01

AWARD NUMBER: W81XWH-14-1-0194 TITLE: DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer PRINCIPAL INVESTIGATOR...SUBTITLE 5a. CONTRACT NUMBER DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer 5b. GRANT NUMBER W81XWH-14-1-0194 5c. PROGRAM...determine the copy number gain and loss for early stage high grade ovarian cancers through IlluminaHumanOmniExpress-FFPE BeadChip system • Subtask 1 DNA

Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets.

PubMed

Cree, L M; Patel, S K; Pyle, A; Lynn, S; Turnbull, D M; Chinnery, P F; Walker, M

2008-08-01

Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.

Allelic recombination between distinct genomic locations generates copy number diversity in human β-defensins

PubMed Central

Bakar, Suhaili Abu; Hollox, Edward J.; Armour, John A. L.

2009-01-01

β-Defensins are small secreted antimicrobial and signaling peptides involved in the innate immune response of vertebrates. In humans, a cluster of at least 7 of these genes shows extensive copy number variation, with a diploid copy number commonly ranging between 2 and 7. Using a genetic mapping approach, we show that this cluster is at not 1 but 2 distinct genomic loci ≈5 Mb apart on chromosome band 8p23.1, contradicting the most recent genome assembly. We also demonstrate that the predominant mechanism of change in β-defensin copy number is simple allelic recombination occurring in the interval between the 2 distinct genomic loci for these genes. In 416 meiotic transmissions, we observe 3 events creating a haplotype copy number not found in the parent, equivalent to a germ-line rate of copy number change of ≈0.7% per gamete. This places it among the fastest-changing copy number variants currently known. PMID:19131514

Low copy number of the salivary amylase gene predisposes to obesity.

PubMed

Falchi, Mario; El-Sayed Moustafa, Julia Sarah; Takousis, Petros; Pesce, Francesco; Bonnefond, Amélie; Andersson-Assarsson, Johanna C; Sudmant, Peter H; Dorajoo, Rajkumar; Al-Shafai, Mashael Nedham; Bottolo, Leonardo; Ozdemir, Erdal; So, Hon-Cheong; Davies, Robert W; Patrice, Alexandre; Dent, Robert; Mangino, Massimo; Hysi, Pirro G; Dechaume, Aurélie; Huyvaert, Marlène; Skinner, Jane; Pigeyre, Marie; Caiazzo, Robert; Raverdy, Violeta; Vaillant, Emmanuel; Field, Sarah; Balkau, Beverley; Marre, Michel; Visvikis-Siest, Sophie; Weill, Jacques; Poulain-Godefroy, Odile; Jacobson, Peter; Sjostrom, Lars; Hammond, Christopher J; Deloukas, Panos; Sham, Pak Chung; McPherson, Ruth; Lee, Jeannette; Tai, E Shyong; Sladek, Robert; Carlsson, Lena M S; Walley, Andrew; Eichler, Evan E; Pattou, Francois; Spector, Timothy D; Froguel, Philippe

2014-05-01

Common multi-allelic copy number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts. Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene (AMY1) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 copy number was positively associated with both amylase gene expression (P = 2.31 × 10(-14)) and serum enzyme levels (P < 2.20 × 10(-16)), whereas reduced AMY1 copy number was associated with increased BMI (change in BMI per estimated copy = -0.15 (0.02) kg/m(2); P = 6.93 × 10(-10)) and obesity risk (odds ratio (OR) per estimated copy = 1.19, 95% confidence interval (CI) = 1.13-1.26; P = 1.46 × 10(-10)). The OR value of 1.19 per copy of AMY1 translates into about an eightfold difference in risk of obesity between subjects in the top (copy number > 9) and bottom (copy number < 4) 10% of the copy number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies.

Aluminum tolerance in maize is associated with higher MATE1 gene copy number

PubMed Central

Maron, Lyza G.; Guimarães, Claudia T.; Kirst, Matias; Albert, Patrice S.; Birchler, James A.; Bradbury, Peter J.; Buckler, Edward S.; Coluccio, Alison E.; Danilova, Tatiana V.; Kudrna, David; Magalhaes, Jurandir V.; Piñeros, Miguel A.; Schatz, Michael C.; Wing, Rod A.; Kochian, Leon V.

2013-01-01

Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world’s potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments. PMID:23479633

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

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

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cellmore » in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

DOE PAGES

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.; ...

2018-01-25

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cellmore » in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

Different Facets of Copy Number Changes: Permanent, Transient, and Adaptive

PubMed Central

Mishra, Sweta

2016-01-01

Chromosomal copy number changes are frequently associated with harmful consequences and are thought of as an underlying mechanism for the development of diseases. However, changes in copy number are observed during development and occur during normal biological processes. In this review, we highlight the causes and consequences of copy number changes in normal physiologic processes as well as cover their associations with cancer and acquired drug resistance. We discuss the permanent and transient nature of copy number gains and relate these observations to a new mechanism driving transient site-specific copy gains (TSSGs). Finally, we discuss implications of TSSGs in generating intratumoral heterogeneity and tumor evolution and how TSSGs can influence the therapeutic response in cancer. PMID:26755558

Peripheral artery disease, calf skeletal muscle mitochondrial DNA copy number, and functional performance.

PubMed

McDermott, Mary M; Peterson, Charlotte A; Sufit, Robert; Ferrucci, Luigi; Guralnik, Jack M; Kibbe, Melina R; Polonsky, Tamar S; Tian, Lu; Criqui, Michael H; Zhao, Lihui; Stein, James H; Li, Lingyu; Leeuwenburgh, Christiaan

2018-05-01

In people without lower extremity peripheral artery disease (PAD), mitochondrial DNA copy number declines with aging, and this decline is associated with declines in mitochondrial activity and functional performance. However, whether lower extremity ischemia is associated with lower mitochondrial DNA copy number and whether mitochondrial DNA copy number is associated with the degree of functional impairment in people with PAD is unknown. In people with and without PAD, age 65 years and older, we studied associations of the ankle-brachial index (ABI) with mitochondrial DNA copy number and associations of mitochondrial DNA copy number with functional impairment. Calf muscle biopsies were obtained from 34 participants with PAD (mean age: 73.5 years (SD 6.4), mean ABI: 0.67 (SD 0.15), mean 6-minute walk distance: 1191 feet (SD 223)) and 10 controls without PAD (mean age: 73.1 years (SD 4.7), mean ABI: 1.14 (SD 0.07), mean 6-minute walk distance: 1387 feet (SD 488)). Adjusting for age and sex, lower ABI values were associated with higher mitochondrial DNA copy number, measured in relative copy number (ABI<0.60: 914, ABI 0.60-0.90: 731, ABI 0.90-1.50: 593; p trend=0.016). The association of mitochondrial DNA copy number with the 6-minute walk distance and 4-meter walking velocity differed significantly between participants with versus without PAD ( p-value for interaction=0.001 and p=0.015, respectively). The correlation coefficient between mitochondrial DNA copy number and the 6-minute walk distance was 0.653 ( p=0.056) among people without PAD and -0.254 ( p=0.154) among people with PAD and ABI < 0.90. In conclusion, lower ABI values are associated with increased mitochondrial DNA copy number. Associations of mitochondrial DNA copy number with the 6-minute walk distance and 4-meter walking velocity significantly differed between people with versus without PAD, with stronger positive associations observed in people without PAD than in people with PAD. The cross-sectional and exploratory nature of the analyses precludes conclusions regarding causal inferences. ClinicalTrials.gov Identifier: NCT02246660.

17 CFR 270.8b-11 - Number of copies; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; signatures... (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.8b-11 Number of copies; signatures... manner prescribed by the appropriate form. Unsigned copies shall be conformed. If the signature of any...

SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR.

PubMed

Stabley, Deborah L; Harris, Ashlee W; Holbrook, Jennifer; Chubbs, Nicholas J; Lozo, Kevin W; Crawford, Thomas O; Swoboda, Kathryn J; Funanage, Vicky L; Wang, Wenlan; Mackenzie, William; Scavina, Mena; Sol-Church, Katia; Butchbach, Matthew E R

2015-07-01

Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples.

Glyoxalase 1 copy number variation in patients with well differentiated gastro-entero-pancreatic neuroendocrine tumours (GEP-NET)

PubMed Central

Xue, Mingzhan; Shafie, Alaa; Qaiser, Talha; Rajpoot, Nasir M.; Kaltsas, Gregory; James, Sean; Gopalakrishnan, Kishore; Fisk, Adrian; Dimitriadis, Georgios K.; Grammatopoulos, Dimitris K.; Rabbani, Naila; Thornalley, Paul J.; Weickert, Martin O.

2017-01-01

Background The glyoxalase-1 gene (GLO1) is a hotspot for copy-number variation (CNV) in human genomes. Increased GLO1 copy-number is associated with multidrug resistance in tumour chemotherapy, but prevalence of GLO1 CNV in gastro-entero-pancreatic neuroendocrine tumours (GEP-NET) is unknown. Methods GLO1 copy-number variation was measured in 39 patients with GEP-NET (midgut NET, n = 25; pancreatic NET, n = 14) after curative or debulking surgical treatment. Primary tumour tissue, surrounding healthy tissue and, where applicable, additional metastatic tumour tissue were analysed, using real time qPCR. Progression and survival following surgical treatment were monitored over 4.2 ± 0.5 years. Results In the pooled GEP-NET cohort, GLO1 copy-number in healthy tissue was 2.0 in all samples but significantly increased in primary tumour tissue in 43% of patients with pancreatic NET and in 72% of patients with midgut NET, mainly driven by significantly higher GLO1 copy-number in midgut NET. In tissue from additional metastases resection (18 midgut NET and one pancreatic NET), GLO1 copy number was also increased, compared with healthy tissue; but was not significantly different compared with primary tumour tissue. During mean 3 - 5 years follow-up, 8 patients died and 16 patients showed radiological progression. In midgut NET, a high GLO1 copy-number was associated with earlier progression. In NETs with increased GLO1 copy number, there was increased Glo1 protein expression compared to non-malignant tissue. Conclusions GLO1 copy-number was increased in a large percentage of patients with GEP-NET and correlated positively with increased Glo1 protein in tumour tissue. Analysis of GLO1 copy-number variation particularly in patients with midgut NET could be a novel prognostic marker for tumour progression. PMID:29100361

Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

PubMed

Ooi, Delicia Shu Qin; Tan, Verena Ming Hui; Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat; Lee, Yung Seng

2017-01-01

The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

Prediction of Response to Therapy and Clinical Outcome through a Pilot Study of Complete Genetic Assessment of Ovarian Cancer

DTIC Science & Technology

2015-12-01

Oncology program supported by this grant consented patients to 11-104. OncoPanel is a cancer genomic assay that detects somatic mutations, copy number...KMT2D, EP300, FANCD2 Sertoli Leydig cell DICER1 Copy number variants: In addition, 219 patients were analyzed for copy-number variations ( CNV ) in...OncoPanel genes. >12,000 total CNV were reported in the cohort (Figure 2). Single- copy deletions (n=5558) and copy-number gains (low amplification) (n

Exonic duplication CNV of NDRG1 associated with autosomal-recessive HMSN-Lom/CMT4D.

PubMed

Okamoto, Yuji; Goksungur, Meryem Tuba; Pehlivan, Davut; Beck, Christine R; Gonzaga-Jauregui, Claudia; Muzny, Donna M; Atik, Mehmed M; Carvalho, Claudia M B; Matur, Zeliha; Bayraktar, Serife; Boone, Philip M; Akyuz, Kaya; Gibbs, Richard A; Battaloglu, Esra; Parman, Yesim; Lupski, James R

2014-05-01

Copy-number variations as a mutational mechanism contribute significantly to human disease. Approximately one-half of the patients with Charcot-Marie-Tooth (CMT) disease have a 1.4 Mb duplication copy-number variation as the cause of their neuropathy. However, non-CMT1A neuropathy patients rarely have causative copy-number variations, and to date, autosomal-recessive disease has not been associated with copy-number variation as a mutational mechanism. We performed Agilent 8 × 60 K array comparative genomic hybridization on DNA from 12 recessive Turkish families with CMT disease. Additional molecular studies were conducted to detect breakpoint junctions and to evaluate gene expression levels in a family in which we detected an intragenic duplication copy-number variation. We detected an ~6.25 kb homozygous intragenic duplication in NDRG1, a gene known to be causative for recessive HMSNL/CMT4D, in three individuals from a Turkish family with CMT neuropathy. Further studies showed that this intragenic copy-number variation resulted in a homozygous duplication of exons 6-8 that caused decreased mRNA expression of NDRG1. Exon-focused high-resolution array comparative genomic hybridization enables the detection of copy-number variation carrier states in recessive genes, particularly small copy-number variations encompassing or disrupting single genes. In families for whom a molecular diagnosis has not been elucidated by conventional clinical assays, an assessment for copy-number variations in known CMT genes might be considered.

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants

PubMed Central

Kwan, Elizabeth X.; Wang, Xiaobin S.; Amemiya, Haley M.; Brewer, Bonita J.; Raghuraman, M. K.

2016-01-01

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. PMID:27449518

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants.

PubMed

Kwan, Elizabeth X; Wang, Xiaobin S; Amemiya, Haley M; Brewer, Bonita J; Raghuraman, M K

2016-09-08

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. Copyright © 2016 Kwan et al.

Gene copy number variation and its significance in cyanobacterial phylogeny

PubMed Central

2012-01-01

Background In eukaryotes, variation in gene copy numbers is often associated with deleterious effects, but may also have positive effects. For prokaryotes, studies on gene copy number variation are rare. Previous studies have suggested that high numbers of rRNA gene copies can be advantageous in environments with changing resource availability, but further association of gene copies and phenotypic traits are not documented. We used one of the morphologically most diverse prokaryotic phyla to test whether numbers of gene copies are associated with levels of cell differentiation. Results We implemented a search algorithm that identified 44 genes with highly conserved copies across 22 fully sequenced cyanobacterial taxa. For two very basal cyanobacterial species, Gloeobacter violaceus and a thermophilic Synechococcus species, distinct phylogenetic positions previously found were supported by identical protein coding gene copy numbers. Furthermore, we found that increased ribosomal gene copy numbers showed a strong correlation to cyanobacteria capable of terminal cell differentiation. Additionally, we detected extremely low variation of 16S rRNA sequence copies within the cyanobacteria. We compared our results for 16S rRNA to three other eubacterial phyla (Chroroflexi, Spirochaetes and Bacteroidetes). Based on Bayesian phylogenetic inference and the comparisons of genetic distances, we could confirm that cyanobacterial 16S rRNA paralogs and orthologs show significantly stronger conservation than found in other eubacterial phyla. Conclusions A higher number of ribosomal operons could potentially provide an advantage to terminally differentiated cyanobacteria. Furthermore, we suggest that 16S rRNA gene copies in cyanobacteria are homogenized by both concerted evolution and purifying selection. In addition, the small ribosomal subunit in cyanobacteria appears to evolve at extraordinary slow evolutionary rates, an observation that has been made previously for morphological characteristics of cyanobacteria. PMID:22894826

Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease.

PubMed

Aldhous, Marian C; Abu Bakar, Suhaili; Prescott, Natalie J; Palla, Raquel; Soo, Kimberley; Mansfield, John C; Mathew, Christopher G; Satsangi, Jack; Armour, John A L

2010-12-15

The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case-control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case-control studies.

A comprehensive profile of DNA copy number variations in a Korean population: identification of copy number invariant regions among Koreans.

PubMed

Jeon, Jae Pil; Shim, Sung Mi; Jung, Jong Sun; Nam, Hye Young; Lee, Hye Jin; Oh, Berm Seok; Kim, Kuchan; Kim, Hyung Lae; Han, Bok Ghee

2009-09-30

To examine copy number variations among the Korean population, we compared individual genomes with the Korean reference genome assembly using the publicly available Korean HapMap SNP 50 k chip data from 90 individuals. Korean individuals exhibited 123 copy number variation regions (CNVRs) covering 27.2 mb, equivalent to 1.0% of the genome in the copy number variation (CNV) analysis using the combined criteria of P value (P<0.01) and standard deviation of copy numbers (SD>or= 0.25) among study subjects. In contrast, when compared to the Affymetrix reference genome assembly from multiple ethnic groups, considerably more CNVRs (n=643) were detected in larger proportions (5.0%) of the genome covering 135.1 mb even by more stringent criteria (P<0.001 and SD>or=0.25), reflecting ethnic diversity of structural variations between Korean and other populations. Some CNVRs were validated by the quantitative multiplex PCR of short fluorescent fragment (QMPSF) method, and then copy number invariant regions were detected among the study subjects. These copy number invariant regions would be used as good internal controls for further CNV studies. Lastly, we demonstrated that the CNV information could stratify even a single ethnic population with a proper reference genome assembly from multiple heterogeneous populations.

Real-time PCR assays for monitoring anaerobic fungal biomass and population size in the rumen.

PubMed

Lwin, Khin Ohnmar; Hayakawa, Mika; Ban-Tokuda, Tomomi; Matsui, Hiroki

2011-04-01

The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.

Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease

PubMed Central

Aldhous, Marian C.; Abu Bakar, Suhaili; Prescott, Natalie J.; Palla, Raquel; Soo, Kimberley; Mansfield, John C.; Mathew, Christopher G.; Satsangi, Jack; Armour, John A.L.

2010-01-01

The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case–control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case–control studies. PMID:20858604

NEDDylation promotes stress granule assembly

PubMed Central

Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum-Yong; Baek, Je-Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

2016-01-01

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly. PMID:27381497

Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting

PubMed Central

Fu, Suneng; Fan, Jason; Blanco, Joshua; Gimenez-Cassina, Alfredo; Danial, Nika N.; Watkins, Steve M.; Hotamisligil, Gökhan S.

2012-01-01

Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)–associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity. PMID:22927828

Polysome profiling in liver identifies dynamic regulation of endoplasmic reticulum translatome by obesity and fasting.

PubMed

Fu, Suneng; Fan, Jason; Blanco, Joshua; Gimenez-Cassina, Alfredo; Danial, Nika N; Watkins, Steve M; Hotamisligil, Gökhan S

2012-08-01

Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)-associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity.

17 CFR 260.7a-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.7a-3 Section 260.7a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.7a-3 Number of copies; filing; signatures; binding. (a) Three copies of the complete application...

17 CFR 260.4c-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.4c-3 Section 260.4c-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.4c-3 Number of copies; filing; signatures; binding. (a) Three copies of every application and of...

17 CFR 260.5a-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.5a-3 Section 260.5a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.5a-3 Number of copies; filing; signatures; binding. (a) Three copies of each statement of...

48 CFR 1852.246-72 - Material inspection and receiving report.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Receiving Report (DD Form 250 series) prepared in __ [Insert number of copies, including original] copies, an original and __ copies [Insert number of copies]. (b) The Contractor shall prepare the DD Form 250 in accordance with NASA FAR Supplement 1846.6. The Contractor shall enclose the copies of the DD Form...

Interpretation of clinical relevance of X-chromosome copy number variations identified in a large cohort of individuals with cognitive disorders and/or congenital anomalies.

PubMed

Willemsen, Marjolein H; de Leeuw, Nicole; de Brouwer, Arjan P M; Pfundt, Rolph; Hehir-Kwa, Jayne Y; Yntema, Helger G; Nillesen, Willy M; de Vries, Bert B A; van Bokhoven, Hans; Kleefstra, Tjitske

2012-11-01

Genome-wide array studies are now routinely being used in the evaluation of patients with cognitive disorders (CD) and/or congenital anomalies (CA). Therefore, inevitably each clinician is confronted with the challenging task of the interpretation of copy number variations detected by genome-wide array platforms in a diagnostic setting. Clinical interpretation of autosomal copy number variations is already challenging, but assessment of the clinical relevance of copy number variations of the X-chromosome is even more complex. This study provides an overview of the X-Chromosome copy number variations that we have identified by genome-wide array analysis in a large cohort of 4407 male and female patients. We have made an interpretation of the clinical relevance of each of these copy number variations based on well-defined criteria and previous reports in literature and databases. The prevalence of X-chromosome copy number variations in this cohort was 57/4407 (∼1.3%), of which 15 (0.3%) were interpreted as (likely) pathogenic. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Statistical tools for transgene copy number estimation based on real-time PCR.

PubMed

Yuan, Joshua S; Burris, Jason; Stewart, Nathan R; Mentewab, Ayalew; Stewart, C Neal

2007-11-01

As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination. These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification.

Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders.

PubMed

Carpenter, Danielle; Walker, Susan; Prescott, Natalie; Schalkwijk, Joost; Armour, John Al

2011-08-18

Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT) method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts. We evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis. Differential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion.

Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders

PubMed Central

2011-01-01

Background Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT) method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts. Results We evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis. Conclusions Differential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion. PMID:21851606

Mitochondrial DNA copy number in peripheral blood cell and hypertension risk among mining workers: a case-control study in Chinese coal miners.

PubMed

Lei, L; Guo, J; Shi, X; Zhang, G; Kang, H; Sun, C; Huang, J; Wang, T

2017-09-01

Alteration of mitochondrial DNA (mtDNA) copy number, which reflects oxidant-induced cell damage, has been observed in a wide range of human diseases. However, whether it correlates with hypertension has not been elucidated. We aimed to explore the association between mtDNA copy number and the risk of hypertension in Chinese coal miners. A case-control study was performed with 378 hypertension patients and 325 healthy controls in a large coal mining group located in North China. Face-to-face interviews were conducted by trained staffs with necessary medical knowledge. The mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral blood. No significant differences in mtDNA copy number were observed between hypertension patients and healthy controls. However, in both case and control groups, the mtDNA copy number was statistically significantly lower in the elder population (≥45 years old) compared with the younger subjects (<45 years old; 7.17 vs 6.64, P=0.005 and 7.21 vs 6.84, P=0.036). A significantly higher mtDNA copy number could be found in hypertension patients consuming alcohol regularly compared with no alcohol consumption patients (7.09 vs 6.69); mtDNA copy number was also positively correlated with age and alcohol consumption. Hypertension was found significantly correlated with factors such as age, work duration, monthly family income and drinking status. Our results suggest that the mtDNA copy number is not associated with hypertension in coal miners.

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes

PubMed Central

Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

2014-01-01

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. PMID:24975239

TEGS-CN: A Statistical Method for Pathway Analysis of Genome-wide Copy Number Profile.

PubMed

Huang, Yen-Tsung; Hsu, Thomas; Christiani, David C

2014-01-01

The effects of copy number alterations make up a significant part of the tumor genome profile, but pathway analyses of these alterations are still not well established. We proposed a novel method to analyze multiple copy numbers of genes within a pathway, termed Test for the Effect of a Gene Set with Copy Number data (TEGS-CN). TEGS-CN was adapted from TEGS, a method that we previously developed for gene expression data using a variance component score test. With additional development, we extend the method to analyze DNA copy number data, accounting for different sizes and thus various numbers of copy number probes in genes. The test statistic follows a mixture of X (2) distributions that can be obtained using permutation with scaled X (2) approximation. We conducted simulation studies to evaluate the size and the power of TEGS-CN and to compare its performance with TEGS. We analyzed a genome-wide copy number data from 264 patients of non-small-cell lung cancer. With the Molecular Signatures Database (MSigDB) pathway database, the genome-wide copy number data can be classified into 1814 biological pathways or gene sets. We investigated associations of the copy number profile of the 1814 gene sets with pack-years of cigarette smoking. Our analysis revealed five pathways with significant P values after Bonferroni adjustment (<2.8 × 10(-5)), including the PTEN pathway (7.8 × 10(-7)), the gene set up-regulated under heat shock (3.6 × 10(-6)), the gene sets involved in the immune profile for rejection of kidney transplantation (9.2 × 10(-6)) and for transcriptional control of leukocytes (2.2 × 10(-5)), and the ganglioside biosynthesis pathway (2.7 × 10(-5)). In conclusion, we present a new method for pathway analyses of copy number data, and causal mechanisms of the five pathways require further study.

Copy Number Variation across European Populations

PubMed Central

Chen, Wanting; Hayward, Caroline; Wright, Alan F.; Hicks, Andrew A.; Vitart, Veronique; Knott, Sara; Wild, Sarah H.; Pramstaller, Peter P.; Wilson, James F.; Rudan, Igor; Porteous, David J.

2011-01-01

Genome analysis provides a powerful approach to test for evidence of genetic variation within and between geographical regions and local populations. Copy number variants which comprise insertions, deletions and duplications of genomic sequence provide one such convenient and informative source. Here, we investigate copy number variants from genome wide scans of single nucleotide polymorphisms in three European population isolates, the island of Vis in Croatia, the islands of Orkney in Scotland and the South Tyrol in Italy. We show that whereas the overall copy number variant frequencies are similar between populations, their distribution is highly specific to the population of origin, a finding which is supported by evidence for increased kinship correlation for specific copy number variants within populations. PMID:21829696

Integrative pipeline for profiling DNA copy number and inferring tumor phylogeny.

PubMed

Urrutia, Eugene; Chen, Hao; Zhou, Zilu; Zhang, Nancy R; Jiang, Yuchao

2018-06-15

Copy number variation is an important and abundant source of variation in the human genome, which has been associated with a number of diseases, especially cancer. Massively parallel next-generation sequencing allows copy number profiling with fine resolution. Such efforts, however, have met with mixed successes, with setbacks arising partly from the lack of reliable analytical methods to meet the diverse and unique challenges arising from the myriad experimental designs and study goals in genetic studies. In cancer genomics, detection of somatic copy number changes and profiling of allele-specific copy number (ASCN) are complicated by experimental biases and artifacts as well as normal cell contamination and cancer subclone admixture. Furthermore, careful statistical modeling is warranted to reconstruct tumor phylogeny by both somatic ASCN changes and single nucleotide variants. Here we describe a flexible computational pipeline, MARATHON, which integrates multiple related statistical software for copy number profiling and downstream analyses in disease genetic studies. MARATHON is publicly available at https://github.com/yuchaojiang/MARATHON. Supplementary data are available at Bioinformatics online.

Survival differences of CIMP subtypes integrated with CNA information in human breast cancer.

PubMed

Wang, Huihan; Yan, Weili; Zhang, Shumei; Gu, Yue; Wang, Yihan; Wei, Yanjun; Liu, Hongbo; Wang, Fang; Wu, Qiong; Zhang, Yan

2017-07-25

CpG island methylator phenotype of breast cancer is associated with widespread aberrant methylation at specified CpG islands and distinct patient outcomes. However, the influence of copy number contributing to the prognosis of tumors with different CpG island methylator phenotypes is still unclear. We analyzed both genetic (copy number) and epigenetic alterations in 765 breast cancers from The Cancer Genome Atlas data portal and got a panel of 15 biomarkers for copy number and methylation status evaluation. The gene panel identified two groups corresponding to distinct copy number profiles. In status of mere-loss copy number, patients were faced with a greater risk if they presented a higher CpG islands methylation pattern in biomarker panels. But for samples presenting merely-gained copy number, higher methylation level of CpG islands was associated with improved viability. In all, the integration of copy number alteration and methylation information enhanced the classification power on prognosis. Moreover, we found the molecular subtypes of breast cancer presented different distributions in two CpG island methylation phenotypes. Generated by the same set of human methylation 450K data, additional copy number information could provide insights into survival prediction of cancers with less heterogeneity and might help to determine the biomarkers for diagnosis and treatment for breast cancer patients in a more personalized approach.

Survival differences of CIMP subtypes integrated with CNA information in human breast cancer

PubMed Central

Wang, Huihan; Yan, Weili; Zhang, Shumei; Gu, Yue; Wang, Yihan; Wei, Yanjun; Liu, Hongbo; Wang, Fang; Wu, Qiong; Zhang, Yan

2017-01-01

CpG island methylator phenotype of breast cancer is associated with widespread aberrant methylation at specified CpG islands and distinct patient outcomes. However, the influence of copy number contributing to the prognosis of tumors with different CpG island methylator phenotypes is still unclear. We analyzed both genetic (copy number) and epigenetic alterations in 765 breast cancers from The Cancer Genome Atlas data portal and got a panel of 15 biomarkers for copy number and methylation status evaluation. The gene panel identified two groups corresponding to distinct copy number profiles. In status of mere-loss copy number, patients were faced with a greater risk if they presented a higher CpG islands methylation pattern in biomarker panels. But for samples presenting merely-gained copy number, higher methylation level of CpG islands was associated with improved viability. In all, the integration of copy number alteration and methylation information enhanced the classification power on prognosis. Moreover, we found the molecular subtypes of breast cancer presented different distributions in two CpG island methylation phenotypes. Generated by the same set of human methylation 450K data, additional copy number information could provide insights into survival prediction of cancers with less heterogeneity and might help to determine the biomarkers for diagnosis and treatment for breast cancer patients in a more personalized approach. PMID:28415743

Integrated genome-wide DNA copy number and expression analysis identifies distinct mechanisms of primary chemoresistance in ovarian carcinomas.

PubMed

Etemadmoghadam, Dariush; deFazio, Anna; Beroukhim, Rameen; Mermel, Craig; George, Joshy; Getz, Gad; Tothill, Richard; Okamoto, Aikou; Raeder, Maria B; Harnett, Paul; Lade, Stephen; Akslen, Lars A; Tinker, Anna V; Locandro, Bianca; Alsop, Kathryn; Chiew, Yoke-Eng; Traficante, Nadia; Fereday, Sian; Johnson, Daryl; Fox, Stephen; Sellers, William; Urashima, Mitsuyoshi; Salvesen, Helga B; Meyerson, Matthew; Bowtell, David

2009-02-15

A significant number of women with serous ovarian cancer are intrinsically refractory to platinum-based treatment. We analyzed somatic DNA copy number variation and gene expression data to identify key mechanisms associated with primary resistance in advanced-stage serous cancers. Genome-wide copy number variation was measured in 118 ovarian tumors using high-resolution oligonucleotide microarrays. A well-defined subset of 85 advanced-stage serous tumors was then used to relate copy number variation to primary resistance to treatment. The discovery-based approach was complemented by quantitative-PCR copy number analysis of 12 candidate genes as independent validation of previously reported associations with clinical outcome. Likely copy number variation targets and tumor molecular subtypes were further characterized by gene expression profiling. Amplification of 19q12, containing cyclin E (CCNE1), and 20q11.22-q13.12, mapping immediately adjacent to the steroid receptor coactivator NCOA3, was significantly associated with poor response to primary treatment. Other genes previously associated with copy number variation and clinical outcome in ovarian cancer were not associated with primary treatment resistance. Chemoresistant tumors with high CCNE1 copy number and protein expression were associated with increased cellular proliferation but so too was a subset of treatment-responsive patients, suggesting a cell-cycle independent role for CCNE1 in modulating chemoresponse. Patients with a poor clinical outcome without CCNE1 amplification overexpressed genes involved in extracellular matrix deposition. We have identified two distinct mechanisms of primary treatment failure in serous ovarian cancer, involving CCNE1 amplification and enhanced extracellular matrix deposition. CCNE1 copy number is validated as a dominant marker of patient outcome in ovarian cancer.

A Simulation Model for Purchasing Duplicate Copies in a Library

ERIC Educational Resources Information Center

Arms, W. Y.; Walter, T. P.

1974-01-01

A method of estimating the number of duplicate copies of books needed based on a computer simulation which takes into account number of copies available, number of loans, total underlying demand, satisfaction level, percentage time on shelf. (LS)

Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus.

PubMed

Black, Holly A; Khan, Fayeza F; Tyson, Jess; Al Armour, John

2014-07-21

The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a region of high linkage disequilibrium, despite its high variability in copy number (n = 3-16); hence, the mechanisms responsible for changes in copy number at this locus are unclear. In this study, a region flanking the DEFA1A3 locus was sequenced across 120 independent haplotypes with European ancestry, identifying five common classes of DEFA1A3 haplotype. Assigning DEFA1A3 class to haplotypes within the 1000 Genomes project highlights a significant difference in DEFA1A3 class frequencies between populations with different ancestry. The features of each DEFA1A3 class, for example, the associated DEFA1A3 copy numbers, were initially assessed in a European cohort (n = 599) and replicated in the 1000 Genomes samples, showing within-class similarity, but between-class and between-population differences in the features of the DEFA1A3 locus. Emulsion haplotype fusion-PCR was used to generate 61 structural haplotypes at the DEFA1A3 locus, showing a high within-class similarity in structure. Structural haplotypes across the DEFA1A3 locus indicate that intra-allelic rearrangement is the predominant mechanism responsible for changes in DEFA1A3 copy number, explaining the conservation of linkage disequilibrium across the locus. The identification of common structural haplotypes at the DEFA1A3 locus could aid studies into how DEFA1A3 copy number influences expression, which is currently unclear.

Interpreting aCGH-defined karyotypic changes in gliomas using copy number status, loss of heterozygosity and allelic ratios

PubMed Central

Cowell, John K; Lo, Ken C; Luce, Jesse; Hawthorn, Lesleyann

2009-01-01

We have used SNP mapping arrays to simultaneously record copy number changes, loss of heterozygosity and allele ratios (ploidy) in a series of 13 gliomas. This combined analysis has defined novel amplification events in this tumor type involving chr1:241544532-243005121 and chr18:54716681-54917277 which contain the AKT3 and ZNF532 genes respectively. The high resolution of this analysis has also identified homozygous deletions involving chr17:25600031-26490848 and Chr19:53883612-55061878. Throughout the karyotypes of these tumors, the combined analysis revealed counter intuitive relationships between copy number and LOH that requires reinterpretation of the significance of copy number gains and losses. It was not uncommon to observe copy number gains that were associated with loss of heterozygosity as well as copy number losses that were not. These events appeared to be related to ploidy status in the tumors as determined using allelic ratio calculations. Overall, this analysis of gliomas provides evidence for the need to perform more comprehensive interpretation of the CGH data beyond copy number analysis alone to evaluate the significance of individual events in the karyotypes. PMID:19818351

Multiply to conquer: Copy number variations at Ppd-B1 and Vrn-A1 facilitate global adaptation in wheat.

PubMed

Würschum, Tobias; Boeven, Philipp H G; Langer, Simon M; Longin, C Friedrich H; Leiser, Willmar L

2015-07-29

Copy number variation was found to be a frequent type of DNA polymorphism in the human genome often associated with diseases but its importance in crops and the effects on agronomic traits are still largely unknown. Here, we employed a large worldwide panel of 1110 winter wheat varieties to assess the frequency and the geographic distribution of copy number variants at the Photoperiod-B1 (Ppd-B1) and the Vernalization-A1 (Vrn-A1) loci as well as their effects on flowering time under field conditions. We identified a novel four copy variant of Vrn-A1 and based on the phylogenetic relationships among the lines show that the higher copy variants at both loci are likely to have arisen independently multiple times. In addition, we found that the frequency of the different copy number variants at both loci reflects the environmental conditions in the varieties' region of origin and based on multi-location field trials show that Ppd-B1 copy number has a substantial effect on the fine-tuning of flowering time. In conclusion, our results show the importance of copy number variation at Ppd-B1 and Vrn-A1 for the global adaptation of wheat making it a key factor for wheat success in a broad range of environments and in a wider context substantiate the significant role of copy number variation in crops.

Selective sweep on human amylase genes postdates the split with Neanderthals

PubMed Central

Inchley, Charlotte E.; Larbey, Cynthia D. A.; Shwan, Nzar A. A.; Pagani, Luca; Saag, Lauri; Antão, Tiago; Jacobs, Guy; Hudjashov, Georgi; Metspalu, Ene; Mitt, Mario; Eichstaedt, Christina A.; Malyarchuk, Boris; Derenko, Miroslava; Wee, Joseph; Abdullah, Syafiq; Ricaut, François-Xavier; Mormina, Maru; Mägi, Reedik; Villems, Richard; Metspalu, Mait; Jones, Martin K.; Armour, John A. L.; Kivisild, Toomas

2016-01-01

Humans have more copies of amylase genes than other primates. It is still poorly understood, however, when the copy number expansion occurred and whether its spread was enhanced by selection. Here we assess amylase copy numbers in a global sample of 480 high coverage genomes and find that regions flanking the amylase locus show notable depression of genetic diversity both in African and non-African populations. Analysis of genetic variation in these regions supports the model of an early selective sweep in the human lineage after the split of humans from Neanderthals which led to the fixation of multiple copies of AMY1 in place of a single copy. We find evidence of multiple secondary losses of copy number with the highest frequency (52%) of a deletion of AMY2A and associated low copy number of AMY1 in Northeast Siberian populations whose diet has been low in starch content. PMID:27853181

Selective sweep on human amylase genes postdates the split with Neanderthals.

PubMed

Inchley, Charlotte E; Larbey, Cynthia D A; Shwan, Nzar A A; Pagani, Luca; Saag, Lauri; Antão, Tiago; Jacobs, Guy; Hudjashov, Georgi; Metspalu, Ene; Mitt, Mario; Eichstaedt, Christina A; Malyarchuk, Boris; Derenko, Miroslava; Wee, Joseph; Abdullah, Syafiq; Ricaut, François-Xavier; Mormina, Maru; Mägi, Reedik; Villems, Richard; Metspalu, Mait; Jones, Martin K; Armour, John A L; Kivisild, Toomas

2016-11-17

Humans have more copies of amylase genes than other primates. It is still poorly understood, however, when the copy number expansion occurred and whether its spread was enhanced by selection. Here we assess amylase copy numbers in a global sample of 480 high coverage genomes and find that regions flanking the amylase locus show notable depression of genetic diversity both in African and non-African populations. Analysis of genetic variation in these regions supports the model of an early selective sweep in the human lineage after the split of humans from Neanderthals which led to the fixation of multiple copies of AMY1 in place of a single copy. We find evidence of multiple secondary losses of copy number with the highest frequency (52%) of a deletion of AMY2A and associated low copy number of AMY1 in Northeast Siberian populations whose diet has been low in starch content.

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes.

PubMed

Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

2014-10-01

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. © 2014 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.

Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

PubMed

Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

2015-04-02

Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

ALK gene copy number gain and immunohistochemical expression status using three antibodies in neuroblastoma.

PubMed

Kim, Eun Kyung; Kim, Sewha

2016-03-17

Anaplastic lymphoma kinase (ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC positive rate in ALK1 and 5A4 antibodies (p= < 0.001 and 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

ALK Gene Copy Number Gain and Immunohistochemical Expression Status Using Three Antibodies in Neuroblastoma.

PubMed

Kim, Eun Kyung; Kim, Sewha

2017-01-01

Anaplastic lymphoma kinase ( ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC-positive rate in ALK1 and 5A4 antibodies ( P < 0.001 and P = 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

Association of mitochondrial DNA in peripheral blood with depression, anxiety and stress- and adjustment disorders in primary health care patients.

PubMed

Wang, Xiao; Sundquist, Kristina; Rastkhani, Hamideh; Palmér, Karolina; Memon, Ashfaque A; Sundquist, Jan

2017-08-01

Mitochondrial dysfunction may result in a variety of diseases. The objectives here were to examine possible differences in mtDNA copy number between healthy controls and patients with depression, anxiety or stress- and adjustment disorders; the association between mtDNA copy number and disease severity at baseline; and the association between mtDNA copy number and response after an 8-week treatment (mindfulness, cognitive based therapy). A total of 179 patients in primary health care (age 20-64 years) with depression, anxiety and stress- and adjustment disorders, and 320 healthy controls (aged 19-70 years) were included in the study. Relative mtDNA copy number was measured using quantitative real-time PCR on peripheral blood samples. We found that the mean mtDNA copy number was significantly higher in patients compared to controls (84.9 vs 75.9, p<0.0001) at baseline. The difference in mtDNA copy number between patients and controls remained significant after controlling for age and sex (ß=8.13, p<0.0001; linear regression analysis). The mtDNA copy number was significantly associated with Patient Health Questionnaire (PHQ-9) scores (β=0.57, p=0.02) at baseline. After treatment, the change in mtDNA copy number was significantly associated with the treatment response, i.e., change in Hospital Anxiety and Depression Scale (HADS-D) and PHQ-9 scores (ß=1.00, p=0.03 and ß=0.65, p=0.04, respectively), after controlling for baseline scores, age, sex, BMI, smoking status, alcohol drinking and medication. Our findings show that mtDNA copy number is associated with symptoms of depression, anxiety and stress- and adjustment disorders and treatment response in these disorders. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins.

PubMed

Wrede, Joanna E; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F

2015-10-01

Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins. Academic clinical research center. 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a "normal" (7-9 h/24) and "short" (< 7 h/24) sleeping twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data structure to assess within-pair effects of sleep duration on mtDNA copy number. Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. © 2015 Associated Professional Sleep Societies, LLC.

DNA replication stress restricts ribosomal DNA copy number.

PubMed

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

PubMed

Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

2017-01-01

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

PubMed Central

Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

2017-01-01

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future. PMID:28085908

Probing the closed-loop model of mRNA translation in living cells

PubMed Central

Archer, Stuart K; Shirokikh, Nikolay E; Hallwirth, Claus V; Beilharz, Traude H; Preiss, Thomas

2015-01-01

The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression. PMID:25826658

Translational Control of Hemoglobin Synthesis by an Initiation Factor Required for Recycling of Ribosomes and for their Binding to Messenger RNA

PubMed Central

Kaempfer, Raymond; Kaufman, Jennifer

1972-01-01

The continued recycling of ribosomes during protein synthesis in rabbit reticulocyte lysates at 37° requires an initiation factor whose activity is rapidly lost in the absence of added heme. Partially purified factor (i) fully maintains the polysomes; (ii) inhibits the association of 40S and 60S ribosomal subunits into single ribosomes; (iii) promotes the quantitative entry of added 60S subunits into polysomes; (iv) allows the accumulation of ribosomal subunits, instead of single ribosomes, when initiation is blocked with aurin tricarboxylate; and (v) is absolutely required for the binding of globin messenger RNA to ribosomes. These properties suggest that this mammalian initiation factor functions analogously to bacterial IF-3. In addition, the translational control of globin synthesis by heme is exerted, directly or indirectly, through this factor. PMID:4508325

aCGH Local Copy Number Aberrations Associated with Overall Copy Number Genomic Instability in Colorectal Cancer: Coordinate Involvement of the Regions Including BCR and ABL

PubMed Central

Bartos, Jeremy D.; Gaile, Daniel P.; McQuaid, Devin E.; Conroy, Jeffrey M.; Darbary, Huferesh; Nowak, Norma J.; Block, Annemarie; Petrelli, Nicholas J.; Mittelman, Arnold; Stoler, Daniel L.; Anderson, Garth R.

2007-01-01

In order to identify small regions of the genome whose specific copy number alteration is associated with high genomic instability in the form of overall genome-wide copy number aberrations, we have analyzed array-based comparative genomic hybridization (aCGH) data from 33 sporadic colorectal carcinomas. Copy number changes of a small number of specific regions were significantly correlated with elevated overall amplifications and deletions scattered throughout the entire genome. One significant region at 9q34 includes the c-ABL gene Another region spanning 22q11–13 includes the breakpoint cluster region (BCR) of the Philadelphia chromosome Coordinate 22q11–13 alterations were observed in nine of eleven tumors with the 9q34 alteration Additional regions on 1q and 14q were associated with overall genome-wide copy number changes, while copy number aberrations on chromosome 7p, 7q, and 13q21.1–31.3 were found associated with this instability only in tumors from patients with a smoking history Our analysis demonstrates there are a small number of regions of the genome where gain or loss is commonly associated with a tumor’s overall level of copy number aberrations Our finding BCR and ABL located within two of the instability-associated regions, and the involvement of these two regions occurring coordinately, suggests a system akin to the BCR-ABL translocation of CML may be involved in genomic instability in about one-third of human colorectal carcinomas. PMID:17196995

Hacking DNA copy number for circuit engineering.

PubMed

Wu, Feilun; You, Lingchong

2017-07-27

DNA copy number represents an essential parameter in the dynamics of synthetic gene circuits but typically is not explicitly considered. A new study demonstrates how dynamic control of DNA copy number can serve as an effective strategy to program robust oscillations in gene expression circuits.

22 CFR 1429.25 - Number of copies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Number of copies. 1429.25 Section 1429.25 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL... AND GENERAL REQUIREMENTS General Requirements § 1429.25 Number of copies. Unless otherwise provided by...

DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer

PubMed Central

Mamlouk, Soulafa; Childs, Liam Harold; Aust, Daniela; Heim, Daniel; Melching, Friederike; Oliveira, Cristiano; Wolf, Thomas; Durek, Pawel; Schumacher, Dirk; Bläker, Hendrik; von Winterfeld, Moritz; Gastl, Bastian; Möhr, Kerstin; Menne, Andrea; Zeugner, Silke; Redmer, Torben; Lenze, Dido; Tierling, Sascha; Möbs, Markus; Weichert, Wilko; Folprecht, Gunnar; Blanc, Eric; Beule, Dieter; Schäfer, Reinhold; Morkel, Markus; Klauschen, Frederick; Leser, Ulf; Sers, Christine

2017-01-01

Genetic heterogeneity between and within tumours is a major factor determining cancer progression and therapy response. Here we examined DNA sequence and DNA copy-number heterogeneity in colorectal cancer (CRC) by targeted high-depth sequencing of 100 most frequently altered genes. In 97 samples, with primary tumours and matched metastases from 27 patients, we observe inter-tumour concordance for coding mutations; in contrast, gene copy numbers are highly discordant between primary tumours and metastases as validated by fluorescent in situ hybridization. To further investigate intra-tumour heterogeneity, we dissected a single tumour into 68 spatially defined samples and sequenced them separately. We identify evenly distributed coding mutations in APC and TP53 in all tumour areas, yet highly variable gene copy numbers in numerous genes. 3D morpho-molecular reconstruction reveals two clusters with divergent copy number aberrations along the proximal–distal axis indicating that DNA copy number variations are a major source of tumour heterogeneity in CRC. PMID:28120820

[Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

PubMed

Zhao, H; Teng, X M; Li, Y F

2017-11-25

Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all P< 0.05]. The copy number of mtDNA and the mitochondrial membrane potential in classⅠ frozen embryo group were significantly higher than those in classⅡ frozen embryo group (both P< 0.05). Conclusion: The mtDNA copy number and the mitochondrial membrane potential of embryos of the better quality embryo are higher.

14 CFR 221.92 - Number of copies required.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Number of copies required. 221.92 Section 221.92 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS TARIFFS Filing Tariff Publications With Department § 221.92 Number of copies...

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

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

Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying

2013-11-15

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletionmore » and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.« less

Copy Number Variations of TBK1 in Australian Patients With Primary Open-Angle Glaucoma

PubMed Central

AWADALLA, MONA S.; FINGERT, JOHN H.; ROOS, BENJAMIN E.; CHEN, SIMON; HOLMES, RICHARD; GRAHAM, STUART L.; CHEHADE, MARK; GALANOPOLOUS, ANNA; RIDGE, BRONWYN; SOUZEAU, EMMANUELLE; ZHOU, TIGER; SIGGS, OWEN M.; HEWITT, ALEX W.; MACKEY, DAVID A.; BURDON, KATHRYN P.; CRAIG, JAMIE E.

2015-01-01

PURPOSE To investigate the presence of TBK1 copy number variations in a large, well-characterized Australian cohort of patients with glaucoma comprising both normal-tension glaucoma and high-tension glaucoma cases. DESIGN A retrospective cohort study. METHODS DNA samples from patients with normal-tension glaucoma and high-tension glaucoma and unaffected controls were screened for TBK1 copy number variations using real-time quantitative polymerase chain reaction. Samples with additional copies of the TBK1 gene were further tested using custom comparative genomic hybridization arrays. RESULTS Four out of 334 normal-tension glaucoma cases (1.2%) were found to carry TBK1 copy number variations using quantitative polymerase chain reaction. One extra dose of the TBK1 gene (duplication) was detected in 3 normal-tension glaucoma patients, while 2 extra doses of the gene (triplication) were detected in a fourth normal-tension glaucoma patient. The results were further confirmed by custom comparative genomic hybridization arrays. Further, the TBK1 copy number variation segregated with normal-tension glaucoma in the family members of the probands, showing an autosomal dominant pattern of inheritance. No TBK1 copy number variations were detected in 1045 Australian patients with high-tension glaucoma or in 254 unaffected controls. CONCLUSION We report the presence of TBK1 copy number variations in our Australian normal-tension glaucoma cohort, including the first example of more than 1 extra copy of this gene in glaucoma patients (gene triplication). These results confirm TBK1 to be an important cause of normal-tension glaucoma, but do not suggest common involvement in high-tension glaucoma. PMID:25284765

Association of Higher Defensin β-4 Genomic Copy Numbers with Behçet's Disease in Iraqi Patients.

PubMed

Hameed, Ammar F; Jaradat, Sameh; Al-Musawi, Bassam M; Sharquie, Khalifa; Ibrahim, Mazin J; Hayani, Raafa K; Norgauer, Johannes

2015-11-01

Behçet's disease (BD) is an immune-mediated small vessel systemic vasculitis. Human β-defensins are antimicrobial peptides associated with many inflammatory diseases and are encoded by the β-defensin family of multiple-copy genes. However, their role in BD necessitates further investigation. The aim of the present study was to investigate the possible association of BD in its various clinical forms with defensin β-4 (DEFB4) genomic copy numbers. This case-control study was conducted from January to September 2011 and included 50 control subjects and 27 unrelated Iraqi BD patients registered at Baghdad Teaching Hospital, Bagdad, Iraq. Copy numbers of the DEFB4 gene were determined using the comparative cycle threshold method by duplex real-time polymerase chain reaction technology at the Department of Dermatology of Jena University Hospital, Jena, Germany. DEFB4 genomic copy numbers were significantly higher in the BD group compared to the control group (P = 0.010). However, no statistically significant association was found between copy numbers and clinical variables within the BD group. The DEFB4 copy number polymorphism may be associated with BD; however, it is not associated with different clinical manifestations of the disease.

Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors.

PubMed

Martin-Trujillo, Alex; Vidal, Enrique; Monteagudo-Sa Nchez, Ana; Sanchez-Delgado, Marta; Moran, Sebastian; Hernandez Mora, Jose Ramon; Heyn, Holger; Guitart, Miriam; Esteller, Manel; Monk, David

2017-09-07

It has been postulated that imprinting aberrations are common in tumors. To understand the role of imprinting in cancer, we have characterized copy-number and methylation in over 280 cancer cell lines and confirm our observations in primary tumors. Imprinted differentially methylated regions (DMRs) regulate parent-of-origin monoallelic expression of neighboring transcripts in cis. Unlike single-copy CpG islands that may be prone to hypermethylation, imprinted DMRs can either loose or gain methylation during tumorigenesis. Here, we show that methylation profiles at imprinted DMRs often not represent genuine epigenetic changes but simply the accumulation of underlying copy-number aberrations (CNAs), which is independent of the genome methylation state inferred from cancer susceptible loci. Our results reveal that CNAs also influence allelic expression as loci with copy-number neutral loss-of-heterozygosity or amplifications may be expressed from the appropriate parental chromosomes, which is indicative of maintained imprinting, although not observed as a single expression foci by RNA FISH.Altered genomic imprinting is frequently reported in cancer. Here, the authors analyze copy number and methylation in cancer cell lines and primary tumors to show that imprinted methylation profiles represent the accumulation of copy number alteration, rather than epigenetic alterations.

Copy number variation of individual cattle genomes using next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

Individualized cattle copy number and segmental duplication maps using next generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

Copy number variation of individual cattle genomes using next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often difficult to track. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angu...

40 CFR 761.209 - Number of copies of a manifest.

Code of Federal Regulations, 2014 CFR

2014-07-01

..., AND USE PROHIBITIONS PCB Waste Disposal Records and Reports § 761.209 Number of copies of a manifest... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Number of copies of a manifest. 761.209 Section 761.209 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...

40 CFR 761.209 - Number of copies of a manifest.

Code of Federal Regulations, 2013 CFR

2013-07-01

..., AND USE PROHIBITIONS PCB Waste Disposal Records and Reports § 761.209 Number of copies of a manifest... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Number of copies of a manifest. 761.209 Section 761.209 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...

Aluminum tolerance is associated with higher MATE1 gene copy-number in maize

USDA-ARS?s Scientific Manuscript database

Genome structure variation, including copy-number (CNV) and presence/absence variation (PAV), comprise a large extent of maize genetic diversity but their effect on phenotypes remains largely unexplored. Here we describe how copy-number variation in a major aluminum (Al) tolerance locus contributes ...

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

PubMed Central

Lyng, Heidi; Lando, Malin; Brøvig, Runar S; Svendsrud, Debbie H; Johansen, Morten; Galteland, Eivind; Brustugun, Odd T; Meza-Zepeda, Leonardo A; Myklebost, Ola; Kristensen, Gunnar B; Hovig, Eivind; Stokke, Trond

2008-01-01

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers. PMID:18500990

Characterization of the Copy Number and Variants of Deformed Wing Virus (DWV) in the Pairs of Honey Bee Pupa and Infesting Varroa destructor or Tropilaelaps mercedesae.

PubMed

Wu, Yunfei; Dong, Xiaofeng; Kadowaki, Tatsuhiko

2017-01-01

Recent honey bee colony losses, particularly during the winter, have been shown to be associated with the presence of both ectoparasitic mites and Deformed Wing Virus (DWV). Whilst the role of Varroa destructor mites as a viral vector is well established, the role of Tropilaelaps mercedesae mites in viral transmission has not been fully investigated. In this study, we tested the effects that V. destructor and T. mercedesae infestation have on fluctuation of the DWV copy number and alteration of the virus variants in honey bees by characterizing individual pupae and their infesting mites. We observed that both mite species were associated with increased viral copy number in honey bee pupae. We found a positive correlation between DWV copy number in pupae and copy number in infesting mites, and the same DWV type A variant was present in either low or high copy number in both honey bee pupae and infesting V. destructor . These data also suggest that variant diversity is similar between honey bee pupae and the mites that infest them. These results support a previously proposed hypothesis that DWV suppresses the honey bee immune system when virus copy number reaches a specific threshold, promoting greater replication.

Characterization of the Copy Number and Variants of Deformed Wing Virus (DWV) in the Pairs of Honey Bee Pupa and Infesting Varroa destructor or Tropilaelaps mercedesae

PubMed Central

Wu, Yunfei; Dong, Xiaofeng; Kadowaki, Tatsuhiko

2017-01-01

Recent honey bee colony losses, particularly during the winter, have been shown to be associated with the presence of both ectoparasitic mites and Deformed Wing Virus (DWV). Whilst the role of Varroa destructor mites as a viral vector is well established, the role of Tropilaelaps mercedesae mites in viral transmission has not been fully investigated. In this study, we tested the effects that V. destructor and T. mercedesae infestation have on fluctuation of the DWV copy number and alteration of the virus variants in honey bees by characterizing individual pupae and their infesting mites. We observed that both mite species were associated with increased viral copy number in honey bee pupae. We found a positive correlation between DWV copy number in pupae and copy number in infesting mites, and the same DWV type A variant was present in either low or high copy number in both honey bee pupae and infesting V. destructor. These data also suggest that variant diversity is similar between honey bee pupae and the mites that infest them. These results support a previously proposed hypothesis that DWV suppresses the honey bee immune system when virus copy number reaches a specific threshold, promoting greater replication. PMID:28878743

Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats

PubMed Central

Armour, John A. L.; Palla, Raquel; Zeeuwen, Patrick L. J. M.; den Heijer, Martin; Schalkwijk, Joost; Hollox, Edward J.

2007-01-01

Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies. PMID:17175532

Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes

PubMed Central

Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M.; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A.A.; Yang, Fengtang; Thomas, Mark G.; Armour, John A.L.

2015-01-01

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. PMID:25788522

Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes.

PubMed

Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A A; Yang, Fengtang; Thomas, Mark G; Armour, John A L

2015-06-15

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. © The Author 2015. Published by Oxford University Press.

14 CFR 221.92 - Number of copies required.

Code of Federal Regulations, 2010 CFR

2010-01-01

... PROCEEDINGS) ECONOMIC REGULATIONS TARIFFS Filing Tariff Publications With Department § 221.92 Number of copies required. Two copies of each paper tariff, tariff revision and adoption notice to be filed shall be sent to...

[Copy number variation of trinucleotide repeat in dynamic mutation sites of autosomal dominant cerebellar ataxias related genes].

PubMed

Chen, Pu; Ma, Mingyi; Shang, Huifang; Su, Dan; Zhang, Sizhong; Yang, Yuan

2009-12-01

To standardize the experimental procedure of the gene test for autosomal dominant cerebellar ataxias (ADCA), and provide the basis for quantitative criteria of the dynamic mutation of spinocerebellar ataxia (SCA) genes in Chinese population. Genotyping of the dynamic mutation loci of the SCA1, SCA2, SCA3, SCA6 and SCA7 genes was performed, using florescence PCR-capillary electrophoresis followed by DNA sequencing, to investigate the variation range of copy number of CAG tandem repeat of the genes in 263 probands of ADCA pedigrees and 261 non-related normal controls. Based on the sequencing result, the bias of the CAG copy number estimation using capillary electrophoresis with different DNA controls was compared to analyze the technical detailes of the electrophresis method in testing the dynamic mutation sites. PCR products containing dynamic mutation loci of the SCA genes showed significantly higher mobility than that of molecular weigh marker with relatively balanced GC content. This was particularly obvious in the SCA2, SCA 6 and SCA7 genes whereas the deviation of copy number could be corrected to +/-1 when known CAG copy number fragments were used as controls. The mobility of PCR products was primarily related to the copy number of CAG repeat when the fragments contained normal CAG repeat. In the 263 ADCA pedigrees, 6 (2.28%) carried SCA1 gene mutation, 8 (3.04%) had SCA2 mutation and 81 (30.80%) harbored SCA3 mutation. The gene mutation of SCA6 and SCA7 was not found. The normal variation range of the CAG repeat was 17-36 copies in SCA1 gene, 13-30 copies in SCA2, 14-39 copies in SCA3, 6-16 copies in SCA6 and 6-13 copies in SCA7. The heterozygosity was 76.1%, 17.7%, 74.4%, 72.1% and 41.3%, respectively. The mutation range of the CAG repeat was 49-56 copies in SCA1 gene, 36-41 copies in SCA2, 59-81 copies in SCA3. Neither homozygous mutation of an SCA gene nor double heterozygous mutation of the SCA genes was observed in the study. The copy number of the CAG repeat in SCA genes could be calculated accurately based on the result of florescence PCR-capillary electrophoresis when limited amount of known repeat copy number controls were used. Our result supported that the notion that SCA3 gene mutation was the most common cause for ADCA, and the obtained data would be helpful for establishing quantitative criteria of the dynamic mutation of the SCA genes in Chinese.

Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins

PubMed Central

Wrede, Joanna E.; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V.; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F.

2015-01-01

Study Objectives: Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins. Setting: Academic clinical research center. Participants: 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Design: Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a “normal” (7–9 h/24) and “short” (< 7 h/24) sleeping twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data structure to assess within-pair effects of sleep duration on mtDNA copy number. Measurements and Results: Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Conclusions: Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. Citation: Wrede JE, Mengel-From J, Buchwald D, Vitiello MV, Bamshad M, Noonan C, Christiansen L, Christensen K, Watson NF. Mitochondrial DNA copy number in sleep duration discordant monozygotic twins. SLEEP 2015;38(10):1655–1658. PMID:26039967

FAS Gene Copy Numbers are Associated with Susceptibility to Behçet Disease and VKH Syndrome in Han Chinese.

PubMed

Yu, Hongsong; Luo, Le; Wu, Lili; Zheng, Minming; Zhang, Lijun; Liu, Yunjia; Li, Hua; Cao, Qingfeng; Kijlstra, Aize; Yang, Peizeng

2015-11-01

Previous studies have identified that disturbed apoptosis was involved in the pathogenesis of Behçet disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome. This study aims to investigate whether copy number variations of apoptosis-related genes, including FAS, CASPASE8, CASPASE3, and BCL2, are associated with BD and VKH syndrome in Han Chinese. A two-stage association study was performed in 1,014 BD patients, 1,051 VKH syndrome patients, and 2,076 healthy controls. TaqMan(®) Copy Number Assays and real-time PCR were performed. The first-stage study showed that increased frequency of high FAS copy number (>2) was found in BD (P = 1.05 × 10(-3) ) and VKH syndrome (P = 2.56 × 10(-3) ). Replication and combined study confirmed the association of high copy number (>2) of FAS with BD (P = 3.35 × 10(-8) ) and VKH syndrome (P = 9.77 × 10(-8) ). A significant upregulated mRNA expression of FAS was observed in anti-CD3/CD28 antibodies-stimulated CD4(+) T cells from individuals carrying a high gene copy number (>2) as compared to normal diploid 2 copy number carriers (P = 0.004). Moreover, the mRNA expression of FAS both in active patients with BD and VKH syndrome was significantly higher than that in controls (P = 0.001 and P = 0.007, respectively). Our findings suggest that a high copy number of FAS gene confers risk for BD and VKH syndrome. © 2015 WILEY PERIODICALS, INC.

10 CFR 205.324 - Form and style; number of copies.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 3 2010-01-01 2010-01-01 false Form and style; number of copies. 205.324 Section 205.324 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System Permits and... Electric Energy at International Boundaries § 205.324 Form and style; number of copies. All applicants...

DETECTION OF STACHYBOTRYS CHARTARUM USING rRNA, tri5, AND Β-TUBULIN PRIMERS AND DETERMINING THEIR RELATIVE COPY NUMBER BY REAL TIME PCR

EPA Science Inventory

This research utilizes the quantitative polymerase chain reaction (qPCR) to determine ribosomal copy number of fungal organisms found in unhealthy indoor environments. Knowing specific copy numbers will allow for greater accuracy in quantification when utilizing current pQCR tec...

10 CFR 205.324 - Form and style; number of copies.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 3 2011-01-01 2011-01-01 false Form and style; number of copies. 205.324 Section 205.324 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System Permits and... Electric Energy at International Boundaries § 205.324 Form and style; number of copies. All applicants...

Copy number of the Adenomatous Polyposis Coli gene is not always neutral in sporadic colorectal cancers with loss of heterozygosity for the gene.

PubMed

Zauber, Peter; Marotta, Stephen; Sabbath-Solitare, Marlene

2016-03-12

Changes in the number of alleles of a chromosome may have an impact upon gene expression. Loss of heterozygosity (LOH) indicates that one allele of a gene has been lost, and knowing the exact copy number of the gene would indicate whether duplication of the remaining allele has occurred. We were interested to determine the copy number of the Adenomatous Polyposis Coli (APC) gene in sporadic colorectal cancers with LOH. We selected 38 carcinomas with LOH for the APC gene region of chromosome 5, as determined by amplification of the CA repeat region within the D5S346 loci. The copy number status of APC was ascertained using the SALSA® MLPA® P043-B1 APC Kit. LOH for the DCC gene, KRAS gene mutation, and microsatellite instability were also evaluated for each tumor, utilizing standard polymerase chain reaction methods. No tumor demonstrated microsatellite instability. LOH of the DCC gene was also present in 33 of 36 (91.7%) informative tumors. A KRAS gene mutation was present in 16 of the 38 (42.1%) tumors. Twenty-four (63.2%) of the tumors were copy number neutral, 10 (26.3%) tumors demonstrated major loss, while two (5.3%) showed partial loss. Two tumors (5.3%) had copy number gain. Results of APC and DCC LOH, KRAS and microsatellite instability indicate our colorectal cancer cases were typical of sporadic cancers following the 'chromosomal instability' pathway. The majority of our colorectal carcinomas with LOH for APC gene are copy number neutral. However, one-third of our cases showed copy number loss, suggesting that duplication of the remaining allele is not required for the development of a colorectal carcinoma.

DUF1220 copy number is linearly associated with increased cognitive function as measured by total IQ and mathematical aptitude scores

PubMed Central

Davis, Jonathon M.; Searles, Veronica B.; Anderson, Nathan; Keeney, Jonathon; Raznahan, Armin; Horwood, L. John; Fergusson, David M.; Kennedy, Martin A.; Giedd, Jay

2014-01-01

DUF1220 protein domains exhibit the greatest human lineage-specific copy number expansion of any protein-coding sequence in the genome, and variation in DUF1220 copy number has been linked to both brain size in humans and brain evolution among primates. Given these findings, we examined associations between DUF1220 subtypes CON1 and CON2 and cognitive aptitude. We identified a linear association between CON2 copy number and cognitive function in two independent populations of European descent. In North American males, an increase in CON2 copy number corresponded with an increase in WISC IQ (R2 = 0.13, p = 0.02), which may be driven by males aged 6–11 (R2 = 0.42, p = 0.003). We utilized ddPCR in a subset as a confirmatory measurement. This group had 26–33 copies of CON2 with a mean of 29, and each copy increase of CON2 was associated with a 3.3-point increase in WISC IQ (R2 = 0.22, p = 0.045). In individuals from New Zealand, an increase in CON2 copy number was associated with an increase in math aptitude ability (R2 = 0.10 p = 0.018). These were not confounded by brain size. To our knowledge, this is the first study to report a replicated association between copy number of a gene coding sequence and cognitive aptitude. Remarkably, dosage variations involving DUF1220 sequences have now been linked to human brain expansion, autism severity and cognitive aptitude, suggesting that such processes may be genetically and mechanistically inter-related. The findings presented here warrant expanded investigations in larger, well-characterized cohorts. PMID:25287832

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis

PubMed Central

2018-01-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that ‘leftover’ proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles. PMID:29518071

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis.

PubMed

Holland, David O; Johnson, Margaret E

2018-03-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that 'leftover' proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles.

DNA replication stress restricts ribosomal DNA copy number

PubMed Central

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

Beta-defensin genomic copy number is not a modifier locus for cystic fibrosis

PubMed Central

Hollox, Edward J; Davies, Jane; Griesenbach, Uta; Burgess, Juliana; Alton, Eric WFW; Armour, John AL

2005-01-01

Human beta-defensin 2 (DEFB4, also known as DEFB2 or hBD-2) is a salt-sensitive antimicrobial protein that is expressed in lung epithelia. Previous work has shown that it is encoded in a cluster of beta-defensin genes at 8p23.1, which varies in copy number between 2 and 12 in different individuals. We determined the copy number of this locus in 355 patients with cystic fibrosis (CF), and tested for correlation between beta-defensin cluster genomic copy number and lung disease associated with CF. No significant association was found. PMID:16336654

Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis)

PubMed Central

Fingert, John H.; Robin, Alan L.; Scheetz, Todd E.; Kwon, Young H.; Liebmann, Jeffrey M.; Ritch, Robert; Alward, Wallace L.M.

2016-01-01

Purpose To investigate the role of TANK-binding kinase 1 (TBK1) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. Methods In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas—juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)—using a quantitative polymerase chain reaction assay. Results No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. Conclusions TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma. PMID:27881886

Higher DEFB4 genomic copy number in SLE and ANCA-associated small vasculitis.

PubMed

Zhou, Xu-Jie; Cheng, Fa-Juan; Lv, Ji-Cheng; Luo, Huan; Yu, Feng; Chen, Min; Zhao, Ming-Hui; Zhang, Hong

2012-06-01

Evidence shows that defensins are involved in the pathogenesis of SLE and ANCA-associated small vasculitis (AASV). The copy number variation of DEFB4 has been proposed to be susceptible to inflammatory disorders. This study aims to investigate whether the DEFB4 genomic copy number variations associate with the susceptibility to these two autoimmune diseases. A total of 1178 Chinese people were enrolled, including panel 1 comprising 240 SLE patients and 275 matched controls, panel 2 comprising 303 SLE patients and 248 matched controls and panel 3 with 112 AASV patients. The DEFB4 copy number was typed by a paralogue ratio test (PRT), and all the subjects in panel 1 were also typed using the restriction enzyme digest variant ratio (REDVR) for validation. The results from PRT and REDVR were highly concordant (R = 0.911, P = 3.85 × 10(-199)) and allowed copy numbers to be assigned into integer classes with high confidence. Comparison of mean DEFB4 copy number revealed a small increase in cases with SLE both in Panel 1 (P = 0.063) and Panel 2 (P = 0.017). When pooling panels 1 and 2 together, the association was reinforced (P = 0.002) in SLE. Such association was also observed in AASV (P = 0.009). We found that a higher DEFB4 gene copy number was associated with both SLE and AASV.

Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis).

PubMed

Fingert, John H; Robin, Alan L; Scheetz, Todd E; Kwon, Young H; Liebmann, Jeffrey M; Ritch, Robert; Alward, Wallace L M

2016-08-01

To investigate the role of TANK-binding kinase 1 ( TBK1 ) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas-juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)-using a quantitative polymerase chain reaction assay. No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma.

A novel approach for copy number variation analysis by combining multiplex PCR with matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Gao, Yonghui; Chen, Xiaoli; Wang, Jianhua; Shangguan, Shaofang; Dai, Yaohua; Zhang, Ting; Liu, Junling

2013-06-20

With the increasing interest in copy number variation as it pertains to human genomic variation, common phenotypes, and disease susceptibility, there is a pressing need for methods to accurately identify copy number. In this study, we developed a simple approach that combines multiplex PCR with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for submicroscopic copy number variation detection. Two pairs of primers were used to simultaneously amplify query and endogenous control regions in the same reaction. Using a base extension reaction, the two amplicons were then distinguished and quantified in a mass spectrometry map. The peak ratio between the test region and the endogenous control region was manually calculated. The relative copy number could be determined by comparing the peak ratio between the test and control samples. This method generated a copy number measurement comparable to those produced by two other commonly used methods - multiplex ligation-dependent probe amplification and quantitative real-time PCR. Furthermore, it can discriminate a wide range of copy numbers. With a typical 384-format SpectroCHIP, at least six loci on 384 samples can be analyzed simultaneously in a hexaplex assay, making this assay adaptable for high throughput, and potentially applicable for large-scale association studies. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum.

PubMed

Son, Yeon Jeong; Ryu, Ae Jin; Li, Ling; Han, Nam Soo; Jeong, Ki Jun

2016-01-15

Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α-amylase. The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.

17 CFR 260.10a-3 - Number of copies-Filing-Signatures.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies-Filing-Signatures. 260.10a-3 Section 260.10a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... Number of copies—Filing—Signatures. (a) Three copies of every application pursuant to rule 10a-1 (§ 260...

17 CFR 240.12b-11 - Number of copies; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; signatures... Under the Securities Exchange Act of 1934 Formal Requirements § 240.12b-11 Number of copies; signatures... bound on the left side in such a manner as to leave the reading matter legible. (d) Signatures. Where...

17 CFR 260.5b-3 - Number of copies-Filing-Signatures.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies-Filing-Signatures. 260.5b-3 Section 260.5b-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... Number of copies—Filing—Signatures. (a) Three copies of every application pursuant to rule 5b-1 (§ 260.5b...

10 CFR 205.307 - Form and style; number of copies

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 3 2010-01-01 2010-01-01 false Form and style; number of copies 205.307 Section 205.307 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System Permits and... Electric Energy to A Foreign Country § 205.307 Form and style; number of copies An original and two...

47 CFR 1.742 - Place of filing, fees, and number of copies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 1 2010-10-01 2010-10-01 false Place of filing, fees, and number of copies. 1..., fees, and number of copies. All applications which do not require a fee shall be filed at the... then forwarded to the Wireline Competition Bureau. All applications accompanied by a fee payment should...

12 CFR 116.40 - Where do I file my application?

Code of Federal Regulations, 2014 CFR

2014-01-01

... application? (a) OCC Office. (1) You must file the original application and the number of copies indicated on.... If the form does not indicate the number of copies you must file or if the OCC has not prescribed a... OCC licensing office at headquarters. You must file the number of copies indicated on the applicable...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2014 CFR

2014-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

12 CFR 116.40 - Where do I file my application?

Code of Federal Regulations, 2012 CFR

2012-01-01

... application? (a) OCC Office. (1) You must file the original application and the number of copies indicated on.... If the form does not indicate the number of copies you must file or if the OCC has not prescribed a... OCC licensing office at headquarters. You must file the number of copies indicated on the applicable...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2012 CFR

2012-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2013 CFR

2013-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

12 CFR 116.40 - Where do I file my application?

Code of Federal Regulations, 2013 CFR

2013-01-01

... application? (a) OCC Office. (1) You must file the original application and the number of copies indicated on.... If the form does not indicate the number of copies you must file or if the OCC has not prescribed a... OCC licensing office at headquarters. You must file the number of copies indicated on the applicable...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2011 CFR

2011-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2010 CFR

2010-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

10 CFR 205.307 - Form and style; number of copies

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 3 2011-01-01 2011-01-01 false Form and style; number of copies 205.307 Section 205.307 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System Permits and... Electric Energy to A Foreign Country § 205.307 Form and style; number of copies An original and two...

Orthogonality and Burdens of Heterologous AND Gate Gene Circuits in E. coli

PubMed Central

2017-01-01

Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal to the host background. However, introduced circuits may interfere with the host’s physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behavior of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guidance for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host. PMID:29240998

Diversity of human copy number variation and multicopy genes.

PubMed

Sudmant, Peter H; Kitzman, Jacob O; Antonacci, Francesca; Alkan, Can; Malig, Maika; Tsalenko, Anya; Sampas, Nick; Bruhn, Laurakay; Shendure, Jay; Eichler, Evan E

2010-10-29

Copy number variants affect both disease and normal phenotypic variation, but those lying within heavily duplicated, highly identical sequence have been difficult to assay. By analyzing short-read mapping depth for 159 human genomes, we demonstrated accurate estimation of absolute copy number for duplications as small as 1.9 kilobase pairs, ranging from 0 to 48 copies. We identified 4.1 million "singly unique nucleotide" positions informative in distinguishing specific copies and used them to genotype the copy and content of specific paralogs within highly duplicated gene families. These data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association.

Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

PubMed

Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

2017-10-15

Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

High BAALC copy numbers in peripheral blood prior to allogeneic transplantation predict early relapse in acute myeloid leukemia patients.

PubMed

Jentzsch, Madlen; Bill, Marius; Grimm, Juliane; Schulz, Julia; Goldmann, Karoline; Beinicke, Stefanie; Häntschel, Janine; Pönisch, Wolfram; Franke, Georg-Nikolaus; Vucinic, Vladan; Behre, Gerhard; Lange, Thoralf; Niederwieser, Dietger; Schwind, Sebastian

2017-10-20

High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of BAALC copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14 BAALC / ABL1 copy numbers was determined and applied to define patients with high or low BAALC / ABL1 copy numbers. High pre-HSCT BAALC / ABL1 copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT BAALC / ABL1 copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT BAALC / ABL1 copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT BAALC / ABL1 copy numbers benefit from additional treatment before or early intervention after HSCT.

High BAALC copy numbers in peripheral blood prior to allogeneic transplantation predict early relapse in acute myeloid leukemia patients

PubMed Central

Jentzsch, Madlen; Bill, Marius; Grimm, Juliane; Schulz, Julia; Goldmann, Karoline; Beinicke, Stefanie; Häntschel, Janine; Pönisch, Wolfram; Franke, Georg-Nikolaus; Vucinic, Vladan; Behre, Gerhard; Lange, Thoralf; Niederwieser, Dietger; Schwind, Sebastian

2017-01-01

High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of BAALC copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14 BAALC/ABL1 copy numbers was determined and applied to define patients with high or low BAALC/ABL1 copy numbers. High pre-HSCT BAALC/ABL1 copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT BAALC/ABL1 copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT BAALC/ABL1 copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT BAALC/ABL1 copy numbers benefit from additional treatment before or early intervention after HSCT. PMID:29152132

DNA Methylation Patterns in Normal Tissue Correlate more Strongly with Breast Cancer Status than Copy-Number Variants.

PubMed

Gao, Yang; Widschwendter, Martin; Teschendorff, Andrew E

2018-05-04

Normal tissue at risk of neoplastic transformation is characterized by somatic mutations, copy-number variation and DNA methylation changes. It is unclear however, which type of alteration may be more informative of cancer risk. We analyzed genome-wide DNA methylation and copy-number calls from the same DNA assay in a cohort of healthy breast samples and age-matched normal samples collected adjacent to breast cancer. Using statistical methods to adjust for cell type heterogeneity, we show that DNA methylation changes can discriminate normal-adjacent from normal samples better than somatic copy-number variants. We validate this important finding in an independent dataset. These results suggest that DNA methylation alterations in the normal cell of origin may offer better cancer risk prediction and early detection markers than copy-number changes. Copyright © 2018. Published by Elsevier B.V.

Allele quantification using molecular inversion probes (MIP)

PubMed Central

Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Falkowski, Matthew; Chen, Chunnuan; Siddiqui, Farooq; Davis, Ronald W.; Willis, Thomas D.; Faham, Malek

2005-01-01

Detection of genomic copy number changes has been an important research area, especially in cancer. Several high-throughput technologies have been developed to detect these changes. Features that are important for the utility of technologies assessing copy number changes include the ability to interrogate regions of interest at the desired density as well as the ability to differentiate the two homologs. In addition, assessing formaldehyde fixed and paraffin embedded (FFPE) samples allows the utilization of the vast majority of cancer samples. To address these points we demonstrate the use of molecular inversion probe (MIP) technology to the study of copy number. MIP is a high-throughput genotyping technology capable of interrogating >20 000 single nucleotide polymorphisms in the same tube. We have shown the ability of MIP at this multiplex level to provide copy number measurements while obtaining the allele information. In addition we have demonstrated a proof of principle for copy number analysis in FFPE samples. PMID:16314297

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas.

PubMed

Rosa-Rosa, Juan M; Leskelä, Susanna; Cristóbal-Lana, Eva; Santón, Almudena; López-García, Ma Ángeles; Muñoz, Gloria; Pérez-Mies, Belen; Biscuola, Michele; Prat, Jaime; Esther, Oliva E; Soslow, Robert A; Matias-Guiu, Xavier; Palacios, Jose

2016-11-01

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumors, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well-differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole-exome sequencing of the endometrioid and undifferentiated components, as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: (a) hypermutated tumors with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); (b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); (c) high copy number alterations (copy-number high) tumors group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%); and (d) low copy number alterations (copy-number low) tumors with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group, whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumors. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumors, which may have prognostic value.

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas

PubMed Central

Rosa-Rosa, J.M.; Leskelä, S.; Cristóbal-Lana, E.; Santón, A.; López-García, M.A.; Muñoz, G.; Pérez-Mies, B.; Biscuola, M; Prat, J.; Oliva, E.; Soslow, R.A.; Matias-Guiu, X.; Palacios, J.

2017-01-01

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumours, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole exome sequencing of the endometrioid and undifferentiated components as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: a) hypermutated tumours with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); c) high copy number alterations (copy-number high) tumours group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%) ; and d) low copy number alterations (copy-number low) tumours with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumours. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumours, which may have prognostic value. PMID:27491810

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

PubMed Central

Cullingford, Timothy E; Markou, Thomais; Fuller, Stephen J; Giraldo, Alejandro; Pikkarainen, Sampsa; Zoumpoulidou, Georgia; Alsafi, Ali; Ekere, Collins; Kemp, Timothy J; Dennis, Jayne L; Game, Laurence; Sugden, Peter H; Clerk, Angela

2008-01-01

Background Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response. PMID:18275597

Mitochondrial DNA in Residual Leukemia Cells in Cerebrospinal Fluid in Children with Acute Lymphoblastic Leukemia

PubMed Central

Egan, Kathryn; Kusao, Ian; Troelstrup, David; Agsalda, Melissa; Shiramizu, Bruce

2010-01-01

This feasibility study was designed to assess the ability to measure mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) cells that contributed to minimal disease/persistent or residual disease (MD/PRD) from children with acute lymphoblastic leukemia (ALL). Increase in mtDNA copies in cancer cells has been suggested to play a role in MD/PRD. CSF as well as blood specimens from 6 children were assayed for MD/PRD and mtDNA copy numbers by quantitative real-time polymerase chain reaction. Of 7 MD/PRD-positive specimens, 6 had increased mtDNA copy numbers; while 11 MD/PRD-negative specimens had no increase in mtDNA copy numbers, p < 0.003. This is the first proof-of-concept study to measure mtDNA copy numbers in MD/PRD-positive CSF specimens from children with ALL. Increase of mtDNA copy numbers in MD/PRD childhood ALL cells and its significance as a mechanism for recurrence requires further investigation. Keywords Minimal residual disease; Acute lymphoblastic leukemia; Central nervous system; Cerebrospinal fluid; Mitochondria PMID:21331151

Molecular inversion probe assay for allelic quantitation

PubMed Central

Ji, Hanlee; Welch, Katrina

2010-01-01

Molecular inversion probe (MIP) technology has been demonstrated to be a robust platform for large-scale dual genotyping and copy number analysis. Applications in human genomic and genetic studies include the possibility of running dual germline genotyping and combined copy number variation ascertainment. MIPs analyze large numbers of specific genetic target sequences in parallel, relying on interrogation of a barcode tag, rather than direct hybridization of genomic DNA to an array. The MIP approach does not replace, but is complementary to many of the copy number technologies being performed today. Some specific advantages of MIP technology include: Less DNA required (37 ng vs. 250 ng), DNA quality less important, more dynamic range (amplifications detected up to copy number 60), allele specific information “cleaner” (less SNP crosstalk/contamination), and quality of markers better (fewer individual MIPs versus SNPs needed to identify copy number changes). MIPs can be considered a candidate gene (targeted whole genome) approach and can find specific areas of interest that otherwise may be missed with other methods. PMID:19488872

Use of autocorrelation scanning in DNA copy number analysis.

PubMed

Zhang, Liangcai; Zhang, Li

2013-11-01

Data quality is a critical issue in the analyses of DNA copy number alterations obtained from microarrays. It is commonly assumed that copy number alteration data can be modeled as piecewise constant and the measurement errors of different probes are independent. However, these assumptions do not always hold in practice. In some published datasets, we find that measurement errors are highly correlated between probes that interrogate nearby genomic loci, and the piecewise-constant model does not fit the data well. The correlated errors cause problems in downstream analysis, leading to a large number of DNA segments falsely identified as having copy number gains and losses. We developed a simple tool, called autocorrelation scanning profile, to assess the dependence of measurement error between neighboring probes. Autocorrelation scanning profile can be used to check data quality and refine the analysis of DNA copy number data, which we demonstrate in some typical datasets. lzhangli@mdanderson.org. Supplementary data are available at Bioinformatics online.

Copy number analysis reveals a novel multiexon deletion of the COLQ gene in congenital myasthenia.

PubMed

Wang, Wei; Wu, Yanhong; Wang, Chen; Jiao, Jinsong; Klein, Christopher J

2016-12-01

Congenital myasthenic syndrome (CMS) is genetically and clinically heterogeneous. 1 Despite a considerable number of causal genes discovered, many patients are left without a specific diagnosis after genetic testing. The presumption is that novel genes yet to be discovered will account for the majority of such patients. However, it is also possible that we are neglecting a type of genetic variation: copy number changes (>50 bp) as causal for some of these patients. Next-generation sequencing (NGS) can simultaneously screen all known causal genes 2 and is increasingly being validated to have a potential to identify copy number changes. 3 We present a CMS case who did not receive a genetic diagnosis from previous Sanger sequencing, but through a novel copy number analysis algorithm integrated into our targeted NGS panel, we discovered a novel copy number mutation in the COLQ gene and made a genetic diagnosis. This discovery expands the genotype-phenotype correlation of CMS, leads to improved genetic counsel, and allows for specific pharmacologic treatment. 1 .

Prognostic Impact of PHIP Copy Number in Melanoma: Linkage to Ulceration

PubMed Central

Nosrati, Mehdi; Tong, Schuyler; Wu, Clayton; Thummala, Suresh; Dar, Altaf A.; Leong, Stanley P.L.; Cleaver, James E.; Sagebiel, Richard W.; Miller, James R.; Kashani-Sabet, Mohammed

2013-01-01

Ulceration is an important prognostic factor in melanoma whose biologic basis is poorly understood. Here we assessed the prognostic impact of pleckstrin homology domain-interacting protein (PHIP) copy number and its relationship to ulceration. PHIP copy number was determined using fluorescence in situ hybridization (FISH) in a tissue microarray cohort of 238 melanomas. Elevated PHIP copy number was associated with significantly reduced DMFS (P = 0.01) and DSS (P = 0.009) by Kaplan-Meier analyses. PHIP FISH scores were independently predictive of DMFS (P = 0.03) and DSS (P = 0.03). Increased PHIP copy number was an independent predictor of ulceration status (P = 0.04). The combined impact of increased PHIP copy number and tumor vascularity on ulceration status was highly significant (P< 0.0001). Stable suppression of PHIP in human melanoma cells resulted in significantly reduced glycolytic activity in vitro, with lower expression of LDH5, HIF1A, and VEGF, and was accompanied by reduced microvessel density in vivo. These results provide further support for PHIP as a molecular prognostic marker of melanoma, and reveal a significant linkage between PHIP levels and ulceration. Moreover, they suggest that ulceration may be driven by increased glycolysis and angiogenesis. PMID:24005052

Chemiluminescent Detection for Estimating Relative Copy Numbers of Porcine Endogenous Retrovirus Proviruses from Chinese Minipigs Based on Magnetic Nanoparticles.

PubMed

Yang, Haowen; Liu, Ming; Zhou, Bingcong; Deng, Yan; He, Nongyue; Jiang, Hesheng; Guo, Yafen; Lan, Ganqiu; Jiang, Qinyang; Yang, Xiurong; Li, Zhiyang

2016-06-01

Chinese Bama minipigs could be potential donors for the supply of xenografts because they are genetically stable, highly inbred, and inexpensive. However, porcine endogenous retrovirus (PERV) is commonly integrated in pig genomes and could cause a cross-species infection by xenotransplantation. For screening out the pigs with low copy numbers of PERV proviruses, we have developed a novel semiquantitative analysis approach based on magnetic nanoparticles (MNPs) and chemiluminescence (CL) for estimating relative copy numbers (RCNs) of PERV proviruses in Chinese Bama minipigs. The CL intensities of PERV proviruses and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were respectively determined with this method, and the RCNs of PERV proviruses were calculated by the equation: RCN of PERV provirus = CL intensity of PERV provirus/CL intensity of GAPDH. The results showed that PERVs were integrated in the genomes of Bama minipigs at different copy numbers, and the copy numbers of PERV-C subtype were greatly low. Two Bama minipigs with low copy numbers of PERV proviruses were detected out and could be considered as xenograft donor candidates. Although only semiquantitation can be achieved, this approach has potential for screening out safe and suitable pig donors for xenotransplantation.

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons.

PubMed

Gervasi, Noreen M; Scott, Shane S; Aschrafi, Armaz; Gale, Jenna; Vohra, Sanah N; MacGibeny, Margaret A; Kar, Amar N; Gioio, Anthony E; Kaplan, Barry B

2016-06-01

Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3'UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

PubMed Central

Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K.; Clemente, Tom E.

2016-01-01

Abstract Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T‐DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided. PMID:26670088

Alfalfa Biomass Germplasms: SFP Detection and Transcriptome Analysis

USDA-ARS?s Scientific Manuscript database

Advances in alfalfa [Medicago sativa (L.) subsp. sativa] breeding, molecular genetics, and genomics have been slow because this crop is an allogamous autotetraploid (2n = 4x = 32) with complex polysomic inheritance. Increasing cellulose and decreasing lignin in alfalfa stem cell walls would improve ...

The Synthesis of Human Placental Lactogen by Ribosomes Derived from Human Placenta

PubMed Central

Boime, Irving; Boguslawski, Sophie

1974-01-01

In a very active cell-free system containing polysomes derived from human placenta and a cell-sap fraction prepared from ascites tumor cells, the synthesis of the hormone human placental lactogen (HPL) was detected. The identification was based on the following: (a) The in vitro synthesized protein labeled with [35S]methionine migrated at the same rate as authentic HPL on sodium dodecyl sulfate-polyacrylamide gels and (b) tryptic fingerprint analysis of the labeled protein yielded peptides having the same mobilities as seen with the same analysis of purified HPL. The amount of HPL synthesized in a cell-free system containing polysomes derived from term placenta was about 10% of the total proteins synthesized and in a comparable system containing first trimester ribosomes the level of synthesis was about 5%. These data suggest the potential for quantitating the HPL mRNA activity as a function of the period of gestation and for isolating the mRNA itself. Images PMID:4524639

Semiconservative quasispecies equations for polysomic genomes: The general case

NASA Astrophysics Data System (ADS)

Itan, Eran; Tannenbaum, Emmanuel

2010-06-01

This paper develops a formulation of the quasispecies equations appropriate for polysomic, semiconservatively replicating genomes. This paper is an extension of previous work on the subject, which considered the case of haploid genomes. Here, we develop a more general formulation of the quasispecies equations that is applicable to diploid and even polyploid genomes. Interestingly, with an appropriate classification of population fractions, we obtain a system of equations that is formally identical to the haploid case. As with the work for haploid genomes, we consider both random and immortal DNA strand chromosome segregation mechanisms. However, in contrast to the haploid case, we have found that an analytical solution for the mean fitness is considerably more difficult to obtain for the polyploid case. Accordingly, whereas for the haploid case we obtained expressions for the mean fitness for the case of an analog of the single-fitness-peak landscape for arbitrary lesion repair probabilities (thereby allowing for noncomplementary genomes), here we solve for the mean fitness for the restricted case of perfect lesion repair.

Further characterization of ribosome binding to thylakoid membranes. [Pisum sativum

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

Hurewitz, J.; Jagendorf, A.T.

1987-05-01

Previous work indicated more polysomes bound to pea (Pisum sativum cv Progress No. 9) thylakoids in light than in the dark, in vivo. With isolated intact chloroplasts incubated in darkness, addition of MgATP had no effect but 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus, the major effect of light on ribosome-binding in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus, cycling of ribosomes is controlled by translation, initiation,more » and termination. Bound RNA accounted for 19 to 24% of the total chloroplast RNA and the incorporation of (/sup 3/H)leucine into thylakoids was proportional to the amount of this bound RNA. These data support the concept that stroma ribosomes are recruited into thylakoid polysomes, which are active in synthesizing thylakoid proteins.« less

Mitochondrial DNA copy numbers in pyramidal neurons are decreased and mitochondrial biogenesis transcriptome signaling is disrupted in Alzheimer's disease hippocampi.

PubMed

Rice, Ann C; Keeney, Paula M; Algarzae, Norah K; Ladd, Amy C; Thomas, Ravindar R; Bennett, James P

2014-01-01

Alzheimer's disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

Copy number variation of human AMY1 is a minor contributor to variation in salivary amylase expression and activity.

PubMed

Carpenter, Danielle; Mitchell, Laura M; Armour, John A L

2017-02-20

Salivary amylase in humans is encoded by the copy variable gene AMY1 in the amylase gene cluster on chromosome 1. Although the role of salivary amylase is well established, the consequences of the copy number variation (CNV) at AMY1 on salivary amylase protein production are less well understood. The amylase gene cluster is highly structured with a fundamental difference between odd and even AMY1 copy number haplotypes. In this study, we aimed to explore, in samples from 119 unrelated individuals, not only the effects of AMY1 CNV on salivary amylase protein expression and amylase enzyme activity but also whether there is any evidence for underlying difference between the common haplotypes containing odd numbers of AMY1 and even copy number haplotypes. AMY1 copy number was significantly correlated with the variation observed in salivary amylase production (11.7% of variance, P < 0.0005) and enzyme activity (13.6% of variance, P < 0.0005) but did not explain the majority of observed variation between individuals. AMY1-odd and AMY1-even haplotypes showed a different relationship between copy number and expression levels, but the difference was not statistically significant (P = 0.052). Production of salivary amylase is correlated with AMY1 CNV, but the majority of interindividual variation comes from other sources. Long-range haplotype structure may affect expression, but this was not significant in our data.

Polycomb repressive complex 1 provides a molecular explanation for repeat copy number dependency in FSHD muscular dystrophy.

PubMed

Casa, Valentina; Runfola, Valeria; Micheloni, Stefano; Aziz, Arif; Dilworth, F Jeffrey; Gabellini, Davide

2017-02-15

Repression of repetitive elements is crucial to preserve genome integrity and has been traditionally ascribed to constitutive heterochromatin pathways. FacioScapuloHumeral Muscular Dystrophy (FSHD), one of the most common myopathies, is characterized by a complex interplay of genetic and epigenetic events. The main FSHD form is linked to a reduced copy number of the D4Z4 macrosatellite repeat on 4q35, causing loss of silencing and aberrant expression of the D4Z4-embedded DUX4 gene leading to disease. By an unknown mechanism, D4Z4 copy-number correlates with FSHD phenotype. Here we show that the DUX4 proximal promoter (DUX4p) is sufficient to nucleate the enrichment of both constitutive and facultative heterochromatin components and to mediate a copy-number dependent gene silencing. We found that both the CpG/GC dense DNA content and the repetitive nature of DUX4p arrays are important for their repressive ability. We showed that DUX4p mediates a copy number-dependent Polycomb Repressive Complex 1 (PRC1) recruitment, which is responsible for the copy-number dependent gene repression. Overall, we directly link genetic and epigenetic defects in FSHD by proposing a novel molecular explanation for the copy number-dependency in FSHD pathogenesis, and offer insight into the molecular functions of repeats in chromatin regulation. © The Author 2016. Published by Oxford University Press.

Mitochondrial DNA copy number is associated with risk of head and neck squamous cell carcinoma in Chinese population.

PubMed

Wang, Lihua; Lv, Hong; Ji, Pei; Zhu, Xun; Yuan, Hua; Jin, Guangfu; Dai, Juncheng; Hu, Zhibin; Su, Yuxiong; Ma, Hongxia

2018-04-19

Mitochondria show the special role in cellular bioenergy and many essential physiological activities. Previous researches have suggested that variations of mitochondrial DNA copy number contribute to development of different types of carcinomas. However, the relationship of mtDNA copy number in peripheral blood leukocytes (PBLs) with the risk of head and neck squamous cell carcinoma (HNSCC) is still inconclusive. We investigated the association of mtDNA with HNSCC risk through a case-control study including 570 HNSCC cases and 597 cancer-free controls. mtDNA copy number in PBLs was measured by real-time qPCR. Logistic regression was performed to estimate the association between the mtDNA copy number in PBLs and HNSCC risk. A U-shaped relation between the mtDNA copy number and HNSCC risk was found. Compared with those in the second quartile group, the adjusted odds ratios (ORs) and 95% confidence interval (CI) for those in the first and the forth quartile groups were 1.95 (1.37-2.76) and 2.16 (1.53-3.04), respectively. Using restricted cubic spline analysis, we confirmed such a significant U-shaped relation. Furthermore, the U-shaped association remained significant in different subgroups stratified by age, gender, tobacco smoking, and alcohol consumption. Both extremely low and high mtDNA copy numbers had significant associations with the increased HNSCC risk. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 17 Commodity and Securities Exchanges 3 2013-04-01 2013-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 17 Commodity and Securities Exchanges 3 2012-04-01 2012-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 17 Commodity and Securities Exchanges 4 2014-04-01 2014-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

Single Color Multiplexed ddPCR Copy Number Measurements and Single Nucleotide Variant Genotyping.

PubMed

Wood-Bouwens, Christina M; Ji, Hanlee P

2018-01-01

Droplet digital PCR (ddPCR) allows for accurate quantification of genetic events such as copy number variation and single nucleotide variants. Probe-based assays represent the current "gold-standard" for detection and quantification of these genetic events. Here, we introduce a cost-effective single color ddPCR assay that allows for single genome resolution quantification of copy number and single nucleotide variation.

Distinct Copy Number, Coding Sequence, and Locus Methylation Patterns Underlie Rhg1-Mediated Soybean Resistance to Soybean Cyst Nematode1[W][OPEN

PubMed Central

Cook, David E.; Bayless, Adam M.; Wang, Kai; Guo, Xiaoli; Song, Qijian; Jiang, Jiming; Bent, Andrew F.

2014-01-01

Copy number variation of kilobase-scale genomic DNA segments, beyond presence/absence polymorphisms, can be an important driver of adaptive traits. Resistance to Heterodera glycines (Rhg1) is a widely utilized quantitative trait locus that makes the strongest known contribution to resistance against soybean cyst nematode (SCN), Heterodera glycines, the most damaging pathogen of soybean (Glycine max). Rhg1 was recently discovered to be a complex locus at which resistance-conferring haplotypes carry up to 10 tandem repeat copies of a 31-kb DNA segment, and three disparate genes present on each repeat contribute to SCN resistance. Here, we use whole-genome sequencing, fiber-FISH (fluorescence in situ hybridization), and other methods to discover the genetic variation at Rhg1 across 41 diverse soybean accessions. Based on copy number variation, transcript abundance, nucleic acid polymorphisms, and differentially methylated DNA regions, we find that SCN resistance is associated with multicopy Rhg1 haplotypes that form two distinct groups. The tested high-copy-number Rhg1 accessions, including plant introduction (PI) 88788, contain a flexible number of copies (seven to 10) of the 31-kb Rhg1 repeat. The identified low-copy-number Rhg1 group, including PI 548402 (Peking) and PI 437654, contains three copies of the Rhg1 repeat and a newly identified allele of Glyma18g02590 (a predicted α-SNAP [α-soluble N-ethylmaleimide–sensitive factor attachment protein]). There is strong evidence for a shared origin of the two resistance-conferring multicopy Rhg1 groups and subsequent independent evolution. Differentially methylated DNA regions also were identified within Rhg1 that correlate with SCN resistance. These data provide insights into copy number variation of multigene segments, using as the example a disease resistance trait of high economic importance. PMID:24733883

18 CFR 33.8 - Number of copies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ENERGY REGULATIONS UNDER THE FEDERAL POWER ACT APPLICATIONS UNDER FEDERAL POWER ACT SECTION 203 § 33.8 Number of copies. An original and eight copies of the application under this part must be submitted. If..., the applicant must submit all such information in electronic format (e.g., on computer diskette or on...

Problem-Solving Test: Attenuation--A Mechanism to Regulate Bacterial Tryptophan Biosynthesis

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2010-01-01

Terms to be familiar with before you start to solve the test: tryptophan, transcription unit, operon, "trp" repressor, corepressor, operator, promoter, palindrome, initiation, elongation, and termination of transcription, open reading frame, coupled transcription/translation, chromosome-polysome complex. (Contains 2 figures and 1 footnote.)

Great Genotypic and Phenotypic Diversities Associated with Copy-Number Variations of Complement C4 and RP-C4-CYP21-TNX (RCCX) Modules: a Comparison of Asian Indian and European American Populations

PubMed Central

Saxena, Kapil; Kitzmiller, Kathryn J.; Wu, Yee Ling; Zhou, Bi; Esack, Nazreen; Hiremath, Leena; Chung, Erwin K.; Yang, Yan; Yu, C. Yung

2009-01-01

Inter-individual gene copy-number variations (CNVs) probably afford human populations the flexibility to respond to a variety of environmental challenges, but also lead to differential disease predispositions. We investigated gene CNVs for complement component C4 and steroid 21-hydroxylase from the RP-C4-CYP21-TNX (RCCX) modules located in the major histocompatibility complex among healthy Asian-Indian Americans (AIA) and compared them to European Americans. A combination of definitive techniques that yielded cross-confirmatory results was used. The medium gene copy-numbers for C4 and its isotypes, acidic C4A and basic C4B, were 4, 2 and 2, respectively, but their frequencies were only 53–56%. The distribution patterns for total C4 and C4A are skewed towards the high copy-number side. For example, the frequency of AIA-subjects with three copies of C4A (30.7%) was 3.92-fold of those with a single copy (7.83%). The monomodular-short haplotype with a single C4B gene and the absence of C4A, which is in linkage- disequilibrium with HLA DRB1*0301 in Europeans and a strong risk factor for autoimmune diseases, has a frequency of 0.012 in AIA but 0.106 among healthy European Americans (p=6.6×10−8). The copy-number and the size of C4 genes strongly determine the plasma C4 protein concentrations. Parallel variations in copy-numbers of CYP21A (CYP21A1P) and TNXA with total C4 were also observed. Notably, 13.1% of AIA-subjects had three copies of the functional CYP21B, which were likely generated by recombinations between monomodular and bimodular RCCX haplotypes. The high copy-numbers of C4 and the high frequency of RCCX recombinants offer important insights to the prevalence of autoimmune and genetic diseases. PMID:19135723

Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem

PubMed Central

Valdivia-Anistro, Jorge A.; Eguiarte-Fruns, Luis E.; Delgado-Sapién, Gabriela; Márquez-Zacarías, Pedro; Gasca-Pineda, Jaime; Learned, Jennifer; Elser, James J.; Olmedo-Alvarez, Gabriela; Souza, Valeria

2016-01-01

The ribosomal RNA (rrn) operon is a key suite of genes related to the production of protein synthesis machinery and thus to bacterial growth physiology. Experimental evidence has suggested an intrinsic relationship between the number of copies of this operon and environmental resource availability, especially the availability of phosphorus (P), because bacteria that live in oligotrophic ecosystems usually have few rrn operons and a slow growth rate. The Cuatro Ciénegas Basin (CCB) is a complex aquatic ecosystem that contains an unusually high microbial diversity that is able to persist under highly oligotrophic conditions. These environmental conditions impose a variety of strong selective pressures that shape the genome dynamics of their inhabitants. The genus Bacillus is one of the most abundant cultivable bacterial groups in the CCB and usually possesses a relatively large number of rrn operon copies (6–15 copies). The main goal of this study was to analyze the variation in the number of rrn operon copies of Bacillus in the CCB and to assess their growth-related properties as well as their stoichiometric balance (N and P content). We defined 18 phylogenetic groups within the Bacilli clade and documented a range of from six to 14 copies of the rrn operon. The growth dynamic of these Bacilli was heterogeneous and did not show a direct relation to the number of operon copies. Physiologically, our results were not consistent with the Growth Rate Hypothesis, since the copies of the rrn operon were decoupled from growth rate. However, we speculate that the diversity of the growth properties of these Bacilli as well as the low P content of their cells in an ample range of rrn copy number is an adaptive response to oligotrophy of the CCB and could represent an ecological mechanism that allows these taxa to coexist. These findings increase the knowledge of the variability in the number of copies of the rrn operon in the genus Bacillus and give insights about the physiology of this bacterial group under extreme oligotrophic conditions. PMID:26779143

Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA.

PubMed

Bartels, Hanni; Luban, Jeremy

2014-09-12

All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated. The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag-only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis. These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA.

Comparison of the copy numbers of bovine leukemia virus in the lymph nodes of cattle with enzootic bovine leukosis and cattle with latent infection.

PubMed

Somura, Yoshiko; Sugiyama, Emi; Fujikawa, Hiroshi; Murakami, Kenji

2014-10-01

To establish a diagnostic index for predicting enzootic bovine leukosis (EBL), proviral bovine leukemia virus (BLV) copies in whole blood, lymph nodes and spleen were examined by quantitative real-time PCR (qPCR). Cattle were divided into two groups, EBL and BLV-infected, based on meat inspection data. The number of BLV copies in all specimens of EBL cattle was significantly higher than those of BLV-infected cattle (p < 0.0001), and the number of BLV copies in the lymph nodes was particularly large. Over 70 % of the superficial cervical, medial iliac and jejunal lymph nodes from EBL cattle had more than 1,000 copies/10 ng DNA, whereas lymph nodes from BLV-infected cattle did not. These findings suggest that the cattle harboring more than 1,000 BLV copies may be diagnosed with EBL.

Genetic Control of L-a and L-(Bc) Dsrna Copy Number in Killer Systems of SACCHAROMYCES CEREVISIAE

PubMed Central

Ball, Steven G.; Tirtiaux, Catherine; Wickner, Reed B.

1984-01-01

M dsRNA in yeast encodes a toxin precursor and immunity protein, whereas L-A dsRNA encodes the 81,000-dalton major protein of the intracellular particles in which both L-A and M are found. L-(BC) dsRNA(s) are found in particles with different coat proteins. We find that M dsRNA lowers the copy number of L-A, but not L-(BC). The SKI gene products lower the copy number of L-(BC), L-A, M1 and M2. This is the first known interaction of L-(BC) with any element of the killer systems. The MAK3, MAK10 and PET18 gene products are necessary for L-A maintenance and replication, but mutations in these genes do not affect L-(BC) copy number. Mutations in MAK1, MAK4, MAK7, MAK17 and MAK24 do not detectably affect copy number of L-(BC) or L-A. PMID:17246214

Copy-number analysis and inference of subclonal populations in cancer genomes using Sclust.

PubMed

Cun, Yupeng; Yang, Tsun-Po; Achter, Viktor; Lang, Ulrich; Peifer, Martin

2018-06-01

The genomes of cancer cells constantly change during pathogenesis. This evolutionary process can lead to the emergence of drug-resistant mutations in subclonal populations, which can hinder therapeutic intervention in patients. Data derived from massively parallel sequencing can be used to infer these subclonal populations using tumor-specific point mutations. The accurate determination of copy-number changes and tumor impurity is necessary to reliably infer subclonal populations by mutational clustering. This protocol describes how to use Sclust, a copy-number analysis method with a recently developed mutational clustering approach. In a series of simulations and comparisons with alternative methods, we have previously shown that Sclust accurately determines copy-number states and subclonal populations. Performance tests show that the method is computationally efficient, with copy-number analysis and mutational clustering taking <10 min. Sclust is designed such that even non-experts in computational biology or bioinformatics with basic knowledge of the Linux/Unix command-line syntax should be able to carry out analyses of subclonal populations.

RUBIC identifies driver genes by detecting recurrent DNA copy number breaks

PubMed Central

van Dyk, Ewald; Hoogstraat, Marlous; ten Hoeve, Jelle; Reinders, Marcel J. T.; Wessels, Lodewyk F. A.

2016-01-01

The frequent recurrence of copy number aberrations across tumour samples is a reliable hallmark of certain cancer driver genes. However, state-of-the-art algorithms for detecting recurrent aberrations fail to detect several known drivers. In this study, we propose RUBIC, an approach that detects recurrent copy number breaks, rather than recurrently amplified or deleted regions. This change of perspective allows for a simplified approach as recursive peak splitting procedures and repeated re-estimation of the background model are avoided. Furthermore, we control the false discovery rate on the level of called regions, rather than at the probe level, as in competing algorithms. We benchmark RUBIC against GISTIC2 (a state-of-the-art approach) and RAIG (a recently proposed approach) on simulated copy number data and on three SNP6 and NGS copy number data sets from TCGA. We show that RUBIC calls more focal recurrent regions and identifies a much larger fraction of known cancer genes. PMID:27396759

Cancer vulnerabilities unveiled by genomic loss

PubMed Central

Nijhawan, Deepak; Zack, Travis I.; Ren, Yin; Strickland, Matthew R.; Lamothe, Rebecca; Schumacher, Steven E.; Tsherniak, Aviad; Besche, Henrike C.; Rosenbluh, Joseph; Shehata, Shyemaa; Cowley, Glenn S.; Weir, Barbara A.; Goldberg, Alfred L.; Mesirov, Jill P.; Root, David E.; Bhatia, Sangeeta N.; Beroukhim, Rameen; Hahn, William C.

2012-01-01

Summary Due to genome instability, most cancers exhibit loss of regions containing tumor suppressor genes and collateral loss of other genes. To identify cancer-specific vulnerabilities that are the result of copy-number losses, we performed integrated analyses of genome-wide copy-number and RNAi profiles and identified 56 genes for which gene suppression specifically inhibited the proliferation of cells harboring partial copy-number loss of that gene. These CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes are enriched for spliceosome, proteasome and ribosome components. One CYCLOPS gene, PSMC2, encodes an essential member of the 19S proteasome. Normal cells express excess PSMC2, which resides in a complex with PSMC1, PSMD2, and PSMD5 and acts as a reservoir protecting cells from PSMC2 suppression. Cells harboring partial PSMC2 copy-number loss lack this complex and die after PSMC2 suppression. These observations define a distinct class of cancer-specific liabilities resulting from genome instability. PMID:22901813

Hidden Markov Model-Based CNV Detection Algorithms for Illumina Genotyping Microarrays.

PubMed

Seiser, Eric L; Innocenti, Federico

2014-01-01

Somatic alterations in DNA copy number have been well studied in numerous malignancies, yet the role of germline DNA copy number variation in cancer is still emerging. Genotyping microarrays generate allele-specific signal intensities to determine genotype, but may also be used to infer DNA copy number using additional computational approaches. Numerous tools have been developed to analyze Illumina genotype microarray data for copy number variant (CNV) discovery, although commonly utilized algorithms freely available to the public employ approaches based upon the use of hidden Markov models (HMMs). QuantiSNP, PennCNV, and GenoCN utilize HMMs with six copy number states but vary in how transition and emission probabilities are calculated. Performance of these CNV detection algorithms has been shown to be variable between both genotyping platforms and data sets, although HMM approaches generally outperform other current methods. Low sensitivity is prevalent with HMM-based algorithms, suggesting the need for continued improvement in CNV detection methodologies.

Application of Nexus copy number software for CNV detection and analysis.

PubMed

Darvishi, Katayoon

2010-04-01

Among human structural genomic variation, copy number variants (CNVs) are the most frequently known component, comprised of gains/losses of DNA segments that are generally 1 kb in length or longer. Array-based comparative genomic hybridization (aCGH) has emerged as a powerful tool for detecting genomic copy number variants (CNVs). With the rapid increase in the density of array technology and with the adaptation of new high-throughput technology, a reliable and computationally scalable method for accurate mapping of recurring DNA copy number aberrations has become a main focus in research. Here we introduce Nexus Copy Number software, a platform-independent tool, to analyze the output files of all types of commercial and custom-made comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays, such as those manufactured by Affymetrix, Agilent Technologies, Illumina, and Roche NimbleGen. It also supports data generated by various array image-analysis software tools such as GenePix, ImaGene, and BlueFuse. (c) 2010 by John Wiley & Sons, Inc.

Functional effects of CCL3L1 copy number.

PubMed

Carpenter, D; McIntosh, R S; Pleass, R J; Armour, J A L

2012-07-01

Copy number variation (CNV) is becoming increasingly important as a feature of human variation in disease susceptibility studies. However, the consequences of CNV are not so well understood. Here, we present data exploring the functional consequences of CNV of CCL3L1 in 55 independent UK samples with no known clinical phenotypes. The copy number of CCL3L1 was determined by the paralogue ratio test, and expression levels of macrophage inflammatory protein-1α (MIP-1α) and mRNA from stimulated monocytes were measured and analysed. The data show no statistically significant association of MIP-1α protein levels with copy number. However, there was a significant correlation between copy number and CCL3L1:CCL3 mRNA ratio. The data also provide evidence that expression of CCL3 predominates in both protein and mRNA, and therefore the observed variation of CCL3 is potentially more important biologically than that of CNV of CCL3L1.

[Detection of the exogenous gene copy number of the transgenic tomato anti-caries vaccine].

PubMed

Bai, Guo-hui; Liu, Jian-guo; Tian, Yuan; Chen, Zhu; Bai, Peng-yuan; Han, Qi; Gu, Yu; Guan, Xiao-yan; Wang, Hai-hui

2013-12-01

To detect the exogenous gene copy number of the transgenic tomato anti-caries vaccine by using the SYBR Green real-time PCR. Recombinant plasmid pEAC10 and pEPC10 were used as standard to detect genome samples of exogenous gene pacA-ctxB and pacP-ctxB by SYBR green fluorescent quantitation, then the average value was calculated as gene copy number. The copy number of the transgenic tomato carrying pacA-ctxB was 1.3 and the pacP-ctxB was 3.2. The transgenic tomato plants which have high stability are low-copy transgenic plants. Supported by National Natural Science Foundation of China (30160086, 81260164), Science and Technical Fund of Guizhou Province (LKZ[2011]41), Project of Technology Innovation Team in Guizhou Province, Leading Academic Discipline Construction Project in Guizhou Province and Excellent Scientific Research Team Cultivation Project in Zunyi Medical College ([2012]12).

Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

PubMed

Tonoike, A; Hori, Y; Inoue-Murayama, M; Konno, A; Fujita, K; Miyado, M; Fukami, M; Nagasawa, M; Mogi, K; Kikusui, T

2015-10-01

A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs. © 2015 Stichting International Foundation for Animal Genetics.

The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

NASA Technical Reports Server (NTRS)

Ludwig, D. L.; Bruschi, C. V.

1991-01-01

The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

Sparse representation and Bayesian detection of genome copy number alterations from microarray data.

PubMed

Pique-Regi, Roger; Monso-Varona, Jordi; Ortega, Antonio; Seeger, Robert C; Triche, Timothy J; Asgharzadeh, Shahab

2008-02-01

Genomic instability in cancer leads to abnormal genome copy number alterations (CNA) that are associated with the development and behavior of tumors. Advances in microarray technology have allowed for greater resolution in detection of DNA copy number changes (amplifications or deletions) across the genome. However, the increase in number of measured signals and accompanying noise from the array probes present a challenge in accurate and fast identification of breakpoints that define CNA. This article proposes a novel detection technique that exploits the use of piece wise constant (PWC) vectors to represent genome copy number and sparse Bayesian learning (SBL) to detect CNA breakpoints. First, a compact linear algebra representation for the genome copy number is developed from normalized probe intensities. Second, SBL is applied and optimized to infer locations where copy number changes occur. Third, a backward elimination (BE) procedure is used to rank the inferred breakpoints; and a cut-off point can be efficiently adjusted in this procedure to control for the false discovery rate (FDR). The performance of our algorithm is evaluated using simulated and real genome datasets and compared to other existing techniques. Our approach achieves the highest accuracy and lowest FDR while improving computational speed by several orders of magnitude. The proposed algorithm has been developed into a free standing software application (GADA, Genome Alteration Detection Algorithm). http://biron.usc.edu/~piquereg/GADA

Population genetics and molecular evolution of DNA sequences in transposable elements. I. A simulation framework.

PubMed

Kijima, T E; Innan, Hideki

2013-11-01

A population genetic simulation framework is developed to understand the behavior and molecular evolution of DNA sequences of transposable elements. Our model incorporates random transposition and excision of transposable element (TE) copies, two modes of selection against TEs, and degeneration of transpositional activity by point mutations. We first investigated the relationships between the behavior of the copy number of TEs and these parameters. Our results show that when selection is weak, the genome can maintain a relatively large number of TEs, but most of them are less active. In contrast, with strong selection, the genome can maintain only a limited number of TEs but the proportion of active copies is large. In such a case, there could be substantial fluctuations of the copy number over generations. We also explored how DNA sequences of TEs evolve through the simulations. In general, active copies form clusters around the original sequence, while less active copies have long branches specific to themselves, exhibiting a star-shaped phylogeny. It is demonstrated that the phylogeny of TE sequences could be informative to understand the dynamics of TE evolution.

Psoriasis is associated with increased beta-defensin genomic copy number

PubMed Central

Hollox, Edward J.; Huffmeier, Ulrike; Zeeuwen, Patrick L.J.M.; Palla, Raquel; Lascorz, Jesús; Rodijk-Olthuis, Diana; van de Kerkhof, Peter C.M.; Traupe, Heiko; de Jongh, Gys; den Heijer, Martin; Reis, André; Armour, John A.L.; Schalkwijk, Joost

2008-01-01

Psoriasis is a common inflammatory skin disease with a strong genetic component. We have analysed the genomic copy number polymorphism of the beta-defensin region on human chromosome 8 in 179 Dutch psoriasis patients and 272 controls, and in 319 German psoriasis patients and 305 controls. Comparisons in both cohorts show a significant association between higher genomic copy number for beta-defensin genes and the risk of psoriasis. PMID:18059266

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.

PubMed

Ziemons, Sandra; Koutsantas, Katerina; Becker, Kordula; Dahlmann, Tim; Kück, Ulrich

2017-02-16

Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster. We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains. Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

Human-Compatible Animal Models for Preclinical Research on Hormones in Breast Cancer

DTIC Science & Technology

2012-09-01

Hormone/Prolactin Family in Biology and Disease” in July, 2012. Several participants inquired as to whether we had determined the number of copies of...in situ hybridization) analysis of both lines to determine the copy number of the transgene. We found that the BAC-h8 line has a single copy of the...transgene and the BAC-h30 line has two copies (Figure 5). Breeding of the hPRL+ mice onto an immunodeficient background: As discussed in last

18 CFR 45.7 - Form of application; number of copies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Form of application; number of copies. 45.7 Section 45.7 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... in accordance with § 131.60 of this chapter. Each copy shall bear the date and signature that appear...

A Coarse-Grained Biophysical Model of E. coli and Its Application to Perturbation of the rRNA Operon Copy Number

PubMed Central

Tadmor, Arbel D.; Tlusty, Tsvi

2008-01-01

We propose a biophysical model of Escherichia coli that predicts growth rate and an effective cellular composition from an effective, coarse-grained representation of its genome. We assume that E. coli is in a state of balanced exponential steady-state growth, growing in a temporally and spatially constant environment, rich in resources. We apply this model to a series of past measurements, where the growth rate and rRNA-to-protein ratio have been measured for seven E. coli strains with an rRNA operon copy number ranging from one to seven (the wild-type copy number). These experiments show that growth rate markedly decreases for strains with fewer than six copies. Using the model, we were able to reproduce these measurements. We show that the model that best fits these data suggests that the volume fraction of macromolecules inside E. coli is not fixed when the rRNA operon copy number is varied. Moreover, the model predicts that increasing the copy number beyond seven results in a cytoplasm densely packed with ribosomes and proteins. Assuming that under such overcrowded conditions prolonged diffusion times tend to weaken binding affinities, the model predicts that growth rate will not increase substantially beyond the wild-type growth rate, as indicated by other experiments. Our model therefore suggests that changing the rRNA operon copy number of wild-type E. coli cells growing in a constant rich environment does not substantially increase their growth rate. Other observations regarding strains with an altered rRNA operon copy number, such as nucleoid compaction and the rRNA operon feedback response, appear to be qualitatively consistent with this model. In addition, we discuss possible design principles suggested by the model and propose further experiments to test its validity. PMID:18437222

Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

PubMed

Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

2014-05-19

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3--high copy number group, TST-1 and PRR-1--medium copy number group, P4H-1, APRT-2 and CYC-2--low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

Comparison of the effects of formaldehyde and gaseous ozone on HBV-contaminated hospital quilts

PubMed Central

Guo, Dan; Li, Ziqiong; Jia, Bei; Che, Xiaoqiong; Song, Tianshuang; Huang, Wenxiang

2015-01-01

Background: Besides being highly infectious, Hepatitis B virus (HBV) is a major cause of liver disease worldwide. In hospital settings, it is easy for the environment and quilts to be contaminated by HBV patient blood and body fluids. Therefore, HBV can be transmitted to other patients via contaminated environmental surfaces or quilts, resulting in an HBV nosocomial infection. Formaldehyde and ozone are commonly used disinfectants that may influence this infectious situation. Objective: To investigate the clinical effectiveness of formaldehyde and gaseous ozone for the terminal cleaning of hospital quilts contaminated by HBV. Methods: Thin cloth and thick cotton soaked with the serum from high HBV copy number patients were prepared and disinfected using formaldehyde fumigation and gaseous ozone at different times. The copy numbers of HBV DNA in the HBV-contaminated cloth and cotton samples were measured quantitatively with fluorescent quantitative polymerase chain reaction (PCR). Results: When gaseous ozone was used to disinfect HBV-contaminated quilts for 23 minutes (min), 36 min, 49 min, and 90 min, the HBV DNA copy number displayed no significant decrease compared with the copy number before disinfection (P > 0.05). In comparison, the copy number of the HBV DNA in the cloth group decreased significantly (P < 0.05) after formaldehyde fumigation disinfection for 1 hour (h), and there was no difference when longer times and increased concentrations were used. In the thick cotton group, there was also a significant decrease (P < 0.05) of the HBV DNA copy numbers, but the decrease was not as dramatic. In addition, in this group, the disinfection effect observed at 4 h was the strongest. Conclusions: The application of ozone to disinfect HBV-contaminated hospital quilts possibly has no effect, whereas, formaldehyde oxide fumigation effectively reduced HBV copy numbers. PMID:26770591

Correlation of Clinical Outcomes with Quantitative Polymerase Chain Reaction DNA Copy Number in Patients with Acute Retinal Necrosis.

PubMed

Calvo, Charles M; Khan, Mohammed Ali; Mehta, Sonia; Garg, Sunir J; Dunn, James P

2017-04-01

To correlate visual acuity outcomes and clinical features with quantitative PCR DNA copy number in patients with acute retinal necrosis (ARN). Retrospective, consecutive case series. In total, 14 eyes of 13 patients were diagnosed with ARN, based on the American Uveitis Society criteria, and were followed for a mean of 324.5 days (median 250.5 days, SD ± 214 days). Anterior chamber fluid analyzed by quantitative PCR identified viral DNA in 11 of 14 eyes (78.5%). Varicella zoster virus (VZV) was identified in seven eyes (50%) and herpes simplex virus (HSV) in four eyes (28.5%). Mean DNA copy number was 7.9 × 10 6 /mL (median 2.10 × 10 6 /mL, range: 0-5.60 × 10 7 /mL). Eyes with quantitative PCR DNA copy number of ≥5.0 × 10 6 /mL (n = 6 eyes) had worse baseline visual acuity (logMAR 1.48 ± 0.71 vs 0.94 ± 0.76, p = 0.196) and final visual acuity (logMAR 2.10 ± 0.60 vs 0.82 ± 0.81, p = 0.007) compared with patients with a DNA copy number <5.0 × 10 6 /mL (n = 8 eyes). Patients with a DNA copy number of ≥5.0 × 10 6 /mL were more likely to have at least 5 clock hours of retinitis on funduscopic exam (p = 0.03) and developed retinal detachment more frequently (p = 0.08). Quantitative DNA copy number of ≥5.0 × 10 6 /mL is associated with more extensive retinitis, worse visual acuity, and development of retinal detachment in patients with acute retinal necrosis.

[Gene copy number, mRNA transcription and protein expression of PD-1 gene in primary hepatocarcinoma patients].

PubMed

Fan, Hui-Min; Wu, Ling-Jie; Hu, Feng-Yu; Yang, Zhan

2012-08-01

To study the gene copy number, mRNA transcription and protien expression of programmed cell death 1 (PD-1) gene in primary hepatocellular carcinoma (PHC) patients and normal control individuals (NC) who are anti-HBs positive, and to investigate the variations in PD-1 gene copy numbers and its relationship with PHC. Real-time PCR was adopted to detect the PD-1 gene copy numbers and their mRNA expressions in peripheral blood mononuclear cells (PBMCs) from 24 samples of PHC patients and 26 of NC. Protein expression level of PD-1 on CD8+ T was analyzed by flow cytometry. In terms of number of PD-1 gene copy numbers, the percentage of cases of haploid (single) was 34.62% and 4.17% in PHC group and control group respectively while the percentage of cases of diploid (double) was 61.54% and 95.83% respectively. The difference between the two was statistically significant (chi2 = 7.639, P = 0.006). The rate of cases with double PD-1 gene copy numbers was found to be higher in patients with PHC than in control group. It was also found that the average expression of PD-1 mRNA was 2.35E-03 in control group and 1.23E-03 in PHC group. The expression level was significant lower in PHC group than that in control group when compared by using Mann-whitey technic (U = 153, P = 0.009). Furthermore, the frequency of PD-1 protein expression on CD8+ T cells was 3.72 +/- 0.32 in control group and 16.13 +/- 1.68 in PHC group. The level of PD-1 mRNA expression was higher in PHC and significant differences was shown between two groups (t = -7.073, P = 0.000). Our study suggests that the variation in PD-1 gene copy number may trigger primary hepatocellular carcinoma to HBV carriers. The relationship between the variation of PD-1 gene copy numbers and its association with primary hepatocellular carcinoma is worth further focus.

Copy Number Variants and Congenital Anomalies Surveillance: A Suggested Coding Strategy Using the Royal College of Paediatrics and Child Health Version of ICD-10.

PubMed

Bedard, Tanya; Lowry, R Brian; Sibbald, Barbara; Thomas, Mary Ann; Innes, A Micheil

2016-01-01

The use of array-based comparative genomic hybridization to assess DNA copy number is increasing in many jurisdictions. Such technology identifies more genetic causes of congenital anomalies; however, the clinical significance of some results may be challenging to interpret. A coding strategy to address cases with copy number variants has recently been implemented by the Alberta Congenital Anomalies Surveillance System and is described.

Constitutional trisomy 8 and Behçet syndrome.

PubMed

Becker, Kristin; Fitzgerald, Oliver; Green, Andrew J; Keogan, Mary; Newbury-Ecob, Ruth; Greenhalgh, Lynn; Withers, Stephen; Hollox, Edward J; Aldred, Patricia M R; Armour, John A L

2009-05-01

The characteristic clinical features of constitutional trisomy 8 include varying degrees of developmental delay, joint contractures and deep palmar and plantar creases. There is an established literature, which describes features of Behçet syndrome occurring in phenotypically normal individuals with myelodysplastic syndromes and trisomy 8 in their bone marrow. In this article, we describe four patients with constitutional trisomy 8, all with varying clinical phenotypes, who developed features of Behçet, in particular but not exclusively mucocutaneous ulceration. In addition, we examined gene copy numbers of the variable-number neutrophil defensin genes DEFA1A3 in one of the cases (case 1) and her parents, together with 14 cases of Behçet syndrome in comparison with 121 normal controls. The gene copy number was highest in case 1 (copy number 14) and was also increased in her parents (both copy number 9). However the mean copy number for DEFA1A3 among the 14 Behçet syndrome patients was actually lower (5.1) than among the controls (mean of 6.8 copies). Thus, we conclude that patients with constitutional trisomy 8 and those with trisomy 8 confined to the bone marrow are both at increased risk of developing features of Behçet syndrome. The mechanism may relate to increased chromosome 8 gene dosage with further analysis of candidate genes on chromosome 8 required.

Dietary Variation and Evolution of Gene Copy Number among Dog Breeds

PubMed Central

Reiter, Taylor; Jagoda, Evelyn; Capellini, Terence D.

2016-01-01

Prolonged human interactions and artificial selection have influenced the genotypic and phenotypic diversity among dog breeds. Because humans and dogs occupy diverse habitats, ecological contexts have likely contributed to breed-specific positive selection. Prior to the advent of modern dog-feeding practices, there was likely substantial variation in dietary landscapes among disparate dog breeds. As such, we investigated one type of genetic variant, copy number variation, in three metabolic genes: glucokinase regulatory protein (GCKR), phytanol-CoA 2-hydroxylase (PHYH), and pancreatic α-amylase 2B (AMY2B). These genes code for proteins that are responsible for metabolizing dietary products that originate from distinctly different food types: sugar, meat, and starch, respectively. After surveying copy number variation among dogs with diverse dietary histories, we found no correlation between diet and positive selection in either GCKR or PHYH. Although it has been previously demonstrated that dogs experienced a copy number increase in AMY2B relative to wolves during or after the dog domestication process, we demonstrate that positive selection continued to act on amylase copy number in dog breeds that consumed starch-rich diets in time periods after domestication. Furthermore, we found that introgression with wolves is not responsible for deterioration of positive selection on AMY2B among diverse dog breeds. Together, this supports the hypothesis that the amylase copy number expansion is found universally in dogs. PMID:26863414

Dietary Variation and Evolution of Gene Copy Number among Dog Breeds.

PubMed

Reiter, Taylor; Jagoda, Evelyn; Capellini, Terence D

2016-01-01

Prolonged human interactions and artificial selection have influenced the genotypic and phenotypic diversity among dog breeds. Because humans and dogs occupy diverse habitats, ecological contexts have likely contributed to breed-specific positive selection. Prior to the advent of modern dog-feeding practices, there was likely substantial variation in dietary landscapes among disparate dog breeds. As such, we investigated one type of genetic variant, copy number variation, in three metabolic genes: glucokinase regulatory protein (GCKR), phytanol-CoA 2-hydroxylase (PHYH), and pancreatic α-amylase 2B (AMY2B). These genes code for proteins that are responsible for metabolizing dietary products that originate from distinctly different food types: sugar, meat, and starch, respectively. After surveying copy number variation among dogs with diverse dietary histories, we found no correlation between diet and positive selection in either GCKR or PHYH. Although it has been previously demonstrated that dogs experienced a copy number increase in AMY2B relative to wolves during or after the dog domestication process, we demonstrate that positive selection continued to act on amylase copy number in dog breeds that consumed starch-rich diets in time periods after domestication. Furthermore, we found that introgression with wolves is not responsible for deterioration of positive selection on AMY2B among diverse dog breeds. Together, this supports the hypothesis that the amylase copy number expansion is found universally in dogs.

Screening for common copy-number variants in cancer genes.

PubMed

Tyson, Jess; Majerus, Tamsin M O; Walker, Susan; Armour, John A L

2010-12-01

For most cases of colorectal cancer that arise without a family history of the disease, it is proposed that an appreciable heritable component of predisposition is the result of contributions from many loci. Although progress has been made in identifying single nucleotide variants associated with colorectal cancer risk, the involvement of low-penetrance copy number variants is relatively unexplored. We have used multiplex amplifiable probe hybridization (MAPH) in a fourfold multiplex (QuadMAPH), positioned at an average resolution of one probe per 2 kb, to screen a total of 1.56 Mb of genomic DNA for copy number variants around the genes APC, AXIN1, BRCA1, BRCA2, CTNNB1, HRAS, MLH1, MSH2, and TP53. Two deletion events were detected, one upstream of MLH1 in a control individual and the other in APC in a colorectal cancer patient, but these do not seem to correspond to copy number polymorphisms with measurably high population frequencies. In summary, by means of our QuadMAPH assay, copy number measurement data were of sufficient resolution and accuracy to detect any copy number variants with high probability. However, this study has demonstrated a very low incidence of deletion and duplication variants within intronic and flanking regions of these nine genes, in both control individuals and colorectal cancer patients. Copyright © 2010 Elsevier Inc. All rights reserved.

Real-Time PCR for the Detection of Precise Transgene Copy Number in Wheat.

PubMed

Giancaspro, Angelica; Gadaleta, Agata; Blanco, Antonio

2017-01-01

Despite the unceasing advances in genetic transformation techniques, the success of common delivery methods still lies on the behavior of the integrated transgenes in the host genome. Stability and expression of the introduced genes are influenced by several factors such as chromosomal location, transgene copy number and interaction with the host genotype. Such factors are traditionally characterized by Southern blot analysis, which can be time-consuming, laborious, and often unable to detect the exact copy number of rearranged transgenes. Recent research in crop field suggests real-time PCR as an effective and reliable tool for the precise quantification and characterization of transgene loci. This technique overcomes most problems linked to phenotypic segregation analysis and can analyze hundreds of samples in a day, making it an efficient method for estimating a gene copy number integrated in a transgenic line. This protocol describes the use of real-time PCR for the detection of transgene copy number in durum wheat transgenic lines by means of two different chemistries (SYBR ® Green I dye and TaqMan ® probes).

An Integrated Approach for RNA-seq Data Normalization.

PubMed

Yang, Shengping; Mercante, Donald E; Zhang, Kun; Fang, Zhide

2016-01-01

DNA copy number alteration is common in many cancers. Studies have shown that insertion or deletion of DNA sequences can directly alter gene expression, and significant correlation exists between DNA copy number and gene expression. Data normalization is a critical step in the analysis of gene expression generated by RNA-seq technology. Successful normalization reduces/removes unwanted nonbiological variations in the data, while keeping meaningful information intact. However, as far as we know, no attempt has been made to adjust for the variation due to DNA copy number changes in RNA-seq data normalization. In this article, we propose an integrated approach for RNA-seq data normalization. Comparisons show that the proposed normalization can improve power for downstream differentially expressed gene detection and generate more biologically meaningful results in gene profiling. In addition, our findings show that due to the effects of copy number changes, some housekeeping genes are not always suitable internal controls for studying gene expression. Using information from DNA copy number, integrated approach is successful in reducing noises due to both biological and nonbiological causes in RNA-seq data, thus increasing the accuracy of gene profiling.

ITALICS: an algorithm for normalization and DNA copy number calling for Affymetrix SNP arrays.

PubMed

Rigaill, Guillem; Hupé, Philippe; Almeida, Anna; La Rosa, Philippe; Meyniel, Jean-Philippe; Decraene, Charles; Barillot, Emmanuel

2008-03-15

Affymetrix SNP arrays can be used to determine the DNA copy number measurement of 11 000-500 000 SNPs along the genome. Their high density facilitates the precise localization of genomic alterations and makes them a powerful tool for studies of cancers and copy number polymorphism. Like other microarray technologies it is influenced by non-relevant sources of variation, requiring correction. Moreover, the amplitude of variation induced by non-relevant effects is similar or greater than the biologically relevant effect (i.e. true copy number), making it difficult to estimate non-relevant effects accurately without including the biologically relevant effect. We addressed this problem by developing ITALICS, a normalization method that estimates both biological and non-relevant effects in an alternate, iterative manner, accurately eliminating irrelevant effects. We compared our normalization method with other existing and available methods, and found that ITALICS outperformed these methods for several in-house datasets and one public dataset. These results were validated biologically by quantitative PCR. The R package ITALICS (ITerative and Alternative normaLIzation and Copy number calling for affymetrix Snp arrays) has been submitted to Bioconductor.

rrndb: the Ribosomal RNA Operon Copy Number Database

PubMed Central

Klappenbach, Joel A.; Saxman, Paul R.; Cole, James R.; Schmidt, Thomas M.

2001-01-01

The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes, information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections. Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme.msu.edu. PMID:11125085

Exploiting rRNA operon copy number to investigate bacterial reproductive strategies.

PubMed

Roller, Benjamin R K; Stoddard, Steven F; Schmidt, Thomas M

2016-09-12

The potential for rapid reproduction is a hallmark of microbial life, but microbes in nature must also survive and compete when growth is constrained by resource availability. Successful reproduction requires different strategies when resources are scarce and when they are abundant 1,2 , but a systematic framework for predicting these reproductive strategies in bacteria has not been available. Here, we show that the number of ribosomal RNA operons (rrn) in bacterial genomes predicts two important components of reproduction-growth rate and growth efficiency-which are favoured under contrasting regimes of resource availability 3,4 . We find that the maximum reproductive rate of bacteria doubles with a doubling of rrn copy number, and the efficiency of carbon use is inversely related to maximal growth rate and rrn copy number. We also identify a feasible explanation for these patterns: the rate and yield of protein synthesis mirror the overall pattern in maximum growth rate and growth efficiency. Furthermore, comparative analysis of genomes from 1,167 bacterial species reveals that rrn copy number predicts traits associated with resource availability, including chemotaxis and genome streamlining. Genome-wide patterns of orthologous gene content covary with rrn copy number, suggesting convergent evolution in response to resource availability. Our findings imply that basic cellular processes adapt in contrasting ways to long-term differences in resource availability. They also establish a basis for predicting changes in bacterial community composition in response to resource perturbations using rrn copy number measurements 5 or inferences 6,7 .

MET amplification as a potential therapeutic target in gastric cancer

PubMed Central

Kawakami, Hisato; Okamoto, Isamu; Arao, Tokuzo; Okamoto, Wataru; Matsumoto, Kazuko; Taniguchi, Hirokazu; Kuwata, Kiyoko; Yamaguchi, Haruka; Nishio, Kazuto; Nakagawa, Kazuhiko; Yamada, Yasuhide

2013-01-01

Our aim was to investigate both the prevalence of MET amplification in gastric cancer as well as the potential of this genetic alteration to serve as a therapeutic target in gastric cancer. MET amplification was assessed by initial screening with a PCR-based copy number assay followed by confirmatory FISH analysis in formalin-fixed, paraffin-embedded specimens of gastric cancer obtained at surgery. The effects of MET tyrosine kinase inhibitors (MET-TKIs) in gastric cancer cells with or without MET amplification were also examined. The median MET copy number in 266 cases of gastric cancer was 1.7, with a range of 0.41 to 21.3. We performed FISH analysis for the 15 cases with the highest MET copy numbers. MET amplification was confirmed in the four assessable cases with a MET copy number of at least 4, whereas MET amplification was not detected in those with a gene copy number of <4. The prevalence of MET amplification was thus 1.5% (4 out of 266 cases). Inhibition of MET by MET-TKIs resulted in the induction of apoptosis accompanied by attenuation of downstream MET signaling in gastric cancer cell lines with MET amplification but not in those without this genetic change. MET amplification identifies a small but clinically important subgroup of gastric cancer patients who are likely to respond to MET-TKIs. Furthermore, screening with a PCR-based copy number assay is an efficient way to reduce the number of patients requiring confirmation of MET amplification by FISH analysis. PMID:23327903

Presence of high-risk human papillomavirus genotype and human immunodeficiency virus DNA in anal high-grade and low-grade squamous intraepithelial lesions.

PubMed

Shiramizu, Bruce; Liang, Chin-Yuan; Agsalda-Garcia, Melissa; Nagata, Ian; Milne, Cris; Zhu, Xuemei; Killeen, Jeffrey; Berry, J Michael; Goodman, Marc T

2013-01-01

Human immunodeficiency virus type 1 (HIV)-infected individuals are at risk for anal cancer, which is caused by human papillomavirus (HPV). The relationship between HIV and HPV that leads to anal cancer remains unclear. Recent data, however, suggest that the continued persistence of HIV DNA in patients treated with combined antiretroviral therapy leads to progression of HIV disease and other HIV-associated complications. Therefore, we investigated the relationship among anal low- and high-grade squamous intraepithelial lesions (LGSIL/HGSIL), high-risk HPV genotypes, and high HIV DNA copy numbers. Anal cytology specimens were assayed for HPV genotype and HIV DNA copy number. High-risk HPV genotypes (odds ratio OR: 3.73; 95% confidence interval CI: 1.08-12.91; p=0.04) and high HIV DNA copy numbers (OR(per 100 HIV DNA copies): 1.13; 95% CI: 1.01-1.27, p=0.04) were both associated with LGSIL/HGSIL. When considering both high-risk HPV genotypes and HIV DNA copy numbers in predicting LGSIL/HGSIL, HIV DNA copy number was significant (OR(per 100 HIV DNA copies): 1.09; 95% CI: 0.96-1.23, p=0.04) but not high-risk HPV genotypes (OR: 2.30, p=0.28), which did not change when adjusted for nadir CD4 cell count and HIV RNA levels. The findings warrant further investigation of HIV DNA and its relationship with HPV in LGSIL/HGSIL pathogenesis.

EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia.

PubMed

Gaines, Todd A; Barker, Abigail L; Patterson, Eric L; Westra, Philip; Westra, Eric P; Wilson, Robert G; Jha, Prashant; Kumar, Vipan; Kniss, Andrew R

2016-01-01

Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number.

Isolation of polysomal RNA for analyzing stress-responsive genes regulated at the translational level in plants

USDA-ARS?s Scientific Manuscript database

Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, in...

Genomic profiling of CHEK2*1100delC-mutated breast carcinomas.

PubMed

Massink, Maarten P G; Kooi, Irsan E; Martens, John W M; Waisfisz, Quinten; Meijers-Heijboer, Hanne

2015-11-09

CHEK2*1100delC is a moderate-risk breast cancer susceptibility allele with a high prevalence in the Netherlands. We performed copy number and gene expression profiling to investigate whether CHEK2*1100delC breast cancers harbor characteristic genomic aberrations, as seen for BRCA1 mutated breast cancers. We performed high-resolution SNP array and gene expression profiling of 120 familial breast carcinomas selected from a larger cohort of 155 familial breast tumors, including BRCA1, BRCA2, and CHEK2 mutant tumors. Gene expression analyses based on a mRNA immune signature was used to identify samples with relative low amounts of tumor infiltrating lymphocytes (TILs), which were previously found to disturb tumor copy number and LOH (loss of heterozygosity) profiling. We specifically compared the genomic and gene expression profiles of CHEK2*1100delC breast cancers (n = 14) with BRCAX (familial non-BRCA1/BRCA2/CHEK2*1100delC mutated) breast cancers (n = 34) of the luminal intrinsic subtypes for which both SNP-array and gene expression data is available. High amounts of TILs were found in a relatively small number of luminal breast cancers as compared to breast cancers of the basal-like subtype. As expected, these samples mostly have very few copy number aberrations and no detectable regions of LOH. By unsupervised hierarchical clustering of copy number data we observed a great degree of heterogeneity amongst the CHEK2*1100delC breast cancers, comparable to the BRCAX breast cancers. Furthermore, copy number aberrations were mostly seen at low frequencies in both the CHEK2*1100delC and BRCAX group of breast cancers. However, supervised class comparison identified copy number loss of chromosomal arm 1p to be associated with CHEK2*1100delC status. In conclusion, in contrast to basal-like BRCA1 mutated breast cancers, no apparent specific somatic copy number aberration (CNA) profile for CHEK2*1100delC breast cancers was found. With the possible exception of copy number loss of chromosomal arm 1p in a subset of tumors, which might be involved in CHEK2 tumorigenesis. This difference in CNAs profiles might be explained by the need for BRCA1-deficient tumor cells to acquire survival factors, by for example specific copy number aberrations, to expand. Such factors may not be needed for breast tumors with a defect in a non-essential gene such as CHEK2.

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

PubMed Central

Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick

2016-01-01

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866

Anti-inflammatory effects of alpinone 3-acetate from Alpinia japonica seeds.

PubMed

Kakegawa, Tomohito; Miyazaki, Aya; Yasukawa, Ken

2016-07-01

We aimed to investigate the bioactive components of Alpinia japonica as anti-inflammatory compounds using searches of the Alpinia genus, and subsequently demonstrated that alpinone 3-acetate markedly inhibits 12-O-tetradecanoyiphorbol 13-acetate-induced inflammation in a mouse model of ear edema. To assess other bioactivities of alpinone 3-acetate, we performed translatome analyses and compared them with those of hydrocortisone. Polysome-associated mRNAs were prepared from alpinone 3-acetate- or hydrocortisone-treated and control cells from 12-O-tetradecanoyiphorbol 13-acetate-induced THP-1-derived macrophages cultured in the presence of Escherichia coli O-111 lipopolysaccharide. Subsequent microarray analysis revealed that alpinone 3-acetate and hydrocortisone upregulated and downregulated the same 155 and 41 genes, respectively. Moreover, direct comparisons of translationally regulated genes indicated 5 and 10 gene probes that were upregulated and downregulated by alpinone 3-acetate and hydrocortisone, respectively. In conclusion, assays of 12-O-tetradecanoyiphorbol 13-acetate-induced inflammation ear edema in mice and polysome profiling of alpinone 3-acetate bioactivities indicated similar medicinal possibilities to those of hydrocortisone.

Combined transcriptome and translatome analyses reveal a role for tryptophan-dependent auxin biosynthesis in the control of DOG1-dependent seed dormancy.

PubMed

Bai, Bing; Novák, Ondřej; Ljung, Karin; Hanson, Johannes; Bentsink, Leónie

2018-02-01

The importance of translational regulation during Arabidopsis seed germination has been shown previously. Here the role of transcriptional and translational regulation during seed imbibition of the very dormant DELAY OF GERMINATION 1 (DOG1) near-isogenic line was investigated. Polysome profiling was performed on dormant and after-ripened seeds imbibed for 6 and 24 h in water and in the transcription inhibitor cordycepin. Transcriptome and translatome changes were investigated. Ribosomal profiles of after-ripened seeds imbibed in cordycepin mimic those of dormant seeds. The polysome occupancy of mRNA species is not affected by germination inhibition, either as a result of seed dormancy or as a result of cordycepin treatment, indicating the importance of the regulation of transcript abundance. The expression of auxin metabolism genes is discriminative during the imbibition of after-ripened and dormant seeds, which is confirmed by altered concentrations of indole-3-acetic acid conjugates and precursors. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

The antidepressant sertraline inhibits translation initiation by curtailing mammalian target of rapamycin signaling.

PubMed

Lin, Chen-Ju; Robert, Francis; Sukarieh, Rami; Michnick, Stephen; Pelletier, Jerry

2010-04-15

Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner. (c) 2010 AACR.

Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia.

PubMed

Lim, Pharath; Alker, Alisa P; Khim, Nimol; Shah, Naman K; Incardona, Sandra; Doung, Socheat; Yi, Poravuth; Bouth, Denis Mey; Bouchier, Christiane; Puijalon, Odile Mercereau; Meshnick, Steven R; Wongsrichanalai, Chansuda; Fandeur, Thierry; Le Bras, Jacques; Ringwald, Pascal; Ariey, Frédéric

2009-01-12

The combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy. Blood samples were collected from P. falciparum-infected patients enrolled in two in vivo efficacy studies in north-western Cambodia: 135 patients were treated with artemether-lumefantrine (AL group) in Sampovloun in 2002 and 2003, and 140 patients with artesunate-mefloquine (AM group) in Sampovloun and Veal Veng in 2003 and 2004. At enrollment, the in vitro IC50 was tested and the strains were genotyped for pfmdr1 copy number by real-time PCR. The pfmdr1 copy number was analysed for 115 isolates in the AM group, and for 109 isolates in the AL group. Parasites with increased pfmdr1 copy number had significantly reduced in vitro susceptibility to mefloquine, lumefantrine and artesunate. There was no association between pfmdr1 polymorphisms and in vitro susceptibilities. In the patients treated with AM, the mean pfmdr1copy number was lower in subjects with adequate clinical and parasitological response compared to those who experienced late treatment failure (n = 112, p < 0.001). This was not observed in the patients treated with AL (n = 96, p = 0.364). The presence of three or more copies of pfmdr1 were associated with recrudescence in artesunate-mefloquine treated patients (hazard ratio (HR) = 7.80 [95%CI: 2.09-29.10], N = 115), p = 0.002) but not with recrudescence in artemether-lumefantrine treated patients (HR = 1.03 [95%CI: 0.24-4.44], N = 109, p = 0.969). This study shows that pfmdr1 copy number is a molecular marker of AM treatment failure in falciparum malaria on the Thai-Cambodian border. However, while it is associated with increased IC50 for lumefantrine, pfmdr1 copy number is not associated with AL treatment failure in the area, suggesting involvement of other molecular mechanisms in AL treatment failures in Cambodia.

Copy number variation of functional RBMY1 is associated with sperm motility: an azoospermia factor-linked candidate for asthenozoospermia.

PubMed

Yan, Yuanlong; Yang, Xiling; Liu, Yunqiang; Shen, Ying; Tu, Wenling; Dong, Qiang; Yang, Dong; Ma, Yongyi; Yang, Yuan

2017-07-01

What is the influence of copy number variation (CNV) in functional RNA binding motif protein Y-linked family 1 (RBMY1) on spermatogenic phenotypes? The RBMY1 functional copy dosage is positively correlated with sperm motility, and dosage insufficiency is an independent risk factor for asthenozoospermia. RBMY1, a multi-copy gene expressed exclusively in the adult testis, is one of the most important candidates for male infertility in the azoospermia factor (AZF) region of the Y-chromosome. RBMY1 encodes an RNA-binding protein that serves as a pre-mRNA splicing regulator during spermatogenesis, and male mice deficient in Rbmy are sterile. A total of 3127 adult males were recruited from 2009 to 2016; of this group, the dosage of RBMY1 functional copy were investigated in 486 fertile males. In the remaining 2641 males with known spermatogenesis status, 1070 Y-chromosome haplogroup (Y-hg) O3* or O3e carriers without chromosomal aberration or known AZF structure mutations responsible for spermatogenic impairment, including 506 men with normozoospermia and 564 men with oligozoospermia or/and asthenozoospermia, were screened, and the RBMY1 functional copy dosage and copy conversion were determined to explore their associations with sperm phenotypes. The correlation between RBMY1 dosage and its mRNA level or RBMY1 protein level and the correlation between sperm RBMY1 level and motility were analysed in 15 testis tissue samples and eight semen samples. Ten additional semen samples were used to confirm the subcellular localization of RBMY1 in individual sperm. All the Han volunteers donating whole blood, semen and testis tissue were from southwest China. RBMY1 copy number, copy conversion, mRNA/protein amount and protein location in sperm were detected using the AccuCopy® assay method, paralog ratio test, quantitative PCR, western blotting and immunofluorescence staining methods, respectively. This study identified Y-hg-independent CNV of functional RBMY1 in the enrolled population. A difference in the distribution of RBMY1 copy number was observed between the group with normal sperm motility and the group with asthenozoospermia. A positive correlation between the RBMY1 copy dosage and sperm motility was identified, and the males with fewer than six copies of RBMY1 showed an elevated risk for asthenozoospermia relative to those with six RBMY1 copies, the most common dosage in the population. The RBMY1 copy dosage was positively correlated with its mRNA and protein level in the testis. Sperm with high motility were found to carry more RBMY1 protein than those with relatively low motility. The RBMY1 protein was confirmed to predominantly localize in the neck and mid-piece region of sperm as well as the principal piece of the sperm tail. Our population study completes a chain of evidence suggesting that RBMY1 influences the susceptibility of males to asthenozoospermia by modulating sperm motility. High sequence similarity between the RBMY1 functional copies and a large number of pseudogenes potentially reduces the accuracy of the copy number detection. The mechanism underlying the CNV in RBMY1 is still unclear, and the effect of the structural variations in the RBMY1 copy cluster on the copy dosage of other protein-coding genes located in the region cannot be excluded, which may potentially bias our observations. Asthenozoospermia is a multi-factor complex disease with a limited number of proven susceptibility genes. This study identified a novel genomic candidate independently contributing to the condition, enriching our understanding of the role of AZF-linked genes in male reproduction. Our finding provides insight into the physiological and pathological characteristics of RBMY1 in terms of sperm motility, supplies persuasive evidence of the significance of RBMY1 copy number analysis in the clinical counselling of male infertility resulting from asthenozoospermia. This work was funded by the National Natural Science Foundation of China (Nos. 81370748 and 30971598). The authors have no conflicts of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants

PubMed Central

Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A.

2016-01-01

Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes. PMID:27622766

Haplotype Phasing and Inheritance of Copy Number Variants in Nuclear Families

PubMed Central

Palta, Priit; Kaplinski, Lauris; Nagirnaja, Liina; Veidenberg, Andres; Möls, Märt; Nelis, Mari; Esko, Tõnu; Metspalu, Andres; Laan, Maris; Remm, Maido

2015-01-01

DNA copy number variants (CNVs) that alter the copy number of a particular DNA segment in the genome play an important role in human phenotypic variability and disease susceptibility. A number of CNVs overlapping with genes have been shown to confer risk to a variety of human diseases thus highlighting the relevance of addressing the variability of CNVs at a higher resolution. So far, it has not been possible to deterministically infer the allelic composition of different haplotypes present within the CNV regions. We have developed a novel computational method, called PiCNV, which enables to resolve the haplotype sequence composition within CNV regions in nuclear families based on SNP genotyping microarray data. The algorithm allows to i) phase normal and CNV-carrying haplotypes in the copy number variable regions, ii) resolve the allelic copies of rearranged DNA sequence within the haplotypes and iii) infer the heritability of identified haplotypes in trios or larger nuclear families. To our knowledge this is the first program available that can deterministically phase null, mono-, di-, tri- and tetraploid genotypes in CNV loci. We applied our method to study the composition and inheritance of haplotypes in CNV regions of 30 HapMap Yoruban trios and 34 Estonian families. For 93.6% of the CNV loci, PiCNV enabled to unambiguously phase normal and CNV-carrying haplotypes and follow their transmission in the corresponding families. Furthermore, allelic composition analysis identified the co-occurrence of alternative allelic copies within 66.7% of haplotypes carrying copy number gains. We also observed less frequent transmission of CNV-carrying haplotypes from parents to children compared to normal haplotypes and identified an emergence of several de novo deletions and duplications in the offspring. PMID:25853576

Haplotype phasing and inheritance of copy number variants in nuclear families.

PubMed

Palta, Priit; Kaplinski, Lauris; Nagirnaja, Liina; Veidenberg, Andres; Möls, Märt; Nelis, Mari; Esko, Tõnu; Metspalu, Andres; Laan, Maris; Remm, Maido

2015-01-01

DNA copy number variants (CNVs) that alter the copy number of a particular DNA segment in the genome play an important role in human phenotypic variability and disease susceptibility. A number of CNVs overlapping with genes have been shown to confer risk to a variety of human diseases thus highlighting the relevance of addressing the variability of CNVs at a higher resolution. So far, it has not been possible to deterministically infer the allelic composition of different haplotypes present within the CNV regions. We have developed a novel computational method, called PiCNV, which enables to resolve the haplotype sequence composition within CNV regions in nuclear families based on SNP genotyping microarray data. The algorithm allows to i) phase normal and CNV-carrying haplotypes in the copy number variable regions, ii) resolve the allelic copies of rearranged DNA sequence within the haplotypes and iii) infer the heritability of identified haplotypes in trios or larger nuclear families. To our knowledge this is the first program available that can deterministically phase null, mono-, di-, tri- and tetraploid genotypes in CNV loci. We applied our method to study the composition and inheritance of haplotypes in CNV regions of 30 HapMap Yoruban trios and 34 Estonian families. For 93.6% of the CNV loci, PiCNV enabled to unambiguously phase normal and CNV-carrying haplotypes and follow their transmission in the corresponding families. Furthermore, allelic composition analysis identified the co-occurrence of alternative allelic copies within 66.7% of haplotypes carrying copy number gains. We also observed less frequent transmission of CNV-carrying haplotypes from parents to children compared to normal haplotypes and identified an emergence of several de novo deletions and duplications in the offspring.

Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases

PubMed Central

Butchbach, Matthew E. R.

2016-01-01

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset, autosomal recessive neurodegenerative disease characterized by the loss of spinal α-motor neurons. This loss of α-motor neurons is associated with muscle weakness and atrophy. SMA can be classified into five clinical grades based on age of onset and severity of the disease. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13. In humans a large tandem chromosomal duplication has lead to a second copy of the SMN gene locus known as SMN2. SMN2 is distinguishable from SMN1 by a single nucleotide difference that disrupts an exonic splice enhancer in exon 7. As a result, most of SMN2 mRNAs lack exon 7 (SMNΔ7) and produce a protein that is both unstable and less than fully functional. Although only 10–20% of the SMN2 gene product is fully functional, increased genomic copies of SMN2 inversely correlates with disease severity among individuals with SMA. Because SMN2 copy number influences disease severity in SMA, there is prognostic value in accurate measurement of SMN2 copy number from patients being evaluated for SMA. This prognostic value is especially important given that SMN2 copy number is now being used as an inclusion criterion for SMA clinical trials. In addition to SMA, copy number variations (CNVs) in the SMN genes can affect the clinical severity of other neurological disorders including amyotrophic lateral sclerosis (ALS) and progressive muscular atrophy (PMA). This review will discuss how SMN1 and SMN2 CNVs are detected and why accurate measurement of SMN1 and SMN2 copy numbers is relevant for SMA and other neurodegenerative diseases. PMID:27014701

Multihormonal induction of hepatic α2u-globulin mRNA as measured by hybridization to complementary DNA

PubMed Central

Kurtz, David T.; Feigelson, Philip

1977-01-01

A procedure is presented for the preparation of a 3H-labeled complementary DNA (cDNA) specific for the mRNA coding for α2u-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to α2u-globulin, which binds to the nascent α2u-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% α2u-globulin mRNA. A labeled cDNA is made to this α2u-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-α2u-globulin sequences, this cDNA preparation is hybridized to an RNA concentration × incubation time (R0t) of 1000 mol of ribonucleotide per liter × sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no α2u-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for α2u-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of α2u-globulin, the level of functional α2u-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of α2u-globulin mRNA sequences as measured by hybridization to the α2u-globulin cDNA. Thus, the hormonal control of hepatic α2u-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of α2u-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis. PMID:73184

Copy Counts

ERIC Educational Resources Information Center

Beaumont, Lee R.

1970-01-01

The level of difficulty of straight copy, which is used to measure typewriting speed, is influenced by syllable intensity (the average number of syllables per word), stroke intensity (average number of strokes per word), and high-frequency words. (CH)

EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia

PubMed Central

Gaines, Todd A.; Barker, Abigail L.; Patterson, Eric L.; Westra, Philip; Westra, Eric P.; Wilson, Robert G.; Jha, Prashant; Kumar, Vipan

2016-01-01

Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number. PMID:27992501

CGHnormaliter: an iterative strategy to enhance normalization of array CGH data with imbalanced aberrations

PubMed Central

van Houte, Bart PP; Binsl, Thomas W; Hettling, Hannes; Pirovano, Walter; Heringa, Jaap

2009-01-01

Background Array comparative genomic hybridization (aCGH) is a popular technique for detection of genomic copy number imbalances. These play a critical role in the onset of various types of cancer. In the analysis of aCGH data, normalization is deemed a critical pre-processing step. In general, aCGH normalization approaches are similar to those used for gene expression data, albeit both data-types differ inherently. A particular problem with aCGH data is that imbalanced copy numbers lead to improper normalization using conventional methods. Results In this study we present a novel method, called CGHnormaliter, which addresses this issue by means of an iterative normalization procedure. First, provisory balanced copy numbers are identified and subsequently used for normalization. These two steps are then iterated to refine the normalization. We tested our method on three well-studied tumor-related aCGH datasets with experimentally confirmed copy numbers. Results were compared to a conventional normalization approach and two more recent state-of-the-art aCGH normalization strategies. Our findings show that, compared to these three methods, CGHnormaliter yields a higher specificity and precision in terms of identifying the 'true' copy numbers. Conclusion We demonstrate that the normalization of aCGH data can be significantly enhanced using an iterative procedure that effectively eliminates the effect of imbalanced copy numbers. This also leads to a more reliable assessment of aberrations. An R-package containing the implementation of CGHnormaliter is available at . PMID:19709427

High Resolution Analysis of Copy Number Mutation in Breast Cancer

DTIC Science & Technology

2005-05-01

tissues and Epstein - Barr sentations and arrays of Hind III probes additional CNPs, as would an increase in the virus -immortalized lymphoblastoid cell...software and laboratory procedures for the design of inter-phase FISH primers. We have also made progress in developing database and data processing...Cancer progression often involves alterations in DNA copy number. Newly developed microarray technologies enable simultane- ous measurement of copy

Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

PubMed

Collier, Ray; Dasgupta, Kasturi; Xing, Yan-Ping; Hernandez, Bryan Tarape; Shao, Min; Rohozinski, Dominica; Kovak, Emma; Lin, Jeanie; de Oliveira, Maria Luiza P; Stover, Ed; McCue, Kent F; Harmon, Frank G; Blechl, Ann; Thomson, James G; Thilmony, Roger

2017-06-01

Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one- and two-copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

[Relationships between the enrichment of ETBF, Fn, Hp in intestinal and colorectal cancer].

PubMed

Zhang, J; Lu, X L; Zhao, G; Shi, H T; Geng, Y; Zhong, W T; Dong, L

2018-02-23

Objective: To explore relationships between the enrichment of ETBF, Fn, Hp in feces, tissues and colorectal cancer. Methods: Feces, lesion tissue and adjacent tissue from 24 patients with colorectal cancer and 31 patients with adenomas were collected, and we collected Feces and tissue of 20 healthy control persons. Then the copy numbers of enterotoxigenic B. fragilis (ETBF), Fusobacterium nucleatum (Fn) and Helicobacter pylori (Hp) were determined by quantitative real-time PCR. Immunohistochemical method was used to examine the expression intensity of EGFR and p53, and the relationships between different expression intensity of EGFR, p53 and the numbers of three bacterias. Results: In the feces, copy numbers of ETBF and Fn were as follous: colorectal cancer group>adenomas group>healthy control group ( P <0.05). Copy numbers of Hp were as follous: colorectal cancer group>healthy control group ( P <0.01); adenomas group>healthy control group ( P <0.01). In the tissue, copy numbers of ETBF, Fn were as follows: colorectal cancer group>adenomas group>healthy control group ( P <0.05). Copy numbers of Hp were as follows: colorectal cancer group>healthy control group ( P <0.01); adenomas group>healthy control group ( P <0.01). Copy numbers of those three bacteria in the lesion tissue and the adjacent tissue had no significant difference. This happened both in colorectal cancer group and adenomas group. The different expression intensity of EGFR, p53 and the number of three bacteria showed no obviously statistical correlation( P >0.05). Conclusion: Adenomatous polyp and colorectal cancer patients show high enrichment of ETBF, Fn and Hp in both feces and tissues. ETBF, Fn and Hp probably contribute to the development of adenomatous polyp and colorectal cancer. Trial registration Chinese Clinical Trial Registry, ChiCTR-BOC-17012509.

Genetic factors affecting EBV copy number in lymphoblastoid cell lines derived from the 1000 Genome Project samples.

PubMed

Mandage, Rajendra; Telford, Marco; Rodríguez, Juan Antonio; Farré, Xavier; Layouni, Hafid; Marigorta, Urko M; Cundiff, Caitlin; Heredia-Genestar, Jose Maria; Navarro, Arcadi; Santpere, Gabriel

2017-01-01

Epstein-Barr virus (EBV), human herpes virus 4, has been classically associated with infectious mononucleosis, multiple sclerosis and several types of cancers. Many of these diseases show marked geographical differences in prevalence, which points to underlying genetic and/or environmental factors. Those factors may include a different susceptibility to EBV infection and viral copy number among human populations. Since EBV is commonly used to transform B-cells into lymphoblastoid cell lines (LCLs) we hypothesize that differences in EBV copy number among individual LCLs may reflect differential susceptibility to EBV infection. To test this hypothesis, we retrieved whole-genome sequenced EBV-mapping reads from 1,753 LCL samples derived from 19 populations worldwide that were sequenced within the context of the 1000 Genomes Project. An in silico methodology was developed to estimate the number of EBV copy number in LCLs and validated these estimations by real-time PCR. After experimentally confirming that EBV relative copy number remains stable over cell passages, we performed a genome wide association analysis (GWAS) to try detecting genetic variants of the host that may be associated with EBV copy number. Our GWAS has yielded several genomic regions suggestively associated with the number of EBV genomes per cell in LCLs, unraveling promising candidate genes such as CAND1, a known inhibitor of EBV replication. While this GWAS does not unequivocally establish the degree to which genetic makeup of individuals determine viral levels within their derived LCLs, for which a larger sample size will be needed, it potentially highlighted human genes affecting EBV-related processes, which constitute interesting candidates to follow up in the context of EBV related pathologies.

DR-Integrator: a new analytic tool for integrating DNA copy number and gene expression data.

PubMed

Salari, Keyan; Tibshirani, Robert; Pollack, Jonathan R

2010-02-01

DNA copy number alterations (CNA) frequently underlie gene expression changes by increasing or decreasing gene dosage. However, only a subset of genes with altered dosage exhibit concordant changes in gene expression. This subset is likely to be enriched for oncogenes and tumor suppressor genes, and can be identified by integrating these two layers of genome-scale data. We introduce DNA/RNA-Integrator (DR-Integrator), a statistical software tool to perform integrative analyses on paired DNA copy number and gene expression data. DR-Integrator identifies genes with significant correlations between DNA copy number and gene expression, and implements a supervised analysis that captures genes with significant alterations in both DNA copy number and gene expression between two sample classes. DR-Integrator is freely available for non-commercial use from the Pollack Lab at http://pollacklab.stanford.edu/ and can be downloaded as a plug-in application to Microsoft Excel and as a package for the R statistical computing environment. The R package is available under the name 'DRI' at http://cran.r-project.org/. An example analysis using DR-Integrator is included as supplemental material. Supplementary data are available at Bioinformatics online.

Novel applications of array comparative genomic hybridization in molecular diagnostics.

PubMed

Cheung, Sau W; Bi, Weimin

2018-05-31

In 2004, the implementation of array comparative genomic hybridization (array comparative genome hybridization [CGH]) into clinical practice marked a new milestone for genetic diagnosis. Array CGH and single-nucleotide polymorphism (SNP) arrays enable genome-wide detection of copy number changes in a high resolution, and therefore microarray has been recognized as the first-tier test for patients with intellectual disability or multiple congenital anomalies, and has also been applied prenatally for detection of clinically relevant copy number variations in the fetus. Area covered: In this review, the authors summarize the evolution of array CGH technology from their diagnostic laboratory, highlighting exonic SNP arrays developed in the past decade which detect small intragenic copy number changes as well as large DNA segments for the region of heterozygosity. The applications of array CGH to human diseases with different modes of inheritance with the emphasis on autosomal recessive disorders are discussed. Expert commentary: An exonic array is a powerful and most efficient clinical tool in detecting genome wide small copy number variants in both dominant and recessive disorders. However, whole-genome sequencing may become the single integrated platform for detection of copy number changes, single-nucleotide changes as well as balanced chromosomal rearrangements in the near future.

Coupling between the basic replicon and the Kis-Kid maintenance system of plasmid R1: modulation by Kis antitoxin levels and involvement in control of plasmid replication.

PubMed

López-Villarejo, Juan; Lobato-Márquez, Damián; Díaz-Orejas, Ramón

2015-02-05

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

PubMed Central

López-Villarejo, Juan; Lobato-Márquez, Damián; Díaz-Orejas, Ramón

2015-01-01

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication. PMID:25664511

Estimation of total bacteria by real-time PCR in patients with periodontal disease.

PubMed

Brajović, Gavrilo; Popović, Branka; Puletić, Miljan; Kostić, Marija; Milasin, Jelena

2016-01-01

Periodontal diseases are associated with the presence of elevated levels of bacteria within the gingival crevice. The aim of this study was to evaluate a total amount of bacteria in subgingival plaque samples in patients with a periodontal disease. A quantitative evaluation of total bacteria amount using quantitative real-time polymerase chain reaction (qRT-PCR) was performed on 20 samples of patients with ulceronecrotic periodontitis and on 10 samples of healthy subjects. The estimation of total bacterial amount was based on gene copy number for 16S rRNA that was determined by comparing to Ct values/gene copy number of the standard curve. A statistically significant difference between average gene copy number of total bacteria in periodontal patients (2.55 x 10⁷) and healthy control (2.37 x 10⁶) was found (p = 0.01). Also, a trend of higher numbers of the gene copy in deeper periodontal lesions (> 7 mm) was confirmed by a positive value of coefficient of correlation (r = 0.073). The quantitative estimation of total bacteria based on gene copy number could be an important additional tool in diagnosing periodontitis.

Phylogenetic Copy-Number Factorization of Multiple Tumor Samples.

PubMed

Zaccaria, Simone; El-Kebir, Mohammed; Klau, Gunnar W; Raphael, Benjamin J

2018-04-16

Cancer is an evolutionary process driven by somatic mutations. This process can be represented as a phylogenetic tree. Constructing such a phylogenetic tree from genome sequencing data is a challenging task due to the many types of mutations in cancer and the fact that nearly all cancer sequencing is of a bulk tumor, measuring a superposition of somatic mutations present in different cells. We study the problem of reconstructing tumor phylogenies from copy-number aberrations (CNAs) measured in bulk-sequencing data. We introduce the Copy-Number Tree Mixture Deconvolution (CNTMD) problem, which aims to find the phylogenetic tree with the fewest number of CNAs that explain the copy-number data from multiple samples of a tumor. We design an algorithm for solving the CNTMD problem and apply the algorithm to both simulated and real data. On simulated data, we find that our algorithm outperforms existing approaches that either perform deconvolution/factorization of mixed tumor samples or build phylogenetic trees assuming homogeneous tumor samples. On real data, we analyze multiple samples from a prostate cancer patient, identifying clones within these samples and a phylogenetic tree that relates these clones and their differing proportions across samples. This phylogenetic tree provides a higher resolution view of copy-number evolution of this cancer than published analyses.

Copy number polymorphism of the salivary amylase gene: implications in human nutrition research.

PubMed

Santos, J L; Saus, E; Smalley, S V; Cataldo, L R; Alberti, G; Parada, J; Gratacòs, M; Estivill, X

2012-01-01

The salivary α-amylase is a calcium-binding enzyme that initiates starch digestion in the oral cavity. The α-amylase genes are located in a cluster on the chromosome that includes salivary amylase genes (AMY1), two pancreatic α-amylase genes (AMY2A and AMY2B) and a related pseudogene. The AMY1 genes show extensive copy number variation which is directly proportional to the salivary α-amylase content in saliva. The α-amylase amount in saliva is also influenced by other factors, such as hydration status, psychosocial stress level, and short-term dietary habits. It has been shown that the average copy number of AMY1 gene is higher in populations that evolved under high-starch diets versus low-starch diets, reflecting an intense positive selection imposed by diet on amylase copy number during evolution. In this context, a number of different aspects can be considered in evaluating the possible impact of copy number variation of the AMY1 gene on nutrition research, such as issues related to human diet gene evolution, action on starch digestion, effect on glycemic response after starch consumption, modulation of the action of α-amylases inhibitors, effect on taste perception and satiety, influence on psychosocial stress and relation to oral health. Copyright © 2012 S. Karger AG, Basel.

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication.

PubMed

Sakudoh, Takashi; Nakashima, Takeharu; Kuroki, Yoko; Fujiyama, Asao; Kohara, Yuji; Honda, Naoko; Fujimoto, Hirofumi; Shimada, Toru; Nakagaki, Masao; Banno, Yutaka; Tsuchida, Kozo

2011-03-01

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time. © 2011 by the Genetics Society of America

Diversity in Copy Number and Structure of a Silkworm Morphogenetic Gene as a Result of Domestication

PubMed Central

Sakudoh, Takashi; Nakashima, Takeharu; Kuroki, Yoko; Fujiyama, Asao; Kohara, Yuji; Honda, Naoko; Fujimoto, Hirofumi; Shimada, Toru; Nakagaki, Masao; Banno, Yutaka; Tsuchida, Kozo

2011-01-01

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time. PMID:21242537

76 FR 59679 - Notice of Intent to Hold Public Meetings and Hear Public Comment on the Proposed New Jersey-New...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-27

... Street, NE, Room 2A, Washington, DC 20426, (202) 502-8371. CD-ROM copies of the draft EIS were mailed to.... A limited number of hard copies and CD-ROMs are available from the Public Reference Room identified above. Please note that copies of the CD-ROM were mailed with a postcard that included a docket number...

Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data.

PubMed

Favero, F; Joshi, T; Marquard, A M; Birkbak, N J; Krzystanek, M; Li, Q; Szallasi, Z; Eklund, A C

2015-01-01

Exome or whole-genome deep sequencing of tumor DNA along with paired normal DNA can potentially provide a detailed picture of the somatic mutations that characterize the tumor. However, analysis of such sequence data can be complicated by the presence of normal cells in the tumor specimen, by intratumor heterogeneity, and by the sheer size of the raw data. In particular, determination of copy number variations from exome sequencing data alone has proven difficult; thus, single nucleotide polymorphism (SNP) arrays have often been used for this task. Recently, algorithms to estimate absolute, but not allele-specific, copy number profiles from tumor sequencing data have been described. We developed Sequenza, a software package that uses paired tumor-normal DNA sequencing data to estimate tumor cellularity and ploidy, and to calculate allele-specific copy number profiles and mutation profiles. We applied Sequenza, as well as two previously published algorithms, to exome sequence data from 30 tumors from The Cancer Genome Atlas. We assessed the performance of these algorithms by comparing their results with those generated using matched SNP arrays and processed by the allele-specific copy number analysis of tumors (ASCAT) algorithm. Comparison between Sequenza/exome and SNP/ASCAT revealed strong correlation in cellularity (Pearson's r = 0.90) and ploidy estimates (r = 0.42, or r = 0.94 after manual inspecting alternative solutions). This performance was noticeably superior to previously published algorithms. In addition, in artificial data simulating normal-tumor admixtures, Sequenza detected the correct ploidy in samples with tumor content as low as 30%. The agreement between Sequenza and SNP array-based copy number profiles suggests that exome sequencing alone is sufficient not only for identifying small scale mutations but also for estimating cellularity and inferring DNA copy number aberrations. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15.

PubMed

Mardanov, Andrey V; Strakhova, Taisia S; Smagin, Vladimir A; Ravin, Nikolai V

2007-06-15

A new Escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. The new pN15E vectors are low copy number plasmids based on the replicon of temperate phage N15, comprising the repA replicase gene and cB repressor gene, controlling the plasmid copy number. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the PBAD promoter. The host strain also carried phage N15 partition operon, sop, allowing stable inheritance of pN15E vectors in the absence of selection pressure. In the first vector, pN15E4, the same PBAD promoter controls expression of a cloned gene. The second vector, pN15E6, carries the phage T5 promoter with a double lac operator repression module thus allowing independent regulation of promoter activity and copy number. Using the lacZ gene to monitor expression in these vectors, we show that the ratio of induction/repression can be about 7600-fold for pN15E4 and more than 15,000-fold for pN15E6. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products that was demonstrated by the stable maintenance of a gene encoding a restriction endonuclease in pN15E4. The tight control of transcription and the potential to regulate gene activities quantitatively over wide ranges will open up new approaches in the study of gene function in vivo and controlled expression of heterologous genes.

Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes.

PubMed

Chan, C C W; Liu, V W S; Lau, E Y L; Yeung, W S B; Ng, E H Y; Ho, P C

2005-12-01

Previous studies analysing the incidences of mitochondrial DNA (mtDNA) deletions and mtDNA content in unfertilized oocytes in relation to donors' age have been controversial. The objective of the study was to compare these two parameters in unfertilized oocytes and relate them to the donors' age. Fifty-two women donated 155 unfertilized metaphase II (MII) oocytes. The incidence of 4977 bp deletion was 34.6%, and the mtDNA copy number was 598 350 +/- 265 862. Women >or=35 years of age had a significantly higher incidence of 4977 bp deletion, lower mtDNA copy number, higher FSH level and poorer ovarian response when compared with younger women. The mtDNA copy number was negatively correlated with the donor's age. The higher incidence of mtDNA deletion and lower mtDNA copy number in older women suggested that these two parameters may reflect ovarian ageing.

Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

PubMed

Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

2013-05-30

Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

Measurement of locus copy number by hybridisation with amplifiable probes

PubMed Central

Armour, John A. L.; Sismani, Carolina; Patsalis, Philippos C.; Cross, Gareth

2000-01-01

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicroscopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader–Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications. PMID:10606661

Measurement of locus copy number by hybridisation with amplifiable probes.

PubMed

Armour, J A; Sismani, C; Patsalis, P C; Cross, G

2000-01-15

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicro-scopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader-Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications.

Bioluminescent symbionts of the Caribbean flashlight fish (Kryptophanaron alfredi) have a single rRNA operon.

PubMed

Wolfe, C J; Haygood, M G

1993-08-01

Ribosomal RNA (rRNA) operon copy number and gene order were determined for the luminous bacterial symbiont of Kryptophanaron alfredi, an anomalopid (flashlight) fish, and estimated for the luminous symbionts of 3 other fish families and of 3 luminous seawater isolates. Compared with the seawater isolates and other fish symbionts, the copy number of rRNA genes in the K. alfredi symbiont was radically reduced, although gene order appeared conserved among all the strains. The K. alfredi symbiont possesses only a single rRNA operon, whereas the other strains examined have minimum copy numbers ranging from 8 to 11. No difference in copy number was observed between light organ and seawater isolates of the same species, or between isolates of the same species from the light organs of 2 different host families. Thus, the anomalopid symbiosis appears unique among characterized light organ symbioses.

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives.

PubMed

Zhao, Min; Wang, Qingguo; Wang, Quan; Jia, Peilin; Zhao, Zhongming

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development.

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives

PubMed Central

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development. PMID:24564169

Copy number variations in patients with electrical status epilepticus in sleep.

PubMed

Kevelam, Sietske H G; Jansen, Floor E; Binsbergen, Ellen van; Braun, Kees P J; Verbeek, Nienke E; Lindhout, Dick; Poot, Martin; Brilstra, Eva H

2012-02-01

Electrical status epilepticus in sleep syndrome is the association of the electroencephalographic pattern and deficits in language or global cognitive function and behavioral problems. The etiology is often unknown, but genetic risk factors have been implicated. Array-based comparative genomic hybridization was used to identify copy number variations in 13 children with electrical status epilepticus in sleep syndrome to identify possible underlying risk factors. Seven copy number variations were detected in 4 of the 13 patients, which consisted of 6 novel gains and 1 loss, the recurrent 15q13.3 microdeletion. Two patients carried a probable pathogenic copy number variation containing a gene involved in the cholinergic pathway. Genetic aberrations in patients with electrical status epilepticus in sleep syndrome can provide an entry in the investigation of the etiology of electrical status epilepticus in sleep. However, further studies are needed to confirm our findings.

Engineered promoters enable constant gene expression at any copy number in bacteria.

PubMed

Segall-Shapiro, Thomas H; Sontag, Eduardo D; Voigt, Christopher A

2018-04-01

The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs). These promoters had near-identical expression in different genome locations and plasmids, even when their copy number was perturbed by genomic mutations or changes in growth medium composition. We applied the stabilized promoters to show that a three-gene metabolic pathway to produce deoxychromoviridans could retain function without re-tuning when the stabilized-promoter-driven genes were moved from a plasmid into the genome.

Genomic DNA Copy-Number Alterations of the let-7 Family in Human Cancers

PubMed Central

Greshock, Joel; Shen, Liang; Yang, Xiaojun; Shao, Zhongjun; Liang, Shun; Tanyi, Janos L.; Sood, Anil K.; Zhang, Lin

2012-01-01

In human cancer, expression of the let-7 family is significantly reduced, and this is associated with shorter survival times in patients. However, the mechanisms leading to let-7 downregulation in cancer are still largely unclear. Since an alteration in copy-number is one of the causes of gene deregulation in cancer, we examined copy number alterations of the let-7 family in 2,969 cancer specimens from a high-resolution SNP array dataset. We found that there was a reduction in the copy number of let-7 genes in a cancer-type specific manner. Importantly, focal deletion of four let-7 family members was found in three cancer types: medulloblastoma (let-7a-2 and let-7e), breast cancer (let-7a-2), and ovarian cancer (let-7a-3/let-7b). For example, the genomic locus harboring let-7a-3/let-7b was deleted in 44% of the specimens from ovarian cancer patients. We also found a positive correlation between the copy number of let-7b and mature let-7b expression in ovarian cancer. Finally, we showed that restoration of let-7b expression dramatically reduced ovarian tumor growth in vitro and in vivo. Our results indicate that copy number deletion is an important mechanism leading to the downregulation of expression of specific let-7 family members in medulloblastoma, breast, and ovarian cancers. Restoration of let-7 expression in tumor cells could provide a novel therapeutic strategy for the treatment of cancer. PMID:22970210

Nucleotide sequences of Dictyostelium discoideum developmentally regulated cDNAs rich in (AAC) imply proteins that contain clusters of asparagine, glutamine, or threonine.

PubMed

Shaw, D R; Richter, H; Giorda, R; Ohmachi, T; Ennis, H L

1989-09-01

A Dictyostelium discoideum repetitive element composed of long repeats of the codon (AAC) is found in developmentally regulated transcripts. The concentration of (AAC) sequences is low in mRNA from dormant spores and growing cells and increases markedly during spore germination and multicellular development. The sequence hybridizes to many different sized Dictyostelium DNA restriction fragments indicating that it is scattered throughout the genome. Four cDNA clones isolated contain (AAC) sequences in the deduced coding region. Interestingly, the (AAC)-rich sequences are present in all three reading frames in the deduced proteins, i.e., AAC (asparagine), ACA (threonine) and CAA (glutamine). Three of the clones contain only one of these in-frame so that the individual proteins carry either asparagine, threonine, or glutamine clusters, not mixtures. However, one clone is both glutamine- and asparagine-rich. The (AAC) portion of the transcripts are reiterated 300 times in the haploid genome while the other portions of the cDNAs represent single copy genes, whose sequences show no similarity other than the (AAC) repeats. The repeated sequence is similar to the opa or M sequence found in Drosophila melanogaster notch and homeo box genes and in fly developmentally regulated transcripts. The transcripts are present on polysomes suggesting that they are translated. Although the function of these repeats is unknown, long amino acid repeats are a characteristic feature of extracellular proteins of lower eukaryotes.

Influenza Virus Aerosols in Human Exhaled Breath: Particle Size, Culturability, and Effect of Surgical Masks

PubMed Central

Milton, Donald K.; Cowling, Benjamin J.; Grantham, Michael L.

2013-01-01

The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza. We collected samples of exhaled particles (one with and one without a facemask) in two size fractions (“coarse”>5 µm, “fine”≤5 µm) from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus. Fine particles contained 8.8 (95% CI 4.1 to 19) fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2) and in the coarse fraction by 25 fold (95% CI 3.5 to 180). Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3) reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (r = 0.17, coarse; r = 0.29, fine fraction). Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples. Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans. PMID:23505369

Polymorphisms in α-Defensin–Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reflux

PubMed Central

Schwaderer, Andrew L.; Wang, Huanyu; Kim, SungHwan; Kline, Jennifer M.; Liang, Dong; Brophy, Pat D.; McHugh, Kirk M.; Tseng, George C.; Saxena, Vijay; Barr-Beare, Evan; Pierce, Keith R.; Shaikh, Nader; Manak, J. Robert; Cohen, Daniel M.; Becknell, Brian; Spencer, John D.; Baker, Peter B.; Yu, Chack-Yung

2016-01-01

The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the α-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real–time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P<0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk. PMID:26940096

The roles of AMY1 copies and protein expression in human salivary α-amylase activity.

PubMed

Yang, Ze-Min; Lin, Jing; Chen, Long-Hui; Zhang, Min; Chen, Wei-Wen; Yang, Xiao-Rong

2015-01-01

Salivary α-amylase (sAA) activity has been extensively investigated in nutrition and psychology. But few studies were performed to assess the role played by sAA gene (AMY1) copies and protein expression in basal and stimulus-induced sAA activity. The sAA activity, amount and AMY1 copy number were determined from 184 saliva samples pre- and post-citric acid stimulation. Our findings showed that citric acid could induce significant increase in sAA activity, total sAA amount, and glycosylated sAA amount, among which the glycosylated sAA amount had the largest response. The correlation analysis showed that AMY1 copy number, total sAA amount and AMY1 copy number×total sAA amount had significantly positive and successively increasing correlations with sAA activity in unstimulated and stimulated saliva, respectively, and furthermore, we observed higher correlations in unstimulated saliva when compared with the corresponding correlations in stimulated saliva. We also observed significant correlations between glycosylated sAA amount and sAA activity in unstimulated and stimulated saliva, respectively. Interestingly, the correlations were higher in stimulated saliva than in unstimulated saliva, and the correlations between glycosylated sAA amount and sAA activity were higher than that of between total sAA amount and sAA activity in stimulated saliva. Moreover, total sAA amount ratio and glycosylated sAA amount ratio showed significantly positive correlation with sAA activity ratio. AMY1 copy number had no correlation with sAA activity ratio. These findings suggested that AMY1 copy number and sAA amount played crucial roles in sAA activity; however, the roles were attenuated after stimulation due to fortified release of glycosylated sAA. Copyright © 2014 Elsevier Inc. All rights reserved.

Gene amplification of the Hps locus in Glycine max

PubMed Central

Gijzen, Mark; Kuflu, Kuflom; Moy, Pat

2006-01-01

Background Hydrophobic protein from soybean (HPS) is an 8 kD cysteine-rich polypeptide that causes asthma in persons allergic to soybean dust. HPS is synthesized in the pod endocarp and deposited on the seed surface during development. Past evidence suggests that the protein may mediate the adherence or dehiscence of endocarp tissues during maturation and affect the lustre, or glossiness of the seed surface. Results A comparison of soybean germplasm by genomic DNA blot hybridization shows that the copy number and structure of the Hps locus is polymorphic among soybean cultivars and related species. Changes in Hps gene copy number were also detected by comparative genomic DNA hybridization using cDNA microarrays. The Hps copy number polymorphisms co-segregated with seed lustre phenotype and HPS surface protein in a cross between dull- and shiny-seeded soybeans. In soybean cultivar Harosoy 63, a minimum of 27 ± 5 copies of the Hps gene were estimated to be present in each haploid genome. The isolation and analysis of genomic clones indicates that the core Hps locus is comprised of a tandem array of reiterated units, with each 8.6 kb unit containing a single HPS open reading frame. Conclusion This study shows that polymorphisms at the Hps locus arise from changes in the gene copy number via gene amplification. We present a model whereby Hps copy number modulates protein expression levels and seed lustre, and we suggest that gene amplification may result from selection pressures imposed on crop plants. PMID:16536872

SSR-enriched genetic linkage maps of bermudagrass (Cynodon dactylon × transvaalensis), and their comparison with allied plant genomes.

PubMed

Khanal, Sameer; Kim, Changsoo; Auckland, Susan A; Rainville, Lisa K; Adhikari, Jeevan; Schwartz, Brian M; Paterson, Andrew H

2017-04-01

We report SSR-enriched genetic maps of bermudagrass that: (1) reveal partial residual polysomic inheritance in the tetraploid species, and (2) provide insights into the evolution of chloridoid genomes. This study describes genetic linkage maps of two bermudagrass species, Cynodon dactylon (T89) and Cynodon transvaalensis (T574), that integrate heterologous microsatellite markers from sugarcane into frameworks built with single-dose restriction fragments (SDRFs). A maximum likelihood approach was used to construct two separate parental maps from a population of 110 F 1 progeny of a cross between the two parents. The T89 map is based on 291 loci on 34 cosegregating groups (CGs), with an average marker spacing of 12.5 cM. The T574 map is based on 125 loci on 14 CGs, with an average marker spacing of 10.7 cM. Six T89 and one T574 CG(s) deviated from disomic inheritance. Furthermore, marker segregation data and linkage phase analysis revealed partial residual polysomic inheritance in T89, suggesting that common bermudagrass is undergoing diploidization following whole genome duplication (WGD). Twenty-six T89 CGs were coalesced into 9 homo(eo)logous linkage groups (LGs), while 12 T574 CGs were assembled into 9 LGs, both putatively representing the basic chromosome complement (x = 9) of the species. Eight T89 and two T574 CGs remain unassigned. The marker composition of bermudagrass ancestral chromosomes was inferred by aligning T89 and T574 homologs, and used in comparisons to sorghum and rice genome sequences based on 108 and 91 significant blast hits, respectively. Two nested chromosome fusions (NCFs) shared by two other chloridoids (i.e., zoysiagrass and finger millet) and at least three independent translocation events were evident during chromosome number reduction from 14 in the polyploid common ancestor of Poaceae to 9 in Cynodon.

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events.

PubMed

Demeke, Tigst; Eng, Monika

2018-05-01

Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences ( HMG-I/Y , FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.

Does Visual Attention Span Relate to Eye Movements during Reading and Copying?

ERIC Educational Resources Information Center

Bosse, Marie-Line; Kandel, Sonia; Prado, Chloé; Valdois, Sylviane

2014-01-01

This research investigated whether text reading and copying involve visual attention-processing skills. Children in grades 3 and 5 read and copied the same text. We measured eye movements while reading and the number of gaze lifts (GL) during copying. The children were also administered letter report tasks that constitute an estimation of the…

Requirements for rapid plasmid ColE1 copy number adjustments: a mathematical model of inhibition modes and RNA turnover rates.

PubMed

Paulsson, J; Nordström, K; Ehrenberg, M

1998-01-01

The random distribution of ColE1 plasmids between the daughter cells at cell division introduces large copy number variations. Statistic variation associated with limited copy number in single cells also causes fluctuations to emerge spontaneously during the cell cycle. Efficient replication control out of steady state is therefore important to tame such stochastic effects of small numbers. In the present model, the dynamic features of copy number control are divided into two parts: first, how sharply the replication frequency per plasmid responds to changes in the concentration of the plasmid-coded inhibitor, RNA I, and second, how tightly RNA I and plasmid concentrations are coupled. Single (hyperbolic)- and multiple (exponential)-step inhibition mechanisms are compared out of steady state and it is shown how the response in replication frequency depends on the mode of inhibition. For both mechanisms, sensitivity of inhibition is "bought" at the expense of a rapid turnover of a replication preprimer, RNA II. Conventional, single-step, inhibition kinetics gives a sloppy replication control even at high RNA II turnover rates, whereas multiple-step inhibition has the potential of working with unlimited precision. When plasmid concentration changes rapidly, RNA I must be degraded rapidly to be "up to date" with the change. Adjustment to steady state is drastically impaired when the turnover rate constants of RNA I decrease below certain thresholds, but is basically unaffected for a corresponding increase. Several features of copy number control that are shown to be crucial for the understanding of ColE1-type plasmids still remain to be experimentally characterized. It is shown how steady-state properties reflect dynamics at the heart of regulation and therefore can be used to discriminate between fundamentally different copy number control mechanisms. The experimental tests of the predictions made require carefully planned assays, and some suggestions for suitable experiments arise naturally from the present work. It is also discussed how the presence of the Rom protein may affect dynamic qualities of copy number control. Copyright 1998 Academic Press.

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting | Office of Cancer Genomics

Cancer.gov

The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest.

Copy number increase of ACTN4 is a prognostic indicator in salivary gland carcinoma

PubMed Central

Watabe, Yukio; Mori, Taisuke; Yoshimoto, Seiichi; Nomura, Takeshi; Shibahara, Takahiko; Yamada, Tesshi; Honda, Kazufumi

2014-01-01

Copy number increase (CNI) of ACTN4 has been associated with poor prognosis and metastatic phenotypes in various human carcinomas. To identify a novel prognostic factor for salivary gland carcinoma, we investigated the copy number of ACTN4. We evaluated DNA copy number of ACTN4 in 58 patients with salivary gland carcinoma by using fluorescent in situ hybridization (FISH). CNI of ACTN4 was recognized in 14 of 58 patients (24.1%) with salivary gland carcinoma. The cases with CNI of ACTN4 were closely associated with histological grade (P = 0.047) and vascular invasion (P = 0.033). The patients with CNI of ACTN4 had a significantly worse prognosis than the patients with normal copy number of ACTN4 (P = 0.0005 log-rank test). Univariate analysis by the Cox proportional hazards model showed that histological grade, vascular invasion, and CNI of ACTN4 were independent risk factors for cancer death. Vascular invasion (hazard ratio [HR]: 7.46; 95% confidence interval [CI]: 1.98–28.06) and CNI of ACTN4 (HR: 3.23; 95% CI: 1.08–9.68) remained as risk factors for cancer death in multivariate analysis. Thus, CNI of ACTN4 is a novel indicator for an unfavorable outcome in patients with salivary gland carcinoma. PMID:24574362

Integrative Genomics Reveals Mechanisms of Copy Number Alterations Responsible for Transcriptional Deregulation in Colorectal Cancer

PubMed Central

Camps, Jordi; Nguyen, Quang Tri; Padilla-Nash, Hesed M.; Knutsen, Turid; McNeil, Nicole E.; Wangsa, Danny; Hummon, Amanda B.; Grade, Marian; Ried, Thomas; Difilippantonio, Michael J.

2016-01-01

To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations. PMID:19691111

Extensive Copy-Number Variation of Young Genes across Stickleback Populations

PubMed Central

Eizaguirre, Christophe; Samonte, Irene E.; Kalbe, Martin; Lenz, Tobias L.; Stoll, Monika; Bornberg-Bauer, Erich; Milinski, Manfred; Reusch, Thorsten B. H.

2014-01-01

Duplicate genes emerge as copy-number variations (CNVs) at the population level, and remain copy-number polymorphic until they are fixed or lost. The successful establishment of such structural polymorphisms in the genome plays an important role in evolution by promoting genetic diversity, complexity and innovation. To characterize the early evolutionary stages of duplicate genes and their potential adaptive benefits, we combine comparative genomics with population genomics analyses to evaluate the distribution and impact of CNVs across natural populations of an eco-genomic model, the three-spined stickleback. With whole genome sequences of 66 individuals from populations inhabiting three distinct habitats, we find that CNVs generally occur at low frequencies and are often only found in one of the 11 populations surveyed. A subset of CNVs, however, displays copy-number differentiation between populations, showing elevated within-population frequencies consistent with local adaptation. By comparing teleost genomes to identify lineage-specific genes and duplications in sticklebacks, we highlight rampant gene content differences among individuals in which over 30% of young duplicate genes are CNVs. These CNV genes are evolving rapidly at the molecular level and are enriched with functional categories associated with environmental interactions, depicting the dynamic early copy-number polymorphic stage of genes during population differentiation. PMID:25474574

nrDNA:mtDNA copy number ratios as a comparative metric for evolutionary and conservation genetics.

PubMed

Goodall-Copestake, William Paul

2018-05-12

Identifying genetic cues of functional relevance is key to understanding the drivers of evolution and increasingly important for the conservation of biodiversity. This study introduces nuclear ribosomal DNA (nrDNA) to mitochondrial DNA (mtDNA) copy number ratios as a metric with which to screen for this functional genetic variation prior to more extensive omics analyses. To illustrate the metric, quantitative PCR was used to estimate nrDNA (18S) to mtDNA (16S) copy number ratios in muscle tissue from samples of two zooplankton species: Salpa thompsoni caught near Elephant Island (Southern Ocean) and S. fusiformis sampled off Gough Island (South Atlantic). Average 18S:16S ratios in these samples were 9:1 and 3:1, respectively. nrDNA 45S arrays and mitochondrial genomes were then deep sequenced to uncover the sources of intra-individual genetic variation underlying these 18S:16S copy number differences. The deep sequencing profiles obtained were consistent with genetic changes resulting from adaptive processes, including an expansion of nrDNA and damage to mtDNA in S. thompsoni, potentially in response to the polar environment. Beyond this example from zooplankton, nrDNA:mtDNA copy number ratios offer a promising metric to help identify genetic variation of functional relevance in animals more broadly.

SG-ADVISER CNV: copy-number variant annotation and interpretation.

PubMed

Erikson, Galina A; Deshpande, Neha; Kesavan, Balachandar G; Torkamani, Ali

2015-09-01

Copy-number variants have been associated with a variety of diseases, especially cancer, autism, schizophrenia, and developmental delay. The majority of clinically relevant events occur de novo, necessitating the interpretation of novel events. In this light, we present the Scripps Genome ADVISER CNV annotation pipeline and Web server, which aims to fill the gap between copy number variant detection and interpretation by performing in-depth annotations and functional predictions for copy number variants. The Scripps Genome ADVISER CNV suite includes a Web server interface to a high-performance computing environment for calculations of annotations and a table-based user interface that allows for the execution of numerous annotation-based variant filtration strategies and statistics. The annotation results include details regarding location, impact on the coding portion of genes, allele frequency information (including allele frequencies from the Scripps Wellderly cohort), and overlap information with other reference data sets (including ClinVar, DGV, DECIPHER). A summary variant classification is produced (ADVISER score) based on the American College of Medical Genetics and Genomics scoring guidelines. We demonstrate >90% sensitivity/specificity for detection of pathogenic events. Scripps Genome ADVISER CNV is designed to allow users with no prior bioinformatics expertise to manipulate large volumes of copy-number variant data. Scripps Genome ADVISER CNV is available at http://genomics.scripps.edu/ADVISER/.

Amplification of the EGFR gene can be maintained and modulated by variation of EGF concentrations in in vitro models of glioblastoma multiforme

PubMed Central

Mokri, Poroshista; Lamp, Nora; Linnebacher, Michael; Classen, Carl Friedrich; Erbersdobler, Andreas; Schneider, Björn

2017-01-01

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults. It is known that amplification of the epidermal growth factor receptor gene (EGFR) occurs in approximately 40% of GBM, leading to enhanced activation of the EGFR signaling pathway and promoting tumor growth. Although GBM mutations are stably maintained in GBM in vitro models, rapid loss of EGFR gene amplification is a common observation during cell culture. To maintain EGFR amplification in vitro, heterotopic GBM xenografts with elevated EGFR copy number were cultured under varying serum conditions and EGF concentrations. EGFR copy numbers were assessed over several passages by quantitative PCR and chromogenic in situ hybridization. As expected, in control assays with 10% FCS, cells lost EGFR amplification with increasing passage numbers. However, cells cultured under serum free conditions stably maintained elevated copy numbers. Furthermore, EGFR protein expression positively correlated with genomic amplification levels. Although elevated EGFR copy numbers could be maintained over several passages in vitro, levels of EGFR amplification were variable and dependent on the EGF concentration in the medium. In vitro cultures of GBM cells with elevated EGFR copy number and corresponding EGFR protein expression should prove valuable preclinical tools to gain a better understanding of EGFR driven glioblastoma and assist in the development of new improved therapies. PMID:28934307

Active ribosomal genes, translational homeostasis and oxidative stress in the pathogenesis of schizophrenia and autism.

PubMed

Porokhovnik, Lev N; Passekov, Vladimir P; Gorbachevskaya, Nataliya L; Sorokin, Alexander B; Veiko, Nataliya N; Lyapunova, Nataliya A

2015-04-01

Infantile autism and schizophrenia are severe multifactorial disorders with a pronounced genetic predisposition. Their pathogeneses are often associated with oxidative stress in the brain. Previously, we established that a cell's resistance to oxidative stress depended on the copy number of transcriptionally active genes for rRNA (ribosomal genes) in the cell's genome. The feature is measured cytogenetically in cultured lymphocytes derived from patients. It varies from 120 up to 190 copies per diploid genome, with an arithmetic mean of 150±4 (SE) copies in a healthy population (n=239), being considerably lower, according to our previous results, in a sample of patients with rheumatoid arthritis (n=49), another multifactorial disease with a proven significant role of oxidative stress in its pathogenesis: from 115 to 165 copies, with a mean of 140±4 (SE). Conversely, a sample of schizophrenic patients (n=42) previously showed a higher value of copy number of active rRNA genes compared with a healthy population: from 145 to 190 copies, with a mean of 170±4. This fact is of special interest in the context of the well-known, but still unexplained phenomenon of the reduced comorbidity rate of schizophrenia and rheumatoid arthritis. The copy number of active ribosomal genes was estimated in a sample of autistic children (n=51). In contrast with the schizophrenic patients studied previously, we found that the values were significantly lower than those in the healthy population: from 125 to 160 copies, with a mean of 142±5. In this work, we suggest a mathematical model of the oxidative stress dynamics on the basis of Lotka-Volterra's approach to predator-prey interactions. In our model, the 'prey' represents reactive oxygen species, whereas the 'predator' simulates molecules of the antioxidant enzymes. The rate of biosynthesis of the latter is limited by the number of ribosomes available, which, in turn, is determined by the copy number of active rRNA genes. Analysis of the model showed the existence of a unique equilibrium point that makes biological sense. The reactive oxygen species level oscillatory approaches this equilibrium value, which inversely depends on the copy number of active rRNA genes. Our findings confirm the hypothesis of disturbance of the 'translational homeostasis' in the pathogeneses of autism and schizophrenia, and would help explain why oxidative stress markers are discovered in most autism studies, whereas similar reports related to schizophrenia are far less consistent.

Integrated analysis of copy number alteration and RNA expression profiles of cancer using a high-resolution whole-genome oligonucleotide array.

PubMed

Jung, Seung-Hyun; Shin, Seung-Hun; Yim, Seon-Hee; Choi, Hye-Sun; Lee, Sug-Hyung; Chung, Yeun-Jun

2009-07-31

Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.

Minimum Copies of Schrödinger’s Cat State in the Multi-Photon System

PubMed Central

Lu, Yiping; Zhao, Qing

2016-01-01

Multi-photon entanglement has been successfully studied by many theoretical and experimental groups. However, as the number of entangled photons increases, some problems are encountered, such as the exponential increase of time necessary to prepare the same number of copies of entangled states in experiment. In this paper, a new scheme is proposed based on the Lagrange multiplier and Feedback, which cuts down the required number of copies of Schrödinger’s Cat state in multi-photon experiment, which is realized with some noise in actual measurements, and still keeps the standard deviation in the error of fidelity unchanged. It reduces about five percent of the measuring time of eight-photon Schrödinger’s Cat state compared with the scheme used in the usual planning of actual measurements, and moreover it guarantees the same low error in fidelity. In addition, we also applied the same approach to the simulation of ten-photon entanglement, and we found that it reduces in priciple about twenty two percent of the required copies of Schrödinger’s Cat state compared with the conventionally used scheme of the uniform distribution; yet the distribution of optimized copies of the ten-photon Schrödinger’s Cat state gives better fidelity estimation than the uniform distribution for the same number of copies of the ten-photon Schrödinger’s Cat state. PMID:27576585

Low copy numbers of complement C4 and homozygous deficiency of C4A may predispose to severe disease and earlier disease onset in patients with systemic lupus erythematosus.

PubMed

Jüptner, M; Flachsbart, F; Caliebe, A; Lieb, W; Schreiber, S; Zeuner, R; Franke, A; Schröder, J O

2018-04-01

Objectives Low copy numbers and deletion of complement C4 genes are potent risk factors for systemic lupus erythematosus (SLE). However, it is not known whether this genetic association affects the clinical outcome. We investigated C4 copy number variation and its relationship to clinical and serological features in a Northern European lupus cohort. Methods We genotyped the C4 gene locus using polymerase chain reaction (PCR)-based TaqMan assays in 169 patients with SLE classified according to the 1997 revised American College of Rheumatology (ACR) criteria and in 520 matched controls. In the patient group the mean C4 serum protein concentrations nephelometrically measured during a 12-month period prior to genetic analysis were compared to C4 gene copy numbers. Severity of disease was classified according to the intensity of the immunosuppressive regimens applied and compared to C4 gene copy numbers, too. In addition, we performed a TaqMan based analysis of three lupus-associated single-nucleotide polymorphisms (SNPs) located inside the major histocompatibility complex (MHC) to investigate the independence of complement C4 in association with SLE. Results Homozygous deficiency of the C4A isotype was identified as the strongest risk factor for SLE (odds ratio (OR) = 5.329; p = 7.7 × 10 -3 ) in the case-control comparison. Moreover, two copies of total C4 were associated with SLE (OR = 3.699; p = 6.8 × 10 -3 ). C4 serum levels were strongly related to C4 gene copy numbers in patients, the mean concentration ranging from 0.110 g/l (two copies) to 0.256 g/l (five to six copies; p = 4.9 × 10 -6 ). Two copies of total C4 and homozygous deletion of C4A were associated with a disease course requiring cyclophosphamide therapy (OR = 4.044; p = 0.040 and OR = 5.798; p = 0.034, respectively). Homozygous deletion of C4A was associated with earlier onset of SLE (median 24 vs. 34 years; p = 0.019) but not significant after correction for multiple testing. SNP analysis revealed a significant association of HLA-DRB1*0301 with SLE (OR = 2.231; p = 1.33 × 10 -5 ). Conclusions Our findings confirm the important role of complement C4 genes in the development of SLE. Beyond the impact on the susceptibility for lupus, C4 copy numbers may be related to earlier onset and a more severe course of the disease. The association of homozygous deletion of C4A and SLE is accompanied by the presence of HLA-DRB1*0301 without a proven pathophysiological mechanism.

Analysis of copy number variations among cattle breeds

USDA-ARS?s Scientific Manuscript database

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in the modern domesticated cattle using array comparative genomic hybridization (array CGH) and quanti...

10 CFR 51.66 - Environmental report-number of copies; distribution.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 2 2013-01-01 2013-01-01 false Environmental report-number of copies; distribution. 51.66 Section 51.66 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) ENVIRONMENTAL PROTECTION REGULATIONS FOR DOMESTIC LICENSING AND RELATED REGULATORY FUNCTIONS National Environmental Policy Act-Regulations...

Gene copy number evolution during tetraploid cotton radiation.

PubMed

Rong, J; Feltus, F A; Liu, L; Lin, L; Paterson, A H

2010-11-01

After polyploid formation, retention or loss of duplicated genes is not random. Genes with some functional domains are convergently restored to 'singleton' state after many independent genome duplications, and have been referred to as 'duplication-resistant' (DR) genes. To further explore the timeframe for their restoration to the singleton state, 27 cotton homologs of genes found to be 'DR' in Arabidopsis were selected based on diagnostic Pfam domains. Their copy numbers were studied using southern hybridization and sequence analysis in five tetraploid species and their ancestral A and D genome diploids. DR genes had significantly lower copy number than gene families hybridizing to randomly selected cotton ESTs. Three DR genes showed complete loss of D genome-derived homoeologs in some or all tetraploid species. Prior analysis has shown gene loss in polyploid cotton to be rare, and herein only one randomly selected gene showed loss of a homoeolog in only one of the five tetraploid species (Gossypium mustelinum). BAC sequencing confirmed two cases of gene loss in tetraploid cotton. Divergence among 5' sequences of DR genes amplified from G. arboreum, G. raimondii, and Gossypioides kirkii was correlated with gene copy number. These results show that genes containing Pfam domains associated with duplication resistance in Arabidopsis have also been preferentially restored to low copy number after a more recent polyploidization event in cotton. In tetraploid cotton, genes from the progenitor D genome seem to experience more gene copy number divergence than genes from the A genome. Together with D subgenome-biased alterations in gene expression, perhaps gene loss may contribute to the relatively larger portion of quantitative trait variation attributable to D than A subgenome chromosomes of tetraploid cotton.

LPA and PLG sequence variation and kringle IV-2 copy number in two populations.

PubMed

Crawford, Dana C; Peng, Ze; Cheng, Jan-Fang; Boffelli, Dario; Ahearn, Magdalena; Nguyen, Dan; Shaffer, Tristan; Yi, Qian; Livingston, Robert J; Rieder, Mark J; Nickerson, Deborah A

2008-01-01

Lp(a) levels have long been recognized as a potential risk factor for coronary heart disease that is almost completely under genetic control. Much of the genetics impacting Lp(a) levels has been attributed to the highly polymorphic LPA kringle IV-2 copy number variant, and most of the variance in Lp(a) levels in populations of European-descent is inversely correlated with kringle IV copy number. However, less of the variance is explained in African-descent populations for the same structural variation. African-descent populations have, on average, higher levels of Lp(a), suggesting other genetic factors contribute to Lp(a) level variability across populations. To identify potential cis-acting factors, we re-sequenced the gene LPA for single nucleotide polymorphism (SNP) discovery in 23 European-Americans and 24 African-Americans. We also re- sequenced the neighboring gene plasminogen (PLG) and genotyped the kringle IV copy number variant in the same reference samples. These data are the most comprehensive description of sequence variation in LPA and its relationship with the kringle IV copy number variant. With these data, we demonstrate that only a fraction of LPA sequence diversity has been previously documented. Also, we identify several high frequency SNPs present in the African-American sample but absent in the European-American sample. Finally, we show that SNPs within PLG are not in linkage disequilibrium with SNPs in LPA, and we show that kringle IV copy number variation is not in linkage disequilibrium with either LPA or PLG SNPs. Together, these data suggest that LPA SNPs could independently contribute to Lp(a) levels in the general population. Copyright (c) 2008 S. Karger AG, Basel.

Influences of AMY1 gene copy number and protein expression on salivary alpha-amylase activity before and after citric acid stimulation in splenic asthenia children.

PubMed

Yang, Zemin; Lin, Jing; Chen, Longhui; Zhang, Min; Yang, Xiaorong; Chen, Weiwen

2015-06-01

To compare the correlations between salivary alpha-amylase (sAA) activity and amylase, alpha 1 (salivary) gene (AMYl) copy number or its gene expression between splenic asthenia and healthy children, and investigate the reasons of attenuated sAA activity ratio before and after citric acid stimulation in splenic asthenia children. Saliva samples from 20 splenic asthenia children and 29 healthy children were collected before and after citric acid stimulation. AMYl copy number, sAA activity, and total sAA and glycosylated sAA contents were determined, and their correlations were analyzed. Although splenic asthenia and healthy children had no differences in AMY1 copy number, splenic asthenia children had positive correlations between AMY1 copy number and sAA activity before or after citric acid stimulation. Splenic asthenia children had a higher sAA glycosylated proportion ratio and glycosylated sAA content ratio, while their total sAA content ratio and sAA activity ratio were lower compared with healthy children. The glycosylated sAA content ratio was higher than the total sAA content ratio in both groups. Splenic asthenia and healthy children had positive correlations between total sAA or glycosylated sAA content and sAA activity. However, the role played by glycosylated sAA content in sAA activity in healthy children increased after citric acid stimulation, while it decreased in splenic asthenia children. Genetic factors like AMY1 copy number variations, and more importantly, sAA glycosylation abnormalities leading to attenuated sAA activity after citric acid stimulation, which were the main reasons of the attenuated sAA activity ratio in splenic asthenia children compared with healthy children.

Independent replication of mitochondrial genes supports the transcriptional program in developing fiber cells of cotton (Gossypium hirsutum L.).

PubMed

Thyssen, Gregory N; Song, Xianliang; Naoumkina, Marina; Kim, Hee-Jin; Fang, David D

2014-07-01

The mitochondrial genomes of flowering plants exist both as a "master circle" chromosome and as numerous subgenomic sublimons that are generated by intramolecular recombination. Differential stability or replication of these sublimons allows individual mitochondrial gene copy numbers to vary independently between different cell types and developmental stages. Our objective was to determine the relationship between mitochondrial gene copy number and transcript abundance in the elongating fiber cells of Upland cotton (Gossypium hirsutum L.). We compared RNA and DNA from cotton fiber cells at five developmental time points from early elongation through secondary cell wall thickening from the Ligon-lintless 2 (Li2) short fiber mutant and its wild type near isogenic line (NIL) DP5690. Mitochondrial gene copy number decreased from 3 to 8-DPA in the developing cotton fiber cells while transcript levels remained low. As secondary cell wall biosynthesis began in developing fibers, the expression levels and copy numbers of mitochondrial genes involved in energy production and respiration were up-regulated in wild type cotton DP5690. However, the short fiber mutant Li2, failed to increase expression of these genes, which include three subunits of ATP synthase, atp1, atp8 and atp9 and two cytochrome genes cox1 and cob. At the same time, Li2 failed to increase the copy numbers of these highly expressed genes. Surprisingly, we found that when mitochondrial genes were highly transcribed, they also had very high copy numbers. This observation suggests that in developing cotton fibers, increased mitochondrial sublimon replication may support increases in gene transcription. Published by Elsevier B.V.

LAPTM4B gene copy number gain is associated with inferior response to anthracycline-based chemotherapy in hormone receptor negative breast carcinomas.

PubMed

Rusz, Orsolya; Papp, Orsolya; Vízkeleti, Laura; Molnár, Béla Ákos; Bende, Kristóf Csaba; Lotz, Gábor; Ács, Balázs; Kahán, Zsuzsanna; Székely, Tamás; Báthori, Ágnes; Szundi, Csilla; Kulka, Janina; Szállási, Zoltán; Tőkés, Anna-Mária

2018-05-16

To determine the associations between lysosomal-associated transmembrane protein 4b (LAPTM4B) gene copy number and response to different chemotherapy regimens in hormone receptor negative (HR-) primary breast carcinomas. Two cohorts were analyzed: (1) 69 core biopsies from HR-breast carcinomas treated with neoadjuvant chemotherapy (anthracycline based in 72.5% of patients and non-anthracycline based in 27.5% of patients). (2) Tissue microarray (TMA) of 74 HR-breast carcinomas treated with adjuvant therapy (77.0% of the patients received anthracycline, 17.6% of the patients non-anthracycline-based therapy, and in 5.4% of the cases, no treatment data are available). Interphase FISH technique was applied on pretreatment core biopsies (cohort I) and on TMAs (cohort II) using custom-made dual-labelled FISH probes (LAPTM4B/CEN8q FISH probe Abnova Corp.). In the neoadjuvant cohort in the anthracycline-treated group, we observed a significant difference (p = 0.029) of average LAPTM4B copy number between the non-responder and pathological complete responder groups (4.1 ± 1.1 vs. 2.6 ± 0.1). In the adjuvant setting, the anthracycline-treated group of metastatic breast carcinomas was characterized by higher LAPTM4B copy number comparing to the non-metastatic ones (p = 0.046). In contrast, in the non-anthracycline-treated group of patients, we did not find any LAPTM4B gene copy number differences between responder vs. non-responder groups or between metastatic vs. non-metastatic groups. Our results confirm the possible role of the LAPTM4B gene in anthracycline resistance in HR- breast cancer. Analyzing LAPTM4B copy number pattern may support future treatment decision.

47 CFR 3.25 - Number of copies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL AUTHORIZATION AND ADMINISTRATION OF ACCOUNTING... copies. One original and one copy of FCC Form 44, “Application For Certification As An Accounting... commencement of settlement activities to allow time for the Commission to review the application and to allow...

Genomic characteristics of cattle copy number variations

USDA-ARS?s Scientific Manuscript database

We performed a systematic analysis of cattle copy number variations (CNVs) using the Bovine HapMap SNP genotyping data, including 539 animals of 21 modern cattle breeds and 6 outgroups. After correcting genomic waves and considering the trio information, we identified 682 candidate CNV regions (CNVR...

Analysis of copy number variations reveals differences among cattle breeds

USDA-ARS?s Scientific Manuscript database

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in the modern domesticated cattle using array comparative genomic hybridization (array CGH) and quanti...

Population-genetic properties of differentiated copy number variations in cattle

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) have been shown to be both common in mammals and important for understanding the relationship between genotype and phenotype. However, CNV differentiation, selection and its population genetic properties are not well understood across diverse populations. We performed a...

Genomic Copy Number Variation in Disorders of Cognitive Development

ERIC Educational Resources Information Center

Morrow, Eric M.

2010-01-01

Objective: To highlight recent discoveries in the area of genomic copy number variation in neuropsychiatric disorders including intellectual disability, autism, and schizophrenia. To emphasize new principles emerging from this area, involving the genetic architecture of disease, pathophysiology, and diagnosis. Method: Review of studies published…

Punctuated Copy Number Evolution and Clonal Stasis in Triple-Negative Breast Cancer

PubMed Central

Gao, Ruli; Davis, Alexander; McDonald, Thomas O.; Sei, Emi; Shi, Xiuqing; Wang, Yong; Tsai, Pei-Ching; Casasent, Anna; Waters, Jill; Zhang, Hong; Meric-Bernstam, Funda; Michor, Franziska; Navin, Nicholas E.

2016-01-01

Aneuploidy is a hallmark of breast cancer; however, our knowledge of how these complex genomic rearrangements evolve during tumorigenesis is limited. In this study we developed a highly multiplexed single-nucleus-sequencing method to investigate copy number evolution in triple-negative breast cancer patients. We sequenced 1000 single cells from 12 patients and identified 1–3 major clonal subpopulations in each tumor that shared a common evolutionary lineage. We also identified a minor subpopulation of non-clonal cells that were classified as: 1) metastable, 2) pseudo-diploid, or 3) chromazemic. Phylogenetic analysis and mathematical modeling suggest that these data are unlikely to be explained by the gradual accumulation of copy number events over time. In contrast, our data challenge the paradigm of gradual evolution, showing that the majority of copy number aberrations are acquired at the earliest stages of tumor evolution, in short punctuated bursts, followed by stable clonal expansions that form the tumor mass. PMID:27526321

A comparative genomic hybridization approach to study gene copy number variations among Chinese hamster cell lines.

PubMed

Vishwanathan, Nandita; Bandyopadhyay, Arpan; Fu, Hsu-Yuan; Johnson, Kathryn C; Springer, Nathan M; Hu, Wei-Shou

2017-08-01

Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The effect of input DNA copy number on genotype call and characterising SNP markers in the humpback whale genome using a nanofluidic array.

PubMed

Bhat, Somanath; Polanowski, Andrea M; Double, Mike C; Jarman, Simon N; Emslie, Kerry R

2012-01-01

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.

Quadruplex MAPH: improvement of throughput in high-resolution copy number screening.

PubMed

Tyson, Jess; Majerus, Tamsin Mo; Walker, Susan; Armour, John Al

2009-09-28

Copy number variation (CNV) in the human genome is recognised as a widespread and important source of human genetic variation. Now the challenge is to screen for these CNVs at high resolution in a reliable, accurate and cost-effective way. Multiplex Amplifiable Probe Hybridisation (MAPH) is a sensitive, high-resolution technology appropriate for screening for CNVs in a defined region, for a targeted population. We have developed MAPH to a highly multiplexed format ("QuadMAPH") that allows the user a four-fold increase in the number of loci tested simultaneously. We have used this method to analyse a genomic region of 210 kb, including the MSH2 gene and 120 kb of flanking DNA. We show that the QuadMAPH probes report copy number with equivalent accuracy to simplex MAPH, reliably demonstrating diploid copy number in control samples and accurately detecting deletions in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) samples. QuadMAPH is an accurate, high-resolution method that allows targeted screening of large numbers of subjects without the expense of genome-wide approaches. Whilst we have applied this technique to a region of the human genome, it is equally applicable to the genomes of other organisms.

Quadruplex MAPH: improvement of throughput in high-resolution copy number screening

PubMed Central

Tyson, Jess; Majerus, Tamsin MO; Walker, Susan; Armour, John AL

2009-01-01

Background Copy number variation (CNV) in the human genome is recognised as a widespread and important source of human genetic variation. Now the challenge is to screen for these CNVs at high resolution in a reliable, accurate and cost-effective way. Results Multiplex Amplifiable Probe Hybridisation (MAPH) is a sensitive, high-resolution technology appropriate for screening for CNVs in a defined region, for a targeted population. We have developed MAPH to a highly multiplexed format ("QuadMAPH") that allows the user a four-fold increase in the number of loci tested simultaneously. We have used this method to analyse a genomic region of 210 kb, including the MSH2 gene and 120 kb of flanking DNA. We show that the QuadMAPH probes report copy number with equivalent accuracy to simplex MAPH, reliably demonstrating diploid copy number in control samples and accurately detecting deletions in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) samples. Conclusion QuadMAPH is an accurate, high-resolution method that allows targeted screening of large numbers of subjects without the expense of genome-wide approaches. Whilst we have applied this technique to a region of the human genome, it is equally applicable to the genomes of other organisms. PMID:19785739

Genomic and evolutionary characteristics of cattle copy number variations

USDA-ARS?s Scientific Manuscript database

We performed a systematic analysis of cattle copy number variations (CNVs) using the Bovine HapMap SNP genotyping data, including 539 animals of 21 modern cattle breeds and 6 outgroups. After correcting genomic waves and considering the trio information, we identified 682 candidate CNV regions (CNVR...

Copy number variation detection in cattle reveals potential breed specific differences

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) are large, common deletions or duplications of genome sequence among individuals of a species that have been linked to diseases and phenotypic traits. For example, a CNV-generating, translocation mechanism encompassing the KIT gene is responsible for color sidedness in ...

Structural and functional impacts of copy number variations on the cattle genome

USDA-ARS?s Scientific Manuscript database

Although there have been significant advances in resolving the pattern and nature of single nucleotide polymorphisms (SNPs), similar realizations for larger, more complex forms of genetic variation have just emerged. Several recent publications reveal that copy number variations (CNVs) are common an...

MERE1, a low-copy-number copia-type retroelement in Medicago truncatula active during tissue culture.

PubMed

Rakocevic, Alexandra; Mondy, Samuel; Tirichine, Leïla; Cosson, Viviane; Brocard, Lysiane; Iantcheva, Anelia; Cayrel, Anne; Devier, Benjamin; Abu El-Heba, Ghada Ahmed; Ratet, Pascal

2009-11-01

We have identified an active Medicago truncatula copia-like retroelement called Medicago RetroElement1-1 (MERE1-1) as an insertion in the symbiotic NSP2 gene. MERE1-1 belongs to a low-copy-number family in the sequenced Medicago genome. These copies are highly related, but only three of them have a complete coding region and polymorphism exists between the long terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but also in other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicago accessions and inserts preferentially in genes.

Performance evaluation of DNA copy number segmentation methods.

PubMed

Pierre-Jean, Morgane; Rigaill, Guillem; Neuvial, Pierre

2015-07-01

A number of bioinformatic or biostatistical methods are available for analyzing DNA copy number profiles measured from microarray or sequencing technologies. In the absence of rich enough gold standard data sets, the performance of these methods is generally assessed using unrealistic simulation studies, or based on small real data analyses. To make an objective and reproducible performance assessment, we have designed and implemented a framework to generate realistic DNA copy number profiles of cancer samples with known truth. These profiles are generated by resampling publicly available SNP microarray data from genomic regions with known copy-number state. The original data have been extracted from dilutions series of tumor cell lines with matched blood samples at several concentrations. Therefore, the signal-to-noise ratio of the generated profiles can be controlled through the (known) percentage of tumor cells in the sample. This article describes this framework and its application to a comparison study between methods for segmenting DNA copy number profiles from SNP microarrays. This study indicates that no single method is uniformly better than all others. It also helps identifying pros and cons of the compared methods as a function of biologically informative parameters, such as the fraction of tumor cells in the sample and the proportion of heterozygous markers. This comparison study may be reproduced using the open source and cross-platform R package jointseg, which implements the proposed data generation and evaluation framework: http://r-forge.r-project.org/R/?group_id=1562. © The Author 2014. Published by Oxford University Press.

Practical guidelines for interpreting copy number gains detected by high-resolution array in routine diagnostics

PubMed Central

Hanemaaijer, Nicolien M; Sikkema-Raddatz, Birgit; van der Vries, Gerben; Dijkhuizen, Trijnie; Hordijk, Roel; van Essen, Anthonie J; Veenstra-Knol, Hermine E; Kerstjens-Frederikse, Wilhelmina S; Herkert, Johanna C; Gerkes, Erica H; Leegte, Lamberta K; Kok, Klaas; Sinke, Richard J; van Ravenswaaij-Arts, Conny M A

2012-01-01

The correct interpretation of copy number gains in patients with developmental delay and multiple congenital anomalies is hampered by the large number of copy number variations (CNVs) encountered in healthy individuals. The variable phenotype associated with copy number gains makes interpretation even more difficult. Literature shows that inheritence, size and presence in healthy individuals are commonly used to decide whether a certain copy number gain is pathogenic, but no general consensus has been established. We aimed to develop guidelines for interpreting gains detected by array analysis using array CGH data of 300 patients analysed with the 105K Agilent oligo array in a diagnostic setting. We evaluated the guidelines in a second, independent, cohort of 300 patients. In the first 300 patients 797 gains of four or more adjacent oligonucleotides were observed. Of these, 45.4% were de novo and 54.6% were familial. In total, 94.8% of all de novo gains and 87.1% of all familial gains were concluded to be benign CNVs. Clinically relevant gains ranged from 288 to 7912 kb in size, and were significantly larger than benign gains and gains of unknown clinical relevance (P<0.001). Our study showed that a threshold of 200 kb is acceptable in a clinical setting, whereas heritability does not exclude a pathogenic nature of a gain. Evaluation of the guidelines in the second cohort of 300 patients revealed that the interpretation guidelines were clear, easy to follow and efficient. PMID:21934709

Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies.

PubMed

Bakker, Bjorn; Taudt, Aaron; Belderbos, Mirjam E; Porubsky, David; Spierings, Diana C J; de Jong, Tristan V; Halsema, Nancy; Kazemier, Hinke G; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline S J M; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M; Colomé-Tatché, Maria; Foijer, Floris

2016-05-31

Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number changes affecting all lymphoma cells. This suggests that chromosome instability is somehow suppressed in the aneuploid lymphomas or that selection for frequently lost/gained chromosomes out-competes the CIN-imposed mis-segregation. To distinguish between these explanations and to examine karyotype dynamics in chromosome instable lymphoma, we use a newly developed single-cell whole genome sequencing (scWGS) platform that provides a complete and unbiased overview of copy number variations (CNV) in individual cells. To analyse these scWGS data, we develop AneuFinder, which allows annotation of copy number changes in a fully automated fashion and quantification of CNV heterogeneity between cells. Single-cell sequencing and AneuFinder analysis reveals high levels of copy number heterogeneity in chromosome instability-driven murine T-cell lymphoma samples, indicating ongoing chromosome instability. Application of this technology to human B cell leukaemias reveals different levels of karyotype heterogeneity in these cancers. Our data show that even though aneuploid tumours select for particular and recurring chromosome combinations, single-cell analysis using AneuFinder reveals copy number heterogeneity. This suggests ongoing chromosome instability that other platforms fail to detect. As chromosome instability might drive tumour evolution, karyotype analysis using single-cell sequencing technology could become an essential tool for cancer treatment stratification.

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

PubMed Central

Pan, W J; Blackburn, E H

1995-01-01

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number. Images PMID:7784211

UGT2B17 and SULT1A1 gene copy number variation (CNV) detection by LabChip microfluidic technology.

PubMed

Gaedigk, Andrea; Gaedigk, Roger; Leeder, J Steven

2010-05-01

Gene copy number variations (CNVs) are increasingly recognized to play important roles in the expression of genes and hence on their respective enzymatic activities. This has been demonstrated for a number of drug metabolizing genes, such as UDP-glucuronosyltransferases 2B17 (UGT2B17) and sulfotransferase 1A1 (SULT1A1), which are subject to genetic heterogeneity, including CNV. Quantitative assays to assess gene copy number are therefore becoming an integral part of accurate genotype assessment and phenotype prediction. In this study, we evaluated a microfluidics-based system, the Bio-Rad Experion system, to determine the power and utility of this platform to detect UGT2B17 and SULT1A1 CNV in DNA samples derived from blood and tissue. UGT2B17 is known to present with 0, 1 or 2 and SULT1A1 with up to 5 gene copies. Distinct clustering (p<0.001) into copy number groups was achieved for both genes. DNA samples derived from blood exhibited less inter-run variability compared to DNA samples obtained from liver tissue. This variability may be caused by tissue-specific PCR inhibitors as it could be overcome by using DNA from another tissue, or after the DNA had undergone whole genome amplification. This method produced results comparable to those reported for other quantitative test platforms.

Copy Number Variation Affecting the Photoperiod-B1 and Vernalization-A1 Genes Is Associated with Altered Flowering Time in Wheat (Triticum aestivum)

PubMed Central

Isaac, Peter; Laurie, David A.

2012-01-01

The timing of flowering during the year is an important adaptive character affecting reproductive success in plants and is critical to crop yield. Flowering time has been extensively manipulated in crops such as wheat (Triticum aestivum L.) during domestication, and this enables them to grow productively in a wide range of environments. Several major genes controlling flowering time have been identified in wheat with mutant alleles having sequence changes such as insertions, deletions or point mutations. We investigated genetic variants in commercial varieties of wheat that regulate flowering by altering photoperiod response (Ppd-B1 alleles) or vernalization requirement (Vrn-A1 alleles) and for which no candidate mutation was found within the gene sequence. Genetic and genomic approaches showed that in both cases alleles conferring altered flowering time had an increased copy number of the gene and altered gene expression. Alleles with an increased copy number of Ppd-B1 confer an early flowering day neutral phenotype and have arisen independently at least twice. Plants with an increased copy number of Vrn-A1 have an increased requirement for vernalization so that longer periods of cold are required to potentiate flowering. The results suggest that copy number variation (CNV) plays a significant role in wheat adaptation. PMID:22457747

High-Resolution SNP/CGH Microarrays Reveal the Accumulation of Loss of Heterozygosity in Commonly Used Candida albicans Strains

PubMed Central

Abbey, Darren; Hickman, Meleah; Gresham, David; Berman, Judith

2011-01-01

Phenotypic diversity can arise rapidly through loss of heterozygosity (LOH) or by the acquisition of copy number variations (CNV) spanning whole chromosomes or shorter contiguous chromosome segments. In Candida albicans, a heterozygous diploid yeast pathogen with no known meiotic cycle, homozygosis and aneuploidy alter clinical characteristics, including drug resistance. Here, we developed a high-resolution microarray that simultaneously detects ∼39,000 single nucleotide polymorphism (SNP) alleles and ∼20,000 copy number variation loci across the C. albicans genome. An important feature of the array analysis is a computational pipeline that determines SNP allele ratios based upon chromosome copy number. Using the array and analysis tools, we constructed a haplotype map (hapmap) of strain SC5314 to assign SNP alleles to specific homologs, and we used it to follow the acquisition of loss of heterozygosity (LOH) and copy number changes in a series of derived laboratory strains. This high-resolution SNP/CGH microarray and the associated hapmap facilitated the phasing of alleles in lab strains and revealed detrimental genome changes that arose frequently during molecular manipulations of laboratory strains. Furthermore, it provided a useful tool for rapid, high-resolution, and cost-effective characterization of changes in allele diversity as well as changes in chromosome copy number in new C. albicans isolates. PMID:22384363

A large-scale survey of genetic copy number variations among Han Chinese residing in Taiwan

PubMed Central

Lin, Chien-Hsing; Li, Ling-Hui; Ho, Sheng-Feng; Chuang, Tzu-Po; Wu, Jer-Yuarn; Chen, Yuan-Tsong; Fann, Cathy SJ

2008-01-01

Background Copy number variations (CNVs) have recently been recognized as important structural variations in the human genome. CNVs can affect gene expression and thus may contribute to phenotypic differences. The copy number inferring tool (CNIT) is an effective hidden Markov model-based algorithm for estimating allele-specific copy number and predicting chromosomal alterations from single nucleotide polymorphism microarrays. The CNIT algorithm, which was constructed using data from 270 HapMap multi-ethnic individuals, was applied to identify CNVs from 300 unrelated Han Chinese individuals in Taiwan. Results Using stringent selection criteria, 230 regions with variable copy numbers were identified in the Han Chinese population; 133 (57.83%) had been reported previously, 64 displayed greater than 1% CNV allele frequency. The average size of the CNV regions was 322 kb (ranging from 1.48 kb to 5.68 Mb) and covered a total of 2.47% of the human genome. A total of 196 of the CNV regions were simple deletions and 27 were simple amplifications. There were 449 genes and 5 microRNAs within these CNV regions; some of these genes are known to be associated with diseases. Conclusion The identified CNVs are characteristic of the Han Chinese population and should be considered when genetic studies are conducted. The CNV distribution in the human genome is still poorly characterized, and there is much diversity among different ethnic populations. PMID:19108714

EGFR molecular profiling in advanced NSCLC: a prospective phase II study in molecularly/clinically selected patients pretreated with chemotherapy.

PubMed

Milella, Michele; Nuzzo, Carmen; Bria, Emilio; Sperduti, Isabella; Visca, Paolo; Buttitta, Fiamma; Antoniani, Barbara; Merola, Roberta; Gelibter, Alain; Cuppone, Federica; D'Alicandro, Valerio; Ceribelli, Anna; Rinaldi, Massimo; Cianciulli, Anna; Felicioni, Lara; Malatesta, Sara; Marchetti, Antonio; Mottolese, Marcella; Cognetti, Francesco

2012-04-01

The optimal use of epidermal growth factor receptor (EGFR)-related molecular markers to prospectively identify tyrosine kinase inhibitor (TKI)-sensitive patients, particularly after a previous chemotherapy treatment, is currently under debate. We designed a prospective phase II study to evaluate the activity of EGFR-TKI in four different patient groups, according to the combination of molecular (EGFR gene mutations, EGFR gene copy number and protein expression, and phosphorylated AKT expression, pAKT) and clinicopathological (histology and smoking habits) factors. Correlations between molecular alterations and clinical outcome were also explored retrospectively for first-line chemotherapy and EGFR-TKI treatment. Patients who had progressed during or after first-line chemotherapy were prospectively assigned to EGFR-TKI treatment as follows: (G1) EGFR mutation (n = 12); (G2) highly polysomic/amplified EGFR (n = 18); (G3) EGFR and/or pAKT positive (n = 41); (G4) adenocarcinoma/bronchoalveolar carcinoma and no smoking history (n = 15). G1 and G4 had the best and second-best overall response rate (25% and 20%, respectively), whereas the worst outcome was observed in G2 (ORR, 6%; p = 0.05). Disease control was highest in G1 and G4 (>50%) and lowest in G3 (<20%) (p = 0.02). Patients selected by EGFR mutation or clinical parameters (G1 and G4) also had significantly better progression-free survival and overall survival (p = 0.02 and p = 0.01, respectively). Multivariate analysis confirmed the impact of sex, smoking history, EGFR/KRAS mutation, and pAKT on outcomes and allowed us to derive an efficient predictive model. Histology, EGFR mutations, and pAKT were independent predictors of response to first-line chemotherapy at retrospective analysis, whereas pAKT and human epidermal growth factor receptor 2 expression were the only independent predictors of progression-free survival and overall survival. Selection of patients based on either EGFR mutation or clinical characteristics seems an effective approach to optimize EGFR-TKI treatment in chemotherapy-pretreated non-small-cell lung cancer patients.

Three Groups of Transposable Elements with Contrasting Copy Number Dynamics and Host Responses in the Maize (Zea mays ssp. mays) Genome

PubMed Central

Diez, Concepcion M.; Meca, Esteban; Tenaillon, Maud I.; Gaut, Brandon S.

2014-01-01

Most angiosperm nuclear DNA is repetitive and derived from silenced transposable elements (TEs). TE silencing requires substantial resources from the plant host, including the production of small interfering RNAs (siRNAs). Thus, the interaction between TEs and siRNAs is a critical aspect of both the function and the evolution of plant genomes. Yet the co-evolutionary dynamics between these two entities remain poorly characterized. Here we studied the organization of TEs within the maize (Zea mays ssp mays) genome, documenting that TEs fall within three groups based on the class and copy numbers. These groups included DNA elements, low copy RNA elements and higher copy RNA elements. The three groups varied statistically in characteristics that included length, location, age, siRNA expression and 24∶22 nucleotide (nt) siRNA targeting ratios. In addition, the low copy retroelements encompassed a set of TEs that had previously been shown to decrease expression within a 24 nt siRNA biogenesis mutant (mop1). To investigate the evolutionary dynamics of the three groups, we estimated their abundance in two landraces, one with a genome similar in size to that of the maize reference and the other with a 30% larger genome. For all three accessions, we assessed TE abundance as well as 22 nt and 24 nt siRNA content within leaves. The high copy number retroelements are under targeted similarly by siRNAs among accessions, appear to be born of a rapid bust of activity, and may be currently transpositionally dead or limited. In contrast, the lower copy number group of retrolements are targeted more dynamically and have had a long and ongoing history of transposition in the maize genome. PMID:24743518

Accurate measure of transgene copy number in crop plants using droplet digital PCR

USDA-ARS?s Scientific Manuscript database

Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy numb...

A network of epigenetic modifiers and DNA repair genes controls tissue-specific copy number alteration preference.

PubMed

Cramer, Dina; Serrano, Luis; Schaefer, Martin H

2016-11-10

Copy number alterations (CNAs) in cancer patients show a large variability in their number, length and position, but the sources of this variability are not known. CNA number and length are linked to patient survival, suggesting clinical relevance. We have identified genes that tend to be mutated in samples that have few or many CNAs, which we term CONIM genes (COpy Number Instability Modulators). CONIM proteins cluster into a densely connected subnetwork of physical interactions and many of them are epigenetic modifiers. Therefore, we investigated how the epigenome of the tissue-of-origin influences the position of CNA breakpoints and the properties of the resulting CNAs. We found that the presence of heterochromatin in the tissue-of-origin contributes to the recurrence and length of CNAs in the respective cancer type.

Diversity and population-genetic properties of copy number variations and multicopy genes in cattle

USDA-ARS?s Scientific Manuscript database

The diversity and population-genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analyzed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, Romagnola), sequenced to 11-fold...

Dana-Farber Cancer Institute (DFCI): Computational Correction of Copy-number Effect in CRISPR-Cas9 Essentiality Screens of Cancer Cells | Office of Cancer Genomics

Cancer.gov

Genome-wide CRISPR-Cas9 screens were performed in 341 cell lines. The results were processed with the CERES algorithm to produce copy-number and guide-efficacy corrected gene-knockout effect estimates.

Dana-Farber Cancer Institute (DFCI): Computational Correction of Copy-number Effect in CRISPR-Cas9 Essentiality Screens of Cancer Cells | Office of Cancer Genomics

Cancer.gov

Genome-wide CRISPR-Cas9 screens were performed in 341 cell lines. The results were processed with the CERES algorithm to produce copy-number and guide-efficacy corrected gene knockout effect estimates.

10 CFR 51.58 - Environmental report-number of copies; distribution.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 2 2010-01-01 2010-01-01 false Environmental report-number of copies; distribution. 51.58 Section 51.58 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) ENVIRONMENTAL PROTECTION REGULATIONS FOR...)(4), each applicant for renewal of an operating or combined license for a nuclear power plant, each...

10 CFR 51.58 - Environmental report-number of copies; distribution.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 2 2012-01-01 2012-01-01 false Environmental report-number of copies; distribution. 51.58 Section 51.58 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) ENVIRONMENTAL PROTECTION REGULATIONS FOR...)(4), each applicant for renewal of an operating or combined license for a nuclear power plant, each...

10 CFR 51.58 - Environmental report-number of copies; distribution.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 2 2014-01-01 2014-01-01 false Environmental report-number of copies; distribution. 51.58 Section 51.58 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) ENVIRONMENTAL PROTECTION REGULATIONS FOR...)(4), each applicant for renewal of an operating or combined license for a nuclear power plant, each...

10 CFR 51.58 - Environmental report-number of copies; distribution.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 2 2013-01-01 2013-01-01 false Environmental report-number of copies; distribution. 51.58 Section 51.58 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) ENVIRONMENTAL PROTECTION REGULATIONS FOR...)(4), each applicant for renewal of an operating or combined license for a nuclear power plant, each...

10 CFR 51.58 - Environmental report-number of copies; distribution.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 2 2011-01-01 2011-01-01 false Environmental report-number of copies; distribution. 51.58 Section 51.58 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) ENVIRONMENTAL PROTECTION REGULATIONS FOR...)(4), each applicant for renewal of an operating or combined license for a nuclear power plant, each...

Rare DNA copy number variants in cardiovascular malformations with extracardiac abnormalities

USDA-ARS?s Scientific Manuscript database

Clinically significant cardiovascular malformations (CVMs) occur in 5-8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be...

18 CFR 33.8 - Number of copies.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ENERGY REGULATIONS UNDER THE FEDERAL POWER ACT APPLICATIONS UNDER FEDERAL POWER ACT SECTION 203 § 33.8 Number of copies. The applicant must submit the application or petition to the Secretary of the..., the applicant must submit all such information in electronic format (e.g., on computer diskette or on...

18 CFR 33.8 - Number of copies.

Code of Federal Regulations, 2011 CFR

2011-04-01

... ENERGY REGULATIONS UNDER THE FEDERAL POWER ACT APPLICATIONS UNDER FEDERAL POWER ACT SECTION 203 § 33.8 Number of copies. The applicant must submit the application or petition to the Secretary of the..., the applicant must submit all such information in electronic format (e.g., on computer diskette or on...

Increased TERC gene copy number and cells in senescence in primary sclerosing cholangitis compared to colitis and control patients.

PubMed

Laish, Ido; Katz, Hila; Sulayev, Yael; Liberman, Meytal; Naftali, Timna; Benjaminov, Fabiana; Stein, Assaf; Kitay-Cohen, Yona; Biron-Shental, Tal; Konikoff, Fred; Amiel, Aliza

2013-10-25

Primary sclerosing cholangitis (PSC) is a chronic cholestatic disorder that involves inflammatory and fibrotic changes in the bile ducts. Up to 80% of patients have concomitant inflammatory bowel disease (IBD) with colitis. PSC patients are predisposed to develop hepatobiliary, colonic and other extrahepatic malignancies, probably related to inflammatory processes that might promote carcinogenesis. Telomerase is an enzyme complex that lengthens telomeres and has enhanced expression in numerous malignancies. In this study, we evaluated the TERC gene copy number, the proportion of cells in senescence and the amount of fragmentation in the senescent state. Fluorescence in situ hybridization (FISH) for the TERC gene was applied to lymphocytes retrieved from PSC (N=19), colitis (N=20) and healthy control patients (N=20) to determine the TERC copy number. On the same FISH slides, cells stained with DAPI were also analyzed for senescence-associated heterochromatin foci (SAHF) status, including the number of cells with fragments and the number of SAHF fragments in each cell. A higher TERC gene copy number was observed in cells from PSC patients compared to colitis and control group patients. It was also higher in the colitis than in the control group. Significantly more cells in the senescent state and more fragmentation in each cell were observed in the PSC group compared to colitis and control groups. The TERC gene copy number and the number of cells in the senescent state were increased in PSC patients compared to the colitis and control groups. These findings are probably related to the genetic instability parameters that reflect the higher tendency of this patient group to develop malignancies. © 2013.

Frequent loss of lineages and deficient duplications accounted for low copy number of disease resistance genes in Cucurbitaceae

PubMed Central

2013-01-01

Background The sequenced genomes of cucumber, melon and watermelon have relatively few R-genes, with 70, 75 and 55 copies only, respectively. The mechanism for low copy number of R-genes in Cucurbitaceae genomes remains unknown. Results Manual annotation of R-genes in the sequenced genomes of Cucurbitaceae species showed that approximately half of them are pseudogenes. Comparative analysis of R-genes showed frequent loss of R-gene loci in different Cucurbitaceae species. Phylogenetic analysis, data mining and PCR cloning using degenerate primers indicated that Cucurbitaceae has limited number of R-gene lineages (subfamilies). Comparison between R-genes from Cucurbitaceae and those from poplar and soybean suggested frequent loss of R-gene lineages in Cucurbitaceae. Furthermore, the average number of R-genes per lineage in Cucurbitaceae species is approximately 1/3 that in soybean or poplar. Therefore, both loss of lineages and deficient duplications in extant lineages accounted for the low copy number of R-genes in Cucurbitaceae. No extensive chimeras of R-genes were found in any of the sequenced Cucurbitaceae genomes. Nevertheless, one lineage of R-genes from Trichosanthes kirilowii, a wild Cucurbitaceae species, exhibits chimeric structures caused by gene conversions, and may contain a large number of distinct R-genes in natural populations. Conclusions Cucurbitaceae species have limited number of R-gene lineages and each genome harbors relatively few R-genes. The scarcity of R-genes in Cucurbitaceae species was due to frequent loss of R-gene lineages and infrequent duplications in extant lineages. The evolutionary mechanisms for large variation of copy number of R-genes in different plant species were discussed. PMID:23682795

Phosphorylation of Ribosomal Protein RPS6 Integrates Light Signals and Circadian Clock Signals

DOE PAGES

Enganti, Ramya; Cho, Sung Ki; Toperzer, Jody D.; ...

2018-01-19

The translation of mRNA into protein is tightly regulated by the light environment as well as by the circadian clock. Although changes in translational efficiency have been well documented at the level of mRNA-ribosome loading, the underlying mechanisms are unclear. The reversible phosphorylation of RIBOSOMAL PROTEIN OF THE SMALL SUBUNIT 6 (RPS6) has been known for 40 years, but the biochemical significance of this event remains unclear to this day. Here, we confirm using a clock-deficient strain of Arabidopsis thaliana that RPS6 phosphorylation (RPS6-P) is controlled by the diel light-dark cycle with a peak during the day. Strikingly, when wild-type,more » clock-enabled, seedlings that have been entrained to a light-dark cycle are placed under free-running conditions, the circadian clock drives a cycle of RPS6-P with an opposite phase, peaking during the subjective night. We show that in wild-type seedlings under a light-dark cycle, the incoherent light and clock signals are integrated by the plant to cause an oscillation in RPS6-P with a reduced amplitude with a peak during the day. Sucrose can stimulate RPS6-P, as seen when sucrose in the medium masks the light response of etiolated seedlings. However, the diel cycles of RPS6-P are observed in the presence of 1% sucrose and in its absence. Sucrose at a high concentration of 3% appears to interfere with the robust integration of light and clock signals at the level of RPS6-P. Finally, we addressed whether RPS6-P occurs uniformly in polysomes, non-polysomal ribosomes and their subunits, and non-ribosomal protein. It is the polysomal RPS6 whose phosphorylation is most highly stimulated by light and repressed by darkness. These data exemplify a striking case of contrasting biochemical regulation between clock signals and light signals. Although the physiological significance of RPS6-P remains unknown, our data provide a mechanistic basis for the future understanding of this enigmatic event.« less

Phosphorylation of Ribosomal Protein RPS6 Integrates Light Signals and Circadian Clock Signals

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

Enganti, Ramya; Cho, Sung Ki; Toperzer, Jody D.

The translation of mRNA into protein is tightly regulated by the light environment as well as by the circadian clock. Although changes in translational efficiency have been well documented at the level of mRNA-ribosome loading, the underlying mechanisms are unclear. The reversible phosphorylation of RIBOSOMAL PROTEIN OF THE SMALL SUBUNIT 6 (RPS6) has been known for 40 years, but the biochemical significance of this event remains unclear to this day. Here, we confirm using a clock-deficient strain of Arabidopsis thaliana that RPS6 phosphorylation (RPS6-P) is controlled by the diel light-dark cycle with a peak during the day. Strikingly, when wild-type,more » clock-enabled, seedlings that have been entrained to a light-dark cycle are placed under free-running conditions, the circadian clock drives a cycle of RPS6-P with an opposite phase, peaking during the subjective night. We show that in wild-type seedlings under a light-dark cycle, the incoherent light and clock signals are integrated by the plant to cause an oscillation in RPS6-P with a reduced amplitude with a peak during the day. Sucrose can stimulate RPS6-P, as seen when sucrose in the medium masks the light response of etiolated seedlings. However, the diel cycles of RPS6-P are observed in the presence of 1% sucrose and in its absence. Sucrose at a high concentration of 3% appears to interfere with the robust integration of light and clock signals at the level of RPS6-P. Finally, we addressed whether RPS6-P occurs uniformly in polysomes, non-polysomal ribosomes and their subunits, and non-ribosomal protein. It is the polysomal RPS6 whose phosphorylation is most highly stimulated by light and repressed by darkness. These data exemplify a striking case of contrasting biochemical regulation between clock signals and light signals. Although the physiological significance of RPS6-P remains unknown, our data provide a mechanistic basis for the future understanding of this enigmatic event.« less

Copy number variation in the region harboring SOX9 gene in dogs with testicular/ovotesticular disorder of sex development (78,XX; SRY-negative).

PubMed

Marcinkowska-Swojak, Malgorzata; Szczerbal, Izabela; Pausch, Hubert; Nowacka-Woszuk, Joanna; Flisikowski, Krzysztof; Dzimira, Stanislaw; Nizanski, Wojciech; Payan-Carreira, Rita; Fries, Ruedi; Kozlowski, Piotr; Switonski, Marek

2015-10-01

Although the disorder of sex development in dogs with female karyotype (XX DSD) is quite common, its molecular basis is still unclear. Among mutations underlying XX DSD in mammals are duplication of a long sequence upstream of the SOX9 gene (RevSex) and duplication of the SOX9 gene (also observed in dogs). We performed a comparative analysis of 16 XX DSD and 30 control female dogs, using FISH and MLPA approaches. Our study was focused on a region harboring SOX9 and a region orthologous to the human RevSex (CanRevSex), which was located by in silico analysis downstream of SOX9. Two highly polymorphic copy number variable regions (CNVRs): CNVR1 upstream of SOX9 and CNVR2 encompassing CanRevSex were identified. Although none of the detected copy number variants were specific to either affected or control animals, we observed that the average number of copies in CNVR1 was higher in XX DSD. No copy variation of SOX9 was observed. Our extensive studies have excluded duplication of SOX9 as the common cause of XX DSD in analyzed samples. However, it remains possible that the causative mutation is hidden in highly polymorphic CNVR1.

Copy number variation in the region harboring SOX9 gene in dogs with testicular/ovotesticular disorder of sex development (78,XX; SRY-negative)

PubMed Central

Marcinkowska-Swojak, Malgorzata; Szczerbal, Izabela; Pausch, Hubert; Nowacka-Woszuk, Joanna; Flisikowski, Krzysztof; Dzimira, Stanislaw; Nizanski, Wojciech; Payan-Carreira, Rita; Fries, Ruedi; Kozlowski, Piotr; Switonski, Marek

2015-01-01

Although the disorder of sex development in dogs with female karyotype (XX DSD) is quite common, its molecular basis is still unclear. Among mutations underlying XX DSD in mammals are duplication of a long sequence upstream of the SOX9 gene (RevSex) and duplication of the SOX9 gene (also observed in dogs). We performed a comparative analysis of 16 XX DSD and 30 control female dogs, using FISH and MLPA approaches. Our study was focused on a region harboring SOX9 and a region orthologous to the human RevSex (CanRevSex), which was located by in silico analysis downstream of SOX9. Two highly polymorphic copy number variable regions (CNVRs): CNVR1 upstream of SOX9 and CNVR2 encompassing CanRevSex were identified. Although none of the detected copy number variants were specific to either affected or control animals, we observed that the average number of copies in CNVR1 was higher in XX DSD. No copy variation of SOX9 was observed. Our extensive studies have excluded duplication of SOX9 as the common cause of XX DSD in analyzed samples. However, it remains possible that the causative mutation is hidden in highly polymorphic CNVR1. PMID:26423656

Copy number variation and microdeletions of the Y chromosome linked genes and loci across different categories of Indian infertile males.

PubMed

Kumari, Anju; Yadav, Sandeep Kumar; Misro, Man Mohan; Ahmad, Jamal; Ali, Sher

2015-12-07

We analyzed 34 azoospermic (AZ), 43 oligospermic (OS), and 40 infertile males with normal spermiogram (INS) together with 55 normal fertile males (NFM) from the Indian population. AZ showed more microdeletions in the AZFa and AZFb regions whereas oligospermic ones showed more microdeletions in the AZFc region. Frequency of the AZF partial deletions was higher in males with spermatogenic impairments than in INS. Significantly, SRY, DAZ and BPY2 genes showed copy number variation across different categories of the patients and much reduced copies of the DYZ1 repeat arrays compared to that in normal fertile males. Likewise, INS showed microdeletions, sequence and copy number variation of several Y linked genes and loci. In the context of infertility, STS deletions and copy number variations both were statistically significant (p = 0.001). Thus, semen samples used during in vitro fertilization (IVF) and assisted reproductive technology (ART) must be assessed for the microdeletions of AZFa, b and c regions in addition to the affected genes reported herein. Present study is envisaged to be useful for DNA based diagnosis of different categories of the infertile males lending support to genetic counseling to the couples aspiring to avail assisted reproductive technologies.

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

PubMed Central

Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F.

2016-01-01

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion. PMID:27031233

Heritable heading time variation in wheat lines with the same number of Ppd-B1 gene copies.

PubMed

Ivaničová, Zuzana; Valárik, Miroslav; Pánková, Kateřina; Trávníčková, Martina; Doležel, Jaroslav; Šafář, Jan; Milec, Zbyněk

2017-01-01

The ability of plants to identify an optimal flowering time is critical for ensuring the production of viable seeds. The main environmental factors that influence the flowering time include the ambient temperature and day length. In wheat, the ability to assess the day length is controlled by photoperiod (Ppd) genes. Due to its allohexaploid nature, bread wheat carries the following three Ppd-1 genes: Ppd-A1, Ppd-B1 and Ppd-D1. While photoperiod (in)sensitivity controlled by Ppd-A1 and Ppd-D1 is mainly determined by sequence changes in the promoter region, the impact of the Ppd-B1 alleles on the heading time has been linked to changes in the copy numbers (and possibly their methylation status) and sequence changes in the promoter region. Here, we report that plants with the same number of Ppd-B1 copies may have different heading times. Differences were observed among F7 lines derived from crossing two spring hexaploid wheat varieties. Several lines carrying three copies of Ppd-B1 headed 16 days later than other plants in the population with the same number of gene copies. This effect was associated with changes in the gene expression level and methylation of the Ppd-B1 gene.

Heritable heading time variation in wheat lines with the same number of Ppd-B1 gene copies

PubMed Central

Ivaničová, Zuzana; Valárik, Miroslav; Pánková, Kateřina; Trávníčková, Martina; Doležel, Jaroslav; Šafář, Jan

2017-01-01

The ability of plants to identify an optimal flowering time is critical for ensuring the production of viable seeds. The main environmental factors that influence the flowering time include the ambient temperature and day length. In wheat, the ability to assess the day length is controlled by photoperiod (Ppd) genes. Due to its allohexaploid nature, bread wheat carries the following three Ppd-1 genes: Ppd-A1, Ppd-B1 and Ppd-D1. While photoperiod (in)sensitivity controlled by Ppd-A1 and Ppd-D1 is mainly determined by sequence changes in the promoter region, the impact of the Ppd-B1 alleles on the heading time has been linked to changes in the copy numbers (and possibly their methylation status) and sequence changes in the promoter region. Here, we report that plants with the same number of Ppd-B1 copies may have different heading times. Differences were observed among F7 lines derived from crossing two spring hexaploid wheat varieties. Several lines carrying three copies of Ppd-B1 headed 16 days later than other plants in the population with the same number of gene copies. This effect was associated with changes in the gene expression level and methylation of the Ppd-B1 gene. PMID:28846721

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells

PubMed Central

Yen-Ting-Liu; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

2014-01-01

Summary The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications. PMID:25541598

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells.

PubMed

Yen-Ting-Liu; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

2014-10-01

The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications.

MixHMM: Inferring Copy Number Variation and Allelic Imbalance Using SNP Arrays and Tumor Samples Mixed with Stromal Cells

PubMed Central

Schulz, Vincent; Chen, Min; Tuck, David

2010-01-01

Background Genotyping platforms such as single nucleotide polymorphism (SNP) arrays are powerful tools to study genomic aberrations in cancer samples. Allele specific information from SNP arrays provides valuable information for interpreting copy number variation (CNV) and allelic imbalance including loss-of-heterozygosity (LOH) beyond that obtained from the total DNA signal available from array comparative genomic hybridization (aCGH) platforms. Several algorithms based on hidden Markov models (HMMs) have been designed to detect copy number changes and copy-neutral LOH making use of the allele information on SNP arrays. However heterogeneity in clinical samples, due to stromal contamination and somatic alterations, complicates analysis and interpretation of these data. Methods We have developed MixHMM, a novel hidden Markov model using hidden states based on chromosomal structural aberrations. MixHMM allows CNV detection for copy numbers up to 7 and allows more complete and accurate description of other forms of allelic imbalance, such as increased copy number LOH or imbalanced amplifications. MixHMM also incorporates a novel sample mixing model that allows detection of tumor CNV events in heterogeneous tumor samples, where cancer cells are mixed with a proportion of stromal cells. Conclusions We validate MixHMM and demonstrate its advantages with simulated samples, clinical tumor samples and a dilution series of mixed samples. We have shown that the CNVs of cancer cells in a tumor sample contaminated with up to 80% of stromal cells can be detected accurately using Illumina BeadChip and MixHMM. Availability The MixHMM is available as a Python package provided with some other useful tools at http://genecube.med.yale.edu:8080/MixHMM. PMID:20532221

Low α-defensin gene copy number increases the risk for IgA nephropathy and renal dysfunction.

PubMed

Ai, Zhen; Li, Ming; Liu, Wenting; Foo, Jia-Nee; Mansouri, Omniah; Yin, Peiran; Zhou, Qian; Tang, Xueqing; Dong, Xiuqing; Feng, Shaozhen; Xu, Ricong; Zhong, Zhong; Chen, Jian; Wan, Jianxin; Lou, Tanqi; Yu, Jianwen; Zhou, Qin; Fan, Jinjin; Mao, Haiping; Gale, Daniel; Barratt, Jonathan; Armour, John A L; Liu, Jianjun; Yu, Xueqing

2016-06-29

Although a major source of genetic variation, copy number variations (CNVs) and their involvement in disease development have not been well studied. Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. We performed association analysis of the DEFA1A3 CNV locus in two independent IgAN cohorts of southern Chinese Han (total of 1189 cases and 1187 controls). We discovered three independent copy number associations within the locus: DEFA1A3 [P = 3.99 × 10(-9); odds ratio (OR), 0.88], DEFA3 (P = 6.55 × 10(-5); OR, 0.82), and a noncoding deletion variant (211bp) (P = 3.50 × 10(-16); OR, 0.75) (OR per copy, fixed-effects meta-analysis). While showing strong association with an increased risk for IgAN (P = 9.56 × 10(-20)), low total copy numbers of the three variants also showed significant association with renal dysfunction in patients with IgAN (P = 0.03; hazards ratio, 3.69; after controlling for the effects of known prognostic factors) and also with increased serum IgA1 (P = 0.02) and galactose-deficient IgA1 (P = 0.03). For replication, we confirmed the associations of DEFA1A3 (P = 4.42 × 10(-4); OR, 0.82) and DEFA3 copy numbers (P = 4.30 × 10(-3); OR, 0.74) with IgAN in a Caucasian cohort (531 cases and 198 controls) and found the 211bp variant to be much rarer in Caucasians. We also observed an association of the 211bp copy number with membranous nephropathy (P = 1.11 × 10(-7); OR, 0.74; in 493 Chinese cases and 500 matched controls), but not with diabetic kidney disease (in 806 Chinese cases and 786 matched controls). By explaining 4.96% of disease risk and influencing renal dysfunction in patients with IgAN, the DEFA1A3 CNV locus may be a potential therapeutic target for developing treatments for this disease. Copyright © 2016, American Association for the Advancement of Science.

Copy-number variations associated with autism spectrum disorder.

PubMed

Kakinuma, Hiroaki; Sato, Hitoshi

2008-08-01

Autism spectrum disorder (ASD) is a clinically heterogeneous developmental disorder with a strong genetic component. Rare genetic disorders and various chromosomal abnormalities are thought to account for approximately 10% of people with ASD. The etiology of the remaining cases remains unknown. Recent advances in array-based technology have increased the resolution in detecting submicroscopic deletions and duplications, referred to as copy-number variations. ASD-associated copy-number variations, which are considered to be present in individuals with ASD but not in unaffected individuals, have been extensively investigated. These data will provide us with an opportunity not only to search for genes causing or contributing to ASDs but also to understand the genetics of ASD.

Genomic regions showing copy number variations associate with resistance or susceptibility to gastrointestinal nematodes in Angus cattle

USDA-ARS?s Scientific Manuscript database

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We previously reported an initial analysis of copy number variations (CNVs) in Angus cattle selected for resistance or susceptibility to gastrointestinal nematodes. In this study, we performed a lar...

Genomic regions showing copy number variations associate with resistance or susceptibility to gastrointestinal nematodes in Angus Cattle

USDA-ARS?s Scientific Manuscript database

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We previously reported an initial analysis of copy number variations (CNVs) in Angus cattle selected for resistance or susceptibility to intestinal nematodes. In this study, we performed a large sca...

Comparative analyses across cattle breeds reveal the pitfalls caused by artificial and lineage-differential copy number variations

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNV) are well known genomic variants, which often complicate structural and functional genomics studies. Here, we integrated the CNV region (CNVR) result detected from 1,682 Nellore cattle with the equivalent result derived from the Bovine HapMap samples. Through comparing CN...

29 CFR 501.39 - Service upon attorneys for the Department of Labor-number of copies.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 3 2010-07-01 2010-07-01 false Service upon attorneys for the Department of Labor-number of copies. 501.39 Section 501.39 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS ENFORCEMENT OF CONTRACTUAL OBLIGATIONS FOR TEMPORARY ALIEN...

29 CFR 502.39 - Service upon attorneys for the Department of Labor-number of copies.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 3 2010-07-01 2010-07-01 false Service upon attorneys for the Department of Labor-number of copies. 502.39 Section 502.39 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS ENFORCEMENT OF CONTRACTUAL OBLIGATIONS FOR TEMPORARY ALIEN...

Subtelomeric Rearrangements and Copy Number Variations in People with Intellectual Disabilities

ERIC Educational Resources Information Center

Christofolini, D. M.; De Paula Ramos, M. A.; Kulikowski, L. D.; Da Silva Bellucco, F. T.; Belangero, S. I. N.; Brunoni, D.; Melaragno, M. I.

2010-01-01

Background: The most prevalent type of structural variation in the human genome is represented by copy number variations that can affect transcription levels, sequence, structure and function of genes. Method: In the present study, we used the multiplex ligation-dependent probe amplification (MLPA) technique and quantitative PCR for the detection…

77 FR 1743 - Facility Operating License Amendment From Florida Power Corporation, Crystal River Nuclear...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-11

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Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-15

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78 FR 31978 - Biweekly Notice; Applications and Amendments to Facility Operating Licenses and Combined Licenses...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-28

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Genome-wide copy number variant analysis reveals variants associated with 10 diverse production traits in Holstein cattle

USDA-ARS?s Scientific Manuscript database

Copy number variation (CNV) is an important type of genetic variation contributing to phenotypic differences among mammals and may serve as an alternative molecular marker to single nucleotide polymorphism (SNP) for genome-wide association study (GWAS). Recently, GWAS analysis using CNV has been app...

Pervasive gene content variation and copy number variation in maize and its undomesticated progenitor

USDA-ARS?s Scientific Manuscript database

Different individuals of the same species are generally thought to have very similar genomes. However, there is growing evidence that structural variation in the form of copy number variation (CNV) and presence-absence variation (PAV) can lead to variation in the genome content of individuals withi...

Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds

USDA-ARS?s Scientific Manuscript database

Genomic structural variations are an important source of genetic diversity. Copy number variations (CNVs), gains and losses of large regions of genomic sequence between individuals of a species, are known to be associated with both diseases and phenotypic traits. Deeply sequenced genomes are often u...

Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

PubMed Central

Pimentel, Belén; Madine, Mark A; de la Cueva-Méndez, Guillermo

2005-01-01

Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plasmid-containing cells when plasmid copy number decreases, cleaving not only host- but also a specific plasmid-encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection. PMID:16163387

Direct and efficient xylitol production from xylan by Saccharomyces cerevisiae through transcriptional level and fermentation processing optimizations.

PubMed

Li, Zhe; Qu, Hongnan; Li, Chun; Zhou, Xiaohong

2013-12-01

In this study, four engineered Saccharomyces cerevisiae carrying xylanase, β-xylosidase and xylose reductase genes by different transcriptional regulations were constructed to directly convert xylan to xylitol. According to the results, the high-copy number plasmid required a rigid selection for promoter characteristics, on the contrast, the selection of promoters could be more flexible for low-copy number plasmid. For cell growth and xylitol production, glucose and galactose were found more efficient than other sugars. The semi-aerobic condition and feeding of co-substrates were taken to improve the yield of xylitol. It was found that the strain containing high-copy number plasmid had the highest xylitol yield, but it was sensitive to the change of fermentation. However, the strain carrying low-copy number plasmid was more adaptable to different processes. By optimization of the transcriptional regulation and fermentation processes, the xylitol concentration could be increased of 1.7 folds and the yield was 0.71 g xylitol/g xylan. Copyright © 2013 Elsevier Ltd. All rights reserved.

Determination of absolute expression profiles using multiplexed miRNA analysis

PubMed Central

Song, Jee Hoon; Cheng, Yulan; Saeui, Christopher T.; Cheung, Douglas G.; Croce, Carlo M.; Yarema, Kevin J.; Meltzer, Stephen J.; Liu, Kelvin J.; Wang, Tza-Huei

2017-01-01

Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR’s ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes. PMID:28704432

Molecular Surveillance as Monitoring Tool for Drug-Resistant Plasmodium falciparum in Suriname

PubMed Central

Adhin, Malti R.; Labadie-Bracho, Mergiory; Bretas, Gustavo

2013-01-01

The aim of this translational study was to show the use of molecular surveillance for polymorphisms and copy number as a monitoring tool to track the emergence and dynamics of Plasmodium falciparum drug resistance. A molecular baseline for Suriname was established in 2005, with P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance (pfmdr1) markers and copy number in 40 samples. The baseline results revealed the existence of a uniformly distributed mutated genotype corresponding with the fully mefloquine-sensitive 7G8-like genotype (Y184F, S1034C, N1042D, and D1246Y) and a fixed pfmdr1 N86 haplotype. All samples harbored the pivotal pfcrtK76T mutation, showing that chloroquine reintroduction should not yet be contemplated in Suriname. After 5 years, 40 samples were assessed to trace temporal changes in the status of pfmdr1 polymorphisms and copy number and showed minor genetic alterations in the pfmdr1 gene and no significant changes in copy number, thus providing scientific support for prolongation of the current drug policy in Suriname. PMID:23836573

Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

PubMed Central

Maggert, Keith A.

2014-01-01

The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments. PMID:24449686

Serum decoy receptor 3 level: a predictive marker for nodal metastasis and survival among oral cavity cancer patients.

PubMed

Tu, Hsi-Feng; Liu, Chung-Ji; Liu, Shyun-Yeu; Chen, Yu-Ping; Yu, En-Hao; Lin, Shu-Chun; Chang, Kuo-Wei

2011-03-01

Validating markers for prediction of nodal metastasis could be beneficial in treatment of oral cavity cancer. Decoy receptor 3 (DcR3), locus on 20q13, functions as a death decoy inhibiting apoptosis mediated by the tumor necrosis factor receptor (TNFR) family. This study analyzed the serum level of DcR3 in relationship to the clinical parameters of oral cavity cancer patients together with detection of DcR3 genomic copy number in primary and recurrent tumors. Elevated serum DcR3 was associated with nodal metastasis and worse prognosis. Gain of DcR3 copy number was detected in 17% of primary tumor tissue but not found in healthy areca chewers. Tissue from recurrent tumors showed more frequent DcR3 copy number alteration (48%) than the paired primary tumor tissue. Serum DcR3 level is a predictor for the nodal metastasis and survival among oral cavity cancer patients and the DcR3 copy number alteration could underlie oral carcinogenesis progression. Copyright © 2010 Wiley Periodicals, Inc.

Change in the plasmid copy number in acetic acid bacteria in response to growth phase and acetic acid concentration.

PubMed

Akasaka, Naoki; Astuti, Wiwik; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke

2015-06-01

Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1-3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Genes commonly deleted in childhood B-cell precursor acute lymphoblastic leukemia: association with cytogenetics and clinical features

PubMed Central

Schwab, Claire J.; Chilton, Lucy; Morrison, Heather; Jones, Lisa; Al-Shehhi, Halima; Erhorn, Amy; Russell, Lisa J.; Moorman, Anthony V.; Harrison, Christine J.

2013-01-01

In childhood B-cell precursor acute lymphoblastic leukemia, cytogenetics is important in diagnosis and as an indicator of response to therapy, thus playing a key role in risk stratification of patients for treatment. Little is known of the relationship between different cytogenetic subtypes in B-cell precursor acute lymphoblastic leukemia and the recently reported copy number abnormalities affecting significant leukemia associated genes. In a consecutive series of 1427 childhood B-cell precursor acute lymphoblastic leukemia patients, we have determined the incidence and type of copy number abnormalities using multiplex ligation-dependent probe amplification. We have shown strong links between certain deletions and cytogenetic subtypes, including the novel association between RB1 deletions and intrachromosomal amplification of chromosome 21. In this study, we characterized the different copy number abnormalities and show heterogeneity of PAX5 and IKZF1 deletions and the recurrent nature of RB1 deletions. Whole gene losses are often indicative of larger deletions, visible by conventional cytogenetics. An increased number of copy number abnormalities is associated with NCI high risk, specifically deletions of IKZF1 and CDKN2A/B, which occur more frequently among these patients. IKZF1 deletions and rearrangements of CRLF2 among patients with undefined karyotypes may point to the poor risk BCR-ABL1-like group. In conclusion, this study has demonstrated in a large representative cohort of children with B-cell precursor acute lymphoblastic leukemia that the pattern of copy number abnormalities is highly variable according to the primary genetic abnormality. PMID:23508010

Lepton number violation in theories with a large number of standard model copies

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

Kovalenko, Sergey; Schmidt, Ivan; Paes, Heinrich

2011-03-01

We examine lepton number violation (LNV) in theories with a saturated black hole bound on a large number of species. Such theories have been advocated recently as a possible solution to the hierarchy problem and an explanation of the smallness of neutrino masses. On the other hand, the violation of the lepton number can be a potential phenomenological problem of this N-copy extension of the standard model as due to the low quantum gravity scale black holes may induce TeV scale LNV operators generating unacceptably large rates of LNV processes. We show, however, that this issue can be avoided bymore » introducing a spontaneously broken U{sub 1(B-L)}. Then, due to the existence of a specific compensation mechanism between contributions of different Majorana neutrino states, LNV processes in the standard model copy become extremely suppressed with rates far beyond experimental reach.« less

Quantification of hookworm ova from wastewater matrices using quantitative PCR.

PubMed

Gyawali, Pradip; Ahmed, Warish; Sidhu, Jatinder P; Jagals, Paul; Toze, Simon

2017-07-01

A quantitative PCR (qPCR) assay was used to quantify Ancylostoma caninum ova in wastewater and sludge samples. We estimated the average gene copy numbers for a single ovum using a mixed population of ova. The average gene copy numbers derived from the mixed population were used to estimate numbers of hookworm ova in A. caninum seeded and unseeded wastewater and sludge samples. The newly developed qPCR assay estimated an average of 3.7×10 3 gene copies per ovum, which was then validated by seeding known numbers of hookworm ova into treated wastewater. The qPCR estimated an average of (1.1±0.1), (8.6±2.9) and (67.3±10.4) ova for treated wastewater that was seeded with (1±0), (10±2) and (100±21) ova, respectively. The further application of the qPCR assay for the quantification of A. caninum ova was determined by seeding a known numbers of ova into the wastewater matrices. The qPCR results indicated that 50%, 90% and 67% of treated wastewater (1L), raw wastewater (1L) and sludge (~4g) samples had variable numbers of A. caninum gene copies. After conversion of the qPCR estimated gene copy numbers to ova for treated wastewater, raw wastewater, and sludge samples, had an average of 0.02, 1.24 and 67 ova, respectively. The result of this study indicated that qPCR can be used for the quantification of hookworm ova from wastewater and sludge samples; however, caution is advised in interpreting qPCR generated data for health risk assessment. Copyright © 2017. Published by Elsevier B.V.

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 implemented using C++, together with R scripts for data formatting and Perl scripts for user interfacing, and it is easy to install and efficient to use. The source code and documentation are freely available at http://www.cbil.ece.vt.edu/software.htm.

GAB2 amplifications refine molecular classification of melanoma.

PubMed

Chernoff, Karen A; Bordone, Lindsey; Horst, Basil; Simon, Katherine; Twadell, William; Lee, Keagan; Cohen, Jason A; Wang, Shuang; Silvers, David N; Brunner, Georg; Celebi, Julide Tok

2009-07-01

Gain-of-function mutations in BRAF, NRAS, or KIT are associated with distinct melanoma subtypes with KIT mutations and/or copy number changes frequently observed among melanomas arising from sun-protected sites, such as acral skin (palms, soles, and nail bed) and mucous membranes. GAB2 has recently been implicated in melanoma pathogenesis, and increased copy numbers are found in a subset of melanomas. We sought to determine the association of increased copy numbers of GAB2 among melanoma subtypes in the context of genetic alterations in BRAF, NRAS, and KIT. A total of 85 melanomas arising from sun-protected (n = 23) and sun-exposed sites (n = 62) were analyzed for copy number changes using array-based comparative genomic hybridization and for gain-of-function mutations in BRAF, NRAS, and KIT. GAB2 amplifications were found in 9% of the cases and were associated with melanomas arising from acral and mucosal sites (P = 0.005). Increased copy numbers of the KIT locus were observed in 6% of the cases. The overall mutation frequencies for BRAF and NRAS were 43.5% and 14%, respectively, and were mutually exclusive. Among the acral and mucosal melanomas studied, the genetic alteration frequency was 26% for GAB2, 13% for KIT, 30% for BRAF, and 4% for NRAS. Importantly, the majority of GAB2 amplifications occurred independent from genetic events in BRAF, NRAS, and KIT. GAB2 amplification is critical for melanomas arising from sun-protected sites. Genetic alterations in GAB2 will help refine the molecular classification of melanomas.

Differentially expressed microRNAs in lung adenocarcinoma invert effects of copy number aberrations of prognostic genes

PubMed Central

Tokar, Tomas; Pastrello, Chiara; Ramnarine, Varune R.; Zhu, Chang-Qi; Craddock, Kenneth J.; Pikor, Larrisa A.; Vucic, Emily A.; Vary, Simon; Shepherd, Frances A.; Tsao, Ming-Sound; Lam, Wan L.; Jurisica, Igor

2018-01-01

In many cancers, significantly down- or upregulated genes are found within chromosomal regions with DNA copy number alteration opposite to the expression changes. Generally, this paradox has been overlooked as noise, but can potentially be a consequence of interference of epigenetic regulatory mechanisms, including microRNA-mediated control of mRNA levels. To explore potential associations between microRNAs and paradoxes in non-small-cell lung cancer (NSCLC) we curated and analyzed lung adenocarcinoma (LUAD) data, comprising gene expressions, copy number aberrations (CNAs) and microRNA expressions. We integrated data from 1,062 tumor samples and 241 normal lung samples, including newly-generated array comparative genomic hybridization (aCGH) data from 63 LUAD samples. We identified 85 “paradoxical” genes whose differential expression consistently contrasted with aberrations of their copy numbers. Paradoxical status of 70 out of 85 genes was validated on sample-wise basis using The Cancer Genome Atlas (TCGA) LUAD data. Of these, 41 genes are prognostic and form a clinically relevant signature, which we validated on three independent datasets. By meta-analysis of results from 9 LUAD microRNA expression studies we identified 24 consistently-deregulated microRNAs. Using TCGA-LUAD data we showed that deregulation of 19 of these microRNAs explains differential expression of the paradoxical genes. Our results show that deregulation of paradoxical genes is crucial in LUAD and their expression pattern is maintained epigenetically, defying gene copy number status. PMID:29507679

The interdependencies of viral load, the innate immune response, and clinical outcome in children presenting to the emergency department with respiratory syncytial virus-associated bronchiolitis

PubMed Central

Mei, Minghua; Mehta, Reena

2017-01-01

Respiratory syncytial virus (RSV) causes significant infant morbidity and mortality. For decades severe RSV-induced disease was thought to result from an uncontrolled host response to viral replication, but recent work suggests that a strong innate immune response early in infection is protective. To shed light on host-virus interactions and the viral determinants of disease, copy numbers of five RSV genes (NS1, NS2, N, G, F) were measured by quantitative real-time polymerase chain reaction (qPCR) in nasal wash samples from children with RSV-associated bronchiolitis. Correlations were sought with host cytokines/chemokines and biomarkers. Associations with disposition from the emergency department (hospitalized or sent home) and pulse oximetry O2 saturation levels were also sought. Additionally, RNase P copy number was measured and used to normalize nasal wash data. RSV gene copy numbers were found to significantly correlate with both cytokine/chemokine and biomarker levels; and RNase P-normalized viral gene copy numbers (NS1, NS2, N and G) were significantly higher in infants with less severe disease. Moreover, three of the normalized viral gene copy numbers (NS1, NS2, and N) correlated significantly with arterial O2 saturation levels. The data support a model where a higher viral load early in infection can promote a robust innate immune response that protects against progression into hypoxic RSV-induced lower respiratory tract illness. PMID:28267794

The interdependencies of viral load, the innate immune response, and clinical outcome in children presenting to the emergency department with respiratory syncytial virus-associated bronchiolitis.

PubMed

Piedra, Felipe-Andrés; Mei, Minghua; Avadhanula, Vasanthi; Mehta, Reena; Aideyan, Letisha; Garofalo, Roberto P; Piedra, Pedro A

2017-01-01

Respiratory syncytial virus (RSV) causes significant infant morbidity and mortality. For decades severe RSV-induced disease was thought to result from an uncontrolled host response to viral replication, but recent work suggests that a strong innate immune response early in infection is protective. To shed light on host-virus interactions and the viral determinants of disease, copy numbers of five RSV genes (NS1, NS2, N, G, F) were measured by quantitative real-time polymerase chain reaction (qPCR) in nasal wash samples from children with RSV-associated bronchiolitis. Correlations were sought with host cytokines/chemokines and biomarkers. Associations with disposition from the emergency department (hospitalized or sent home) and pulse oximetry O2 saturation levels were also sought. Additionally, RNase P copy number was measured and used to normalize nasal wash data. RSV gene copy numbers were found to significantly correlate with both cytokine/chemokine and biomarker levels; and RNase P-normalized viral gene copy numbers (NS1, NS2, N and G) were significantly higher in infants with less severe disease. Moreover, three of the normalized viral gene copy numbers (NS1, NS2, and N) correlated significantly with arterial O2 saturation levels. The data support a model where a higher viral load early in infection can promote a robust innate immune response that protects against progression into hypoxic RSV-induced lower respiratory tract illness.

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication.

PubMed

Hori, Akiko; Yoshida, Minoru; Shibata, Takehiko; Ling, Feng

2009-02-01

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho(-)] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho(-)] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.

Copy number of ArsR reporter plasmid determines its arsenite response and metal specificity.

PubMed

Fang, Yun; Zhu, Chunjie; Chen, Xingjuan; Wang, Yan; Xu, Meiying; Sun, Guoping; Guo, Jun; Yoo, Jinnon; Tie, Cuijuan; Jiang, Xin; Li, Xianqiang

2018-05-16

The key component in bacteria-based biosensors is a transcriptional reporter employed to monitor induction or repression of a reporter gene corresponding to environmental change. In this study, we made a series of reporters in order to achieve highly sensitive detection of arsenite. From these reporters, two biosensors were developed by transformation of Escherichia coli DH5α with pLHPars9 and pLLPars9, consisting of either a high or low copy number plasmid, along with common elements of ArsR-luciferase fusion and addition of two binding sequences, one each from E. coli and Acidithiobacillus ferrooxidans chromosome, in front of the R773 ArsR operon. Both of them were highly sensitive to arsenite, with a low detection limit of 0.04 μM arsenite (~ 5 μg/L). They showed a wide dynamic range of detection up to 50 μM using high copy number pLHPars9 and 100 μM using low copy number pLLPars9. Significantly, they differ in metal specificity, pLLPars9 more specific to arsenite, while pLHPars9 to both arsenite and antimonite. The only difference between pLHPars9 and pLLPars9 is their copy numbers of plasmid and corresponding ratios of ArsR to its binding promoter/operator sequence. Therefore, we propose a working model in which DNA bound-ArsR is different from its free form in metal specificity.

Genomic copy number gains of ErbB family members predict poor clinical outcomes in glioma patients

PubMed Central

Liu, Rui; Qu, Yiping; Chen, Lihong; Pu, Jun; Ma, Sharui; Zhang, Xiaozhi; Yang, Qi; Shi, Bingyin; Hou, Peng; Ji, Meiju

2017-01-01

The aim of this study was to investigate copy number of ErbB family members (including EGFR, HER2, HER3 and HER4) in a cohort of gliomas and benign meningiomas (control subjects), and explore the associations of their copy number with clinicopathological characteristics and clinical outcomes of glioma patients. Using real-time quantitative PCR assay, we demonstrated that copy number of EGFR, HER2, HER3 and HER4 in glioma patients was significantly increased compared to control subjects. Moreover, our data also showed that the risk of cancer-related death was positively associated with copy number gain (CNG) of EGFR, HER3 and HER4, but not HER2. CNG of EGFR and HER2 was positively related to radiotherapy, while CNG of HER3 and HER4 was negatively related to chemotherapy. Importantly, EGFR CNG significantly shortened median survival times of glioma patients regardless of gender, tumor grade and therapeutic regimens. Stratified analysis showed that CNG of HER2-4 almost did not influence the survival of male patients, patients with high-grade tumors and patients receiving chemotherapy, but dramatically shortened median survival times of female patients, those with low-grade tumors and those receiving radiotherapy. Collectively, our data not only demonstrate that the members of ErbB family are frequently amplified in gliomas, but also suggest that these common genetic events may be prognostic factors for poor clinical outcomes in glioma patients. PMID:29190914

Genetic and intermediate phenotypic susceptibility markers of gastric cancer in Hispanic Americans: a case-control study.

PubMed

Sun, Yuhui; Gu, Jian; Ajani, Jaffer A; Chang, David W; Wu, Xifeng; Stroehlein, John R

2014-10-01

Hispanics are the largest nonwhite ethnic group in the US population, and they have higher incidence and mortality rates for gastric cancer (GC) than whites and Asians. Studies have identified several genetic susceptibility loci and intermediate phenotypic biomarkers for GC in whites and Asians. No studies have evaluated genetic susceptibility and intermediate phenotypic biomarkers in Hispanics. In a case-control study of 132 Hispanic patients with GC (cases) and a control group of 125 Hispanics (controls), the authors evaluated the association of 5 single nucleotide polymorphisms (SNPs) that predispose whites and/or Asians to GC and of 2 intermediate phenotypic markers in peripheral blood leukocytes, ie, telomere length and mitochondrial DNA (mtDNA) copy number, with the GC risk. The variant C allele of the reference SNP rs2294008 in the PSCA gene was associated with a significantly reduced risk of GC (per allele-adjusted odds ratio [aOR], 0.51; 95% confidence interval [CI], 0.33-0.77; P = .002). Leukocyte mtDNA copy numbers were significantly lower in GC cases (mean ± standard deviation, 0.91 ± 0.28) than in controls (1.29 ± 0.42; P < .001). When individuals were dichotomized into high and low mtDNA copy number groups based on the median mtDNA copy number value in the controls, those who had a low mtDNA copy number had a significantly increased risk of GC (aOR, 11.00; 95% CI, 4.79-25.23; P < .001) compared with those who had a high mtDNA copy number. Telomere length was not associated significantly with the risk of GC (aOR, 1.21; 95% CI, 0.65-2.27; P = .551). Hispanics share certain genetic susceptibility loci and intermediate phenotypic GC biomarkers with whites and Asians and may also have distinct genetic susceptibility factors. © 2014 American Cancer Society.

A remark on copy number variation detection methods.

PubMed

Li, Shuo; Dou, Xialiang; Gao, Ruiqi; Ge, Xinzhou; Qian, Minping; Wan, Lin

2018-01-01

Copy number variations (CNVs) are gain and loss of DNA sequence of a genome. High throughput platforms such as microarrays and next generation sequencing technologies (NGS) have been applied for genome wide copy number losses. Although progress has been made in both approaches, the accuracy and consistency of CNV calling from the two platforms remain in dispute. In this study, we perform a deep analysis on copy number losses on 254 human DNA samples, which have both SNP microarray data and NGS data publicly available from Hapmap Project and 1000 Genomes Project respectively. We show that the copy number losses reported from Hapmap Project and 1000 Genome Project only have < 30% overlap, while these reports are required to have cross-platform (e.g. PCR, microarray and high-throughput sequencing) experimental supporting by their corresponding projects, even though state-of-art calling methods were employed. On the other hand, copy number losses are found directly from HapMap microarray data by an accurate algorithm, i.e. CNVhac, almost all of which have lower read mapping depth in NGS data; furthermore, 88% of which can be supported by the sequences with breakpoint in NGS data. Our results suggest the ability of microarray calling CNVs and the possible introduction of false negatives from the unessential requirement of the additional cross-platform supporting. The inconsistency of CNV reports from Hapmap Project and 1000 Genomes Project might result from the inadequate information containing in microarray data, the inconsistent detection criteria, or the filtration effect of cross-platform supporting. The statistical test on CNVs called from CNVhac show that the microarray data can offer reliable CNV reports, and majority of CNV candidates can be confirmed by raw sequences. Therefore, the CNV candidates given by a good caller could be highly reliable without cross-platform supporting, so additional experimental information should be applied in need instead of necessarily.

18 CFR 156.3 - Applications; number of copies; general requirements.

Code of Federal Regulations, 2010 CFR

2010-04-01

... copies; general requirements. 156.3 Section 156.3 Conservation of Power and Water Resources FEDERAL... requirements. (a) Applicable rules. An original and 7 conformed copies of an application under this part shall... other respects applications shall conform to the requirements of §§ 156.1 through 156.5. Amendments to...

17 CFR 230.472 - Filing of amendments; number of copies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... exhibits and all other papers and documents filed as part of the amendment, and eight additional copies of... include the consent of the certifying accountant to the use of his certificate in connection with the... Commission three complete, unmarked copies of every amendment, including exhibits and all other papers and...

17 CFR 230.472 - Filing of amendments; number of copies.

Code of Federal Regulations, 2014 CFR

2014-04-01

... exhibits and all other papers and documents filed as part of the amendment, and eight additional copies of... include the consent of the certifying accountant to the use of his certificate in connection with the... Commission three complete, unmarked copies of every amendment, including exhibits and all other papers and...

17 CFR 230.472 - Filing of amendments; number of copies.

Code of Federal Regulations, 2011 CFR

2011-04-01

... exhibits and all other papers and documents filed as part of the amendment, and eight additional copies of... include the consent of the certifying accountant to the use of his certificate in connection with the... Commission three complete, unmarked copies of every amendment, including exhibits and all other papers and...

17 CFR 230.472 - Filing of amendments; number of copies.

Code of Federal Regulations, 2013 CFR

2013-04-01

... exhibits and all other papers and documents filed as part of the amendment, and eight additional copies of... include the consent of the certifying accountant to the use of his certificate in connection with the... Commission three complete, unmarked copies of every amendment, including exhibits and all other papers and...

17 CFR 230.472 - Filing of amendments; number of copies.

Code of Federal Regulations, 2012 CFR

2012-04-01

... exhibits and all other papers and documents filed as part of the amendment, and eight additional copies of... include the consent of the certifying accountant to the use of his certificate in connection with the... Commission three complete, unmarked copies of every amendment, including exhibits and all other papers and...

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number

PubMed Central

Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

2015-01-01

Summary Background The borders of Thailand harbour the world’s most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. Methods The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Findings Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6·3 (95% CI 2·9–13·8, p<0·001) after mefloquine monotherapy and 5·4 (2·0-14·6, p=0·001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Interpretation Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Relevance to practice Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days’ artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients. PMID:15288742

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

PubMed

Price, Ric N; Uhlemann, Anne-Catrin; Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

Multiple-copy entanglement transformation and entanglement catalysis

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

Duan Runyao; Feng Yuan; Li Xin

2005-04-01

We prove that any multiple-copy entanglement transformation [S. Bandyopadhyay, V. Roychowdhury, and U. Sen, Phys. Rev. A 65, 052315 (2002)] can be implemented by a suitable entanglement-assisted local transformation [D. Jonathan and M. B. Plenio, Phys. Rev. Lett. 83, 3566 (1999)]. Furthermore, we show that the combination of multiple-copy entanglement transformation and the entanglement-assisted one is still equivalent to the pure entanglement-assisted one. The mathematical structure of multiple-copy entanglement transformations then is carefully investigated. Many interesting properties of multiple-copy entanglement transformations are presented, which exactly coincide with those satisfied by the entanglement-assisted ones. Most interestingly, we show that an arbitrarilymore » large number of copies of state should be considered in multiple-copy entanglement transformations.« less

Effects of a petunia scaffold/matrix attachment region on copy number dependency and stability of transgene expression in Nicotiana tabacum.

PubMed

Dietz-Pfeilstetter, Antje; Arndt, Nicola; Manske, Ulrike

2016-04-01

Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region.

EVIDENCE FOR REGULATION OF TYROSINE HYDROXLASE mRNA TRANSLATION BY STRESS IN RAT ADRENAL MEDULLA

PubMed Central

Xu, Lu; Chen, Xiqun; Sun, Baoyong; Sterling, Carol; Tank, A. William

2009-01-01

Long-term stress leads to induction of tyrosine hydroxylase (TH) protein and enzymatic activity in the adrenal medulla. This adaptive response is necessary to maintain the catecholamine biosynthetic capacity of adrenal chromaffin cells during periods of sustained catecholamine secretion. In this report we demonstrate that when rats are subjected to short-term stress, TH mRNA is induced for at least 24 hr, but TH protein and TH activity (assayed under Vmax conditions) are not increased. In contrast, adrenal TH mRNA, TH protein and TH activity are induced in rats subjected to long-term stress. Using sucrose gradient fractionation, we show that the lack of induction of TH protein after one type of short-term stressor, a single 2 hr immobilization stress is associated with a decrease in the percentage of TH mRNA molecules associated with polysomes. In contrast, after repeated immobilizations the polysome profile of TH mRNA is identical to that observed in control animals, even though TH mRNA is induced 2–3 fold. These results are consistent with the hypothesis that even though TH mRNA is induced by short-term stressors, mechanisms that control TH mRNA translation must also be appropriately regulated for TH protein to be induced. PMID:17543899

Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.

PubMed Central

Mullinix, K P; Wetekam, W; Deeley, R G; Gordon, J I; Meyers, M; Kent, K A; Goldberger, R F

1976-01-01

We have investigated the estrogen-mediated induction of vitellogenin synthesis in rooster liver. We compared the concentrations of vitellogenin messenger RNA (mRNA) in the liver with the concentrations of vitellogenin in the sera of roosters that had recieved various treatments with estrogen. We found no vitellogenin mRNA in the livers of the unstimulated roosters. An initial injection of estrogen was attended by de novo synthesis of vitellogenin mRNA in the liver and accumulation of vitellogenin in the serum. When vitellogenin was no longer present in the serum or liver (the "post-estrogen-serum-negative" state), the liver was found to contain appreciable amounts of vitellogenin mRNA. This mRNA was of the same size as that found in the liver of the rooster actively synthesizing vitellogenin in response to estrogen. Whereas vitellogenin mRNA was in large polysomes in the livers of the roosters actively synthesizing vitellogenin, the vitellogenin mRNA in the liver of the post-estrogen-serum-negative rooster was not associated with polysomes. The possible relevance of these findings to the fact that the rooster responds differently to a primary stimulation with estrogen than to subsequent stimulations is discussed. PMID:1064017

Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis

PubMed Central

Grgic, Ivica; Krautzberger, A. Michaela; Hofmeister, Andreas; Lalli, Matthew; DiRocco, Derek P.; Fleig, Susanne V.; Liu, Jing; Duffield, Jeremy S.; McMahon, Andrew P.; Aronow, Bruce

2014-01-01

Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)–tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies. PMID:24652793

Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells.

PubMed

Tan, Thomas C J; Knight, John; Sbarrato, Thomas; Dudek, Kate; Willis, Anne E; Zamoyska, Rose

2017-07-25

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

Staufen recruitment into stress granules does not affect early mRNA transport in oligodendrocytes.

PubMed

Thomas, María G; Martinez Tosar, Leandro J; Loschi, Mariela; Pasquini, Juana M; Correale, Jorge; Kindler, Stefan; Boccaccio, Graciela L

2005-01-01

Staufen is a conserved double-stranded RNA-binding protein required for mRNA localization in Drosophila oocytes and embryos. The mammalian homologues Staufen 1 and Staufen 2 have been implicated in dendritic RNA targeting in neurons. Here we show that in rodent oligodendrocytes, these two proteins are present in two independent sets of RNA granules located at the distal myelinating processes. A third kind of RNA granules lacks Staufen and contains major myelin mRNAs. Myelin Staufen granules associate with microfilaments and microtubules, and their subcellular distribution is affected by polysome-disrupting drugs. Under oxidative stress, both Staufen 1 and Staufen 2 are recruited into stress granules (SGs), which are stress-induced organelles containing transiently silenced messengers. Staufen SGs contain the poly(A)-binding protein (PABP), the RNA-binding proteins HuR and TIAR, and small but not large ribosomal subunits. Staufen recruitment into perinuclear SGs is paralleled by a similar change in the overall localization of polyadenylated RNA. Under the same conditions, the distribution of recently transcribed and exported mRNAs is not affected. Our results indicate that Staufen 1 and Staufen 2 are novel and ubiquitous SG components and suggest that Staufen RNPs are involved in repositioning of most polysomal mRNAs, but not of recently synthesized transcripts, during the stress response.

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation

PubMed Central

Schepetilnikov, Mikhail; Kobayashi, Kappei; Geldreich, Angèle; Caranta, Carole; Robaglia, Christophe; Keller, Mario; Ryabova, Lyubov A

2011-01-01

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator–viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV–TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP—a novel and specific target of TOR/S6K1—in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes. PMID:21343906

iCopyDAV: Integrated platform for copy number variations—Detection, annotation and visualization

PubMed Central

Vogeti, Sriharsha

2018-01-01

Discovery of copy number variations (CNVs), a major category of structural variations, have dramatically changed our understanding of differences between individuals and provide an alternate paradigm for the genetic basis of human diseases. CNVs include both copy gain and copy loss events and their detection genome-wide is now possible using high-throughput, low-cost next generation sequencing (NGS) methods. However, accurate detection of CNVs from NGS data is not straightforward due to non-uniform coverage of reads resulting from various systemic biases. We have developed an integrated platform, iCopyDAV, to handle some of these issues in CNV detection in whole genome NGS data. It has a modular framework comprising five major modules: data pre-treatment, segmentation, variant calling, annotation and visualization. An important feature of iCopyDAV is the functional annotation module that enables the user to identify and prioritize CNVs encompassing various functional elements, genomic features and disease-associations. Parallelization of the segmentation algorithms makes the iCopyDAV platform even accessible on a desktop. Here we show the effect of sequencing coverage, read length, bin size, data pre-treatment and segmentation approaches on accurate detection of the complete spectrum of CNVs. Performance of iCopyDAV is evaluated on both simulated data and real data for different sequencing depths. It is an open-source integrated pipeline available at https://github.com/vogetihrsh/icopydav and as Docker’s image at http://bioinf.iiit.ac.in/icopydav/. PMID:29621297

Herpesviruses viral loads and levels of proinflammatory cytokines in apical periodontitis.

PubMed

Jakovljevic, A; Knezevic, A; Nikolic, N; Soldatovic, I; Jovanovic, T; Milasin, J; Andric, M

2018-07-01

This study aimed to analyse Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) viral loads in symptomatic and asymptomatic apical periodontitis lesions, to determine levels of TNF-α, IL-1β and IL-6 in these lesions and to investigate a possible correlation between herpesviral copy numbers and levels of proinflammatory cytokines. A total of 100 samples of apical periodontitis were subjected to HCMV and EBV copy numbers analysis by nested polymerase chain reaction (PCR) and TaqMan real-time PCR. The concentrations of TNF-α, IL-1β and IL-6 were determined by ELISA method. SPSS software was used for statistical analysis. There were no significant differences in the occurrence of EBV and HCMV between symptomatic and asymptomatic periapical lesions (p = .686, p = .879, respectively). Only 12 of 74 EBV (16.2%) and four of 54 HCMV (13.5%) nested PCR-positive samples showed increased viral copy numbers above the limit of 125 copies/ml. There was no significant correlation between the levels of analysed proinflammatory cytokines and herpesviral copy numbers in our sample. The observed low viral loads point to a relatively rare occurrence of active EBV and HCMV infection in our sample. Latent herpesviral infection does not enhance the production of investigated proinflammatory cytokines. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti.

PubMed

Richardson, Jason; Molina-Cruz, Alvaro; Salazar, Ma Isabel; Black, William

2006-01-01

Dengue virus-2 (DENV-2) RNA was quantified from the midgut and legs of individual Aedes aegypti at each of 14 days postinfectious blood meal (dpi) in a DENV-2 susceptible strain from Chetumal, Mexico. A SYBR Green I based strand-specific, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed. The lower detection and quantitation limits were 20 and 200 copies per reaction, respectively. Amounts of positive and negative strand viral RNA strands were correlated. Numbers of plaque-forming units (PFU) were correlated with DENV-2 RNA copy number in both C6/36 cell cultures and mosquitoes. PFU were consistently lower than RNA copy number by 2-3 log(10). Midgut levels of DENV-2 RNA peaked 8 dpi and fluctuated erratically between 6 and 9 dpi. Copies of DENV-2 RNA varied significantly among infected mosquitoes at each time point. Quantitative real-time RT-PCR is a convenient and reliable method that provides new insights into virus-vector interactions.

Impact of parental Bos taurus and Bos indicus origins on copy number variation in traditional Chinese cattle breeds

USDA-ARS?s Scientific Manuscript database

Copy number variation (CNV) is an important component of genomic structural variation and plays a role not only in evolutionary diversification but also domestication. Chinese cattle were derived from Bos taurus and Bos indicus, and several breeds presumably are of hybrid origin, but the evolution o...

Distribution of Disease-Associated Copy Number Variants across Distinct Disorders of Cognitive Development

ERIC Educational Resources Information Center

Pescosolido, Matthew F.; Gamsiz, Ece D.; Nagpal, Shailender; Morrow, Eric M.

2013-01-01

Objective: The purpose of the present study was to discover the extent to which distinct "DSM" disorders share large, highly recurrent copy number variants (CNVs) as susceptibility factors. We also sought to identify gene mechanisms common to groups of diagnoses and/or specific to a given diagnosis based on associations with CNVs. Method:…

Identification of a low copy number plasmid in Xylella fastidiosa Strain Stag’s Leap

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa (Xf) causes Pierce’s Disease (PD) in grapevine. The Stag’s Leap strain is known for its high virulence level and is a model for PD research. Research on Xf has been difficult due to its nutritional fastidiousness. One difficult research issue is the low copy number plasmid. Plasmi...

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Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-16

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Exploring the feasibility of using copy number variants as genetic markers through large-scale whole genome sequencing experiments

USDA-ARS?s Scientific Manuscript database

Copy number variants (CNV) are large scale duplications or deletions of genomic sequence that are caused by a diverse set of molecular phenomena that are distinct from single nucleotide polymorphism (SNP) formation. Due to their different mechanisms of formation, CNVs are often difficult to track us...

Genome-wide identification of copy number variations between two chicken lines that differ in genetic resistance to Marek’s disease

USDA-ARS?s Scientific Manuscript database

Background: Copy number variation (CNV) is a major source of genome polymorphism that directly contributes to phenotypic variation such as resistance to infectious diseases. Lines 63 and 72 are two highly inbred experimental chicken lines that differ greatly in susceptibility to Marek’s disease (MD)...

Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) are large insertions, deletions or duplications in the genome that vary between members of a species and are known to affect a wide variety of phenotypic traits. In this study, we identified CNVs in a population of bulls using low coverage next-generation sequence data....

Cover-Copy-Compare and Spelling: One versus Three Repetitions

ERIC Educational Resources Information Center

Erion, Joel; Davenport, Cindy; Rodax, Nicole; Scholl, Bethany; Hardy, Jennifer

2009-01-01

Cover, copy, compare (CCC) has been used with success to improve spelling skills. This study adds to existing research by completing an analysis of the rewriting component of the intervention. The impact of varying the number of times a subject copied a word following an error was examined with four elementary age students. An adaptive alternating…

Transcriptional insulation of the human keratin 18 gene in transgenic mice.

PubMed Central

Neznanov, N; Thorey, I S; Ceceña, G; Oshima, R G

1993-01-01

Expression of the 10-kb human keratin 18 (K18) gene in transgenic mice results in efficient and appropriate tissue-specific expression in a variety of internal epithelial organs, including liver, lung, intestine, kidney, and the ependymal epithelium of brain, but not in spleen, heart, or skeletal muscle. Expression at the RNA level is directly proportional to the number of integrated K18 transgenes. These results indicate that the K18 gene is able to insulate itself both from the commonly observed cis-acting effects of the sites of integration and from the potential complications of duplicated copies of the gene arranged in head-to-tail fashion. To begin to identify the K18 gene sequences responsible for this property of transcriptional insulation, additional transgenic mouse lines containing deletions of either the 5' or 3' distal end of the K18 gene have been characterized. Deletion of 1.5 kb of the distal 5' flanking sequence has no effect upon either the tissue specificity or the copy number-dependent behavior of the transgene. In contrast, deletion of the 3.5-kb 3' flanking sequence of the gene results in the loss of the copy number-dependent behavior of the gene in liver and intestine. However, expression in kidney, lung, and brain remains efficient and copy number dependent in these transgenic mice. Furthermore, herpes simplex virus thymidine kinase gene expression is copy number dependent in transgenic mice when the gene is located between the distal 5'- and 3'-flanking sequences of the K18 gene. Each adult transgenic male expressed the thymidine kinase gene in testes and brain and proportionally to the number of integrated transgenes. We conclude that the characteristic of copy number-dependent expression of the K18 gene is tissue specific because the sequence requirements for transcriptional insulation in adult liver and intestine are different from those for lung and kidney. In addition, the behavior of the transgenic thymidine kinase gene in testes and brain suggests that the property of transcriptional insulation of the K18 gene may be conferred by the distal flanking sequences of the K18 gene and, additionally, may function for other genes. Images PMID:7681143

A dynamic replication management strategy in distributed GIS

NASA Astrophysics Data System (ADS)

Pan, Shaoming; Xiong, Lian; Xu, Zhengquan; Chong, Yanwen; Meng, Qingxiang

2018-03-01

Replication strategy is one of effective solutions to meet the requirement of service response time by preparing data in advance to avoid the delay of reading data from disks. This paper presents a brand-new method to create copies considering the selection of replicas set, the number of copies for each replica and the placement strategy of all copies. First, the popularities of all data are computed considering both the historical access records and the timeliness of the records. Then, replica set can be selected based on their recent popularities. Also, an enhanced Q-value scheme is proposed to assign the number of copies for each replica. Finally, a reasonable copies placement strategy is designed to meet the requirement of load balance. In addition, we present several experiments that compare the proposed method with techniques that use other replication management strategies. The results show that the proposed model has better performance than other algorithms in all respects. Moreover, the experiments based on different parameters also demonstrated the effectiveness and adaptability of the proposed algorithm.

Plasmid P1 replication: negative control by repeated DNA sequences.

PubMed Central

Chattoraj, D; Cordes, K; Abeles, A

1984-01-01

The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pieces of the incA fragment that each contain only three repeats destabilize P1 plasmids efficiently. This result makes it unlikely that incA specifies a regulatory product. Our in vivo results suggest that the repeating DNA sequence itself negatively controls replication by titrating a P1-determined protein, RepA, that is essential for replication. Consistent with this hypothesis is the observation that the RepA protein binds to the incA fragment in vitro. Images PMID:6387706

High throughput screening of human subtelomeric DNA for copy number changes using multiplex amplifiable probe hybridisation (MAPH).

PubMed

Hollox, E J; Atia, T; Cross, G; Parkin, T; Armour, J A L

2002-11-01

Subtelomeric regions of the human genome are gene rich, with a high level of sequence polymorphism. A number of clinical conditions, including learning disability, have been attributed to subtelomeric deletions or duplications, but screening for deletion in these regions using conventional cytogenetic methods and fluorescence in situ hybridisation (FISH) is laborious. Here we report that a new method, multiplex amplifiable probe hybridisation (MAPH), can be used to screen for copy number at subtelomeric regions. We have constructed a set of MAPH probes with each subtelomeric region represented at least once, so that one gel lane can assay copy number at all chromosome ends in one person. Each probe has been sequenced and, where possible, its position relative to the telomere determined by comparison with mapped clones. The sensitivity of the probes has been characterised on a series of cytogenetically verified positive controls and 83 normal controls were used to assess the frequency of polymorphic copy number with no apparent phenotypic effect. We have also used MAPH to test a cohort of 37 people selected from males referred for fragile X syndrome testing and found six changes that were confirmed by dosage PCR. MAPH can be used to screen subtelomeric regions of chromosomes for deletions and duplications before confirmation by FISH or dosage PCR. The high throughput nature of this technique allows it to be used for large scale screening of subtelomeric copy number, before confirmation by FISH. In practice, the availability of a rapid and efficient screen may allow subtelomeric analysis to be applied to a wider selection of patients than is currently possible using FISH alone.

High throughput screening of human subtelomeric DNA for copy number changes using multiplex amplifiable probe hybridisation (MAPH)

PubMed Central

Hollox, E; Atia, T; Cross, G; Parkin, T; Armour, J

2002-01-01

Background: Subtelomeric regions of the human genome are gene rich, with a high level of sequence polymorphism. A number of clinical conditions, including learning disability, have been attributed to subtelomeric deletions or duplications, but screening for deletion in these regions using conventional cytogenetic methods and fluorescence in situ hybridisation (FISH) is laborious. Here we report that a new method, multiplex amplifiable probe hybridisation (MAPH), can be used to screen for copy number at subtelomeric regions. Methods: We have constructed a set of MAPH probes with each subtelomeric region represented at least once, so that one gel lane can assay copy number at all chromosome ends in one person. Each probe has been sequenced and, where possible, its position relative to the telomere determined by comparison with mapped clones. Results: The sensitivity of the probes has been characterised on a series of cytogenetically verified positive controls and 83 normal controls were used to assess the frequency of polymorphic copy number with no apparent phenotypic effect. We have also used MAPH to test a cohort of 37 people selected from males referred for fragile X syndrome testing and found six changes that were confirmed by dosage PCR. Conclusions: MAPH can be used to screen subtelomeric regions of chromosomes for deletions and duplications before confirmation by FISH or dosage PCR. The high throughput nature of this technique allows it to be used for large scale screening of subtelomeric copy number, before confirmation by FISH. In practice, the availability of a rapid and efficient screen may allow subtelomeric analysis to be applied to a wider selection of patients than is currently possible using FISH alone. PMID:12414816

A Multi-Megabase Copy Number Gain Causes Maternal Transmission Ratio Distortion on Mouse Chromosome 2

PubMed Central

Didion, John P.; Morgan, Andrew P.; Clayshulte, Amelia M.-F.; Mcmullan, Rachel C.; Yadgary, Liran; Petkov, Petko M.; Bell, Timothy A.; Gatti, Daniel M.; Crowley, James J.; Hua, Kunjie; Aylor, David L.; Bai, Ling; Calaway, Mark; Chesler, Elissa J.; French, John E.; Geiger, Thomas R.; Gooch, Terry J.; Garland, Theodore; Harrill, Alison H.; Hunter, Kent; McMillan, Leonard; Holt, Matt; Miller, Darla R.; O'Brien, Deborah A.; Paigen, Kenneth; Pan, Wenqi; Rowe, Lucy B.; Shaw, Ginger D.; Simecek, Petr; Sullivan, Patrick F.; Svenson, Karen L; Weinstock, George M.; Threadgill, David W.; Pomp, Daniel; Churchill, Gary A.; Pardo-Manuel de Villena, Fernando

2015-01-01

Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 – 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2WSBdel1) that eliminates the majority of the R2d2WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2WSBdel1, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2WSBdel1, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system. PMID:25679959

A retrospective analysis of RET translocation, gene copy number gain and expression in NSCLC patients treated with vandetanib in four randomized Phase III studies.

PubMed

Platt, Adam; Morten, John; Ji, Qunsheng; Elvin, Paul; Womack, Chris; Su, Xinying; Donald, Emma; Gray, Neil; Read, Jessica; Bigley, Graham; Blockley, Laura; Cresswell, Carl; Dale, Angela; Davies, Amanda; Zhang, Tianwei; Fan, Shuqiong; Fu, Haihua; Gladwin, Amanda; Harrod, Grace; Stevens, James; Williams, Victoria; Ye, Qingqing; Zheng, Li; de Boer, Richard; Herbst, Roy S; Lee, Jin-Soo; Vasselli, James

2015-03-23

To determine the prevalence of RET rearrangement genes, RET copy number gains and expression in tumor samples from four Phase III non-small-cell lung cancer (NSCLC) trials of vandetanib, a selective inhibitor of VEGFR, RET and EGFR signaling, and to determine any association with outcome to vandetanib treatment. Archival tumor samples from the ZODIAC ( NCT00312377 , vandetanib ± docetaxel), ZEAL ( NCT00418886 , vandetanib ± pemetrexed), ZEPHYR ( NCT00404924 , vandetanib vs placebo) and ZEST ( NCT00364351 , vandetanib vs erlotinib) studies were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in 944 and 1102 patients. The prevalence of RET rearrangements by FISH was 0.7% (95% CI 0.3-1.5%) among patients with a known result. Seven tumor samples were positive for RET rearrangements (vandetanib, n = 3; comparator, n = 4). 2.8% (n = 26) of samples had RET amplification (innumerable RET clusters, or ≥7 copies in > 10% of tumor cells), 8.1% (n = 76) had low RET gene copy number gain (4-6 copies in ≥40% of tumor cells) and 8.3% (n = 92) were RET expression positive (signal intensity ++ or +++ in >10% of tumor cells). Of RET-rearrangement-positive patients, none had an objective response in the vandetanib arm and one patient responded in the comparator arm. Radiologic evidence of tumor shrinkage was observed in two patients treated with vandetanib and one treated with comparator drug. The objective response rate was similar in the vandetanib and comparator arms for patients positive for RET copy number gains or RET protein expression. We have identified prevalence for three RET biomarkers in a population predominated by non-Asians and smokers. RET rearrangement prevalence was lower than previously reported. We found no evidence of a differential benefit for efficacy by IHC and RET gene copy number gains. The low prevalence of RET rearrangements (0.7%) prevents firm conclusions regarding association of vandetanib treatment with efficacy in the RET rearrangement NSCLC subpopulation. Randomized Phase III clinical trials ( NCT00312377 , ZODIAC; NCT00418886 , ZEAL; NCT00364351 , ZEST; NCT00404924 , ZEPHYR).

Rapid evolution and copy number variation of primate RHOXF2, an X-linked homeobox gene involved in male reproduction and possibly brain function.

PubMed

Niu, Ao-lei; Wang, Yin-qiu; Zhang, Hui; Liao, Cheng-hong; Wang, Jin-kai; Zhang, Rui; Che, Jun; Su, Bing

2011-10-12

Homeobox genes are the key regulators during development, and they are in general highly conserved with only a few reported cases of rapid evolution. RHOXF2 is an X-linked homeobox gene in primates. It is highly expressed in the testicle and may play an important role in spermatogenesis. As male reproductive system is often the target of natural and/or sexual selection during evolution, in this study, we aim to dissect the pattern of molecular evolution of RHOXF2 in primates and its potential functional consequence. We studied sequences and copy number variation of RHOXF2 in humans and 16 nonhuman primate species as well as the expression patterns in human, chimpanzee, white-browed gibbon and rhesus macaque. The gene copy number analysis showed that there had been parallel gene duplications/losses in multiple primate lineages. Our evidence suggests that 11 nonhuman primate species have one RHOXF2 copy, and two copies are present in humans and four Old World monkey species, and at least 6 copies in chimpanzees. Further analysis indicated that the gene duplications in primates had likely been mediated by endogenous retrovirus (ERV) sequences flanking the gene regions. In striking contrast to non-human primates, humans appear to have homogenized their two RHOXF2 copies by the ERV-mediated non-allelic recombination mechanism. Coding sequence and phylogenetic analysis suggested multi-lineage strong positive selection on RHOXF2 during primate evolution, especially during the origins of humans and chimpanzees. All the 8 coding region polymorphic sites in human populations are non-synonymous, implying on-going selection. Gene expression analysis demonstrated that besides the preferential expression in the reproductive system, RHOXF2 is also expressed in the brain. The quantitative data suggests expression pattern divergence among primate species. RHOXF2 is a fast-evolving homeobox gene in primates. The rapid evolution and copy number changes of RHOXF2 had been driven by Darwinian positive selection acting on the male reproductive system and possibly also on the central nervous system, which sheds light on understanding the role of homeobox genes in adaptive evolution.

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens.

PubMed

Wood, Henry M; Belvedere, Ornella; Conway, Caroline; Daly, Catherine; Chalkley, Rebecca; Bickerdike, Melissa; McKinley, Claire; Egan, Phil; Ross, Lisa; Hayward, Bruce; Morgan, Joanne; Davidson, Leslie; MacLennan, Ken; Ong, Thian K; Papagiannopoulos, Kostas; Cook, Ian; Adams, David J; Taylor, Graham R; Rabbitts, Pamela

2010-08-01

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

Microarray profile of human kidney from diabetes, renal cell carcinoma and renal cell carcinoma with diabetes

PubMed Central

Kosti, Adam; Harry Chen, Hung-I; Mohan, Sumathy; Liang, Sitai; Chen, Yidong; Habib, Samy L.

2015-01-01

Recent study from our laboratory showed that patients with diabetes are at a higher risk of developing kidney cancer. In the current study, we have screened whole human DNA genome from healthy control, patients with diabetes or renal cell carcinoma (RCC) or RCC+diabetes. We found that 883 genes gain/163 genes loss of copy number in RCC+diabetes group, 669 genes gain/307 genes loss in RCC group and 458 genes gain/38 genes loss of copy number in diabetes group, after removing gain/loss genes obtained from healthy control group. Data analyzed for functional annotation enrichment pathways showed that control group had the highest number (280) of enriched pathways, 191 in diabetes+RCC group, 148 in RCC group, and 81 in diabetes group. The overlap GO pathways between RCC+diabetes and RCC groups showed that nine were enriched, between RCC+diabetes and diabetes groups was four and between diabetes and RCC groups was eight GO pathways. Overall, we observed majority of DNA alterations in patients from RCC+diabetes group. Interestingly, insulin receptor (INSR) is highly expressed and had gains in copy number in RCC+diabetes and diabetes groups. The changes in INSR copy number may use as a biomarker for predicting RCC development in diabetic patients. PMID:25821562

The consequences of chromosomal aneuploidy on the transcriptome of cancer cells☆

PubMed Central

Ried, Thomas; Hu, Yue; Difilippantonio, Michael J.; Ghadimi, B. Michael; Grade, Marian; Camps, Jordi

2016-01-01

Chromosomal aneuploidies are a defining feature of carcinomas, i.e., tumors of epithelial origin. Such aneuploidies result in tumor specific genomic copy number alterations. The patterns of genomic imbalances are tumor specific, and to a certain extent specific for defined stages of tumor development. Genomic imbalances occur already in premalignant precursor lesions, i.e., before the transition to invasive disease, and their distribution is maintained in metastases, and in cell lines derived from primary tumors. These observations are consistent with the interpretation that tumor specific genomic imbalances are drivers of malignant transformation. Naturally, this precipitates the question of how such imbalances influence the expression of resident genes. A number of laboratories have systematically integrated copy number alterations with gene expression changes in primary tumors and metastases, cell lines, and experimental models of aneuploidy to address the question as to whether genomic imbalances deregulate the expression of one or few key genes, or rather affect the cancer transcriptome more globally. The majority of these studies showed that gene expression levels follow genomic copy number. Therefore, gross genomic copy number changes, including aneuploidies of entire chromosome arms and chromosomes, result in a massive deregulation of the transcriptome of cancer cells. This article is part of a Special Issue entitled: Chromatin in time and space. PMID:22426433

Clinical significance of ESR1 gene copy number changes in breast cancer as measured by fluorescence in situ hybridisation.

PubMed

Lin, Ching-Hung; Liu, Jacqueline M; Lu, Yen-Shen; Lan, Chieh; Lee, Wei-Chung; Kuo, Kuan-Ting; Wang, Chung-Chieh; Chang, Dwan-Ying; Huang, Chiun-Sheng; Cheng, Ann-Lii

2013-02-01

The ESR1 gene encodes for oestrogen receptor (ER) α, which plays a crucial role in mammary carcinogenesis and clinical outcome in patients with breast cancer. However, the clinical significance of the ESR1 gene copy number change for breast cancer has not been clarified. ESR1 gene copy number was determined by fluorescence in situ hybridisation (FISH) on tissue sections. A minimum of 20 tumour cells were counted per section, and a FISH ratio of ESR1 gene to CEP6 ≥ 2.0 was considered ESR1 amplification. A ratio >1.2 but <2.0 was considered ESR1 gain. The ESR1 copy number was further measured by quantitative real-time PCR (Q-PCR) with ASXL2 as a reference. FISH revealed ESR1 amplification in six cases (4.0%) and ESR1 gain in 13 cases (8.7%) from a total of 150 cases. ESR1 gain and amplification were more common in older patients (p<0.001), and correlated well with ER protein expression (p=0.03) measured by immunohistochemistry, and ESR1 copy number (p<0.001) measured by Q-PCR. Furthermore, the multivariate analysis revealed that ESR1 amplification was associated with a shorter disease-free survival (HR=5.56, p=0.03) and a shorter overall survival (HR=5.11, p=0.04). In general, the frequency of ESR1 amplification in breast cancer is low when measured by FISH in large sections. ESR1 gain and amplification in breast cancer may be associated with older age and poorer outcomes.

Rare copy number variations in congenital heart disease patients identify unique genes in left-right patterning

PubMed Central

Fakhro, Khalid A.; Choi, Murim; Ware, Stephanie M.; Belmont, John W.; Towbin, Jeffrey A.; Lifton, Richard P.; Khokha, Mustafa K.; Brueckner, Martina

2011-01-01

Dominant human genetic diseases that impair reproductive fitness and have high locus heterogeneity constitute a problem for gene discovery because the usual criterion of finding more mutations in specific genes than expected by chance may require extremely large populations. Heterotaxy (Htx), a congenital heart disease resulting from abnormalities in left-right (LR) body patterning, has features suggesting that many cases fall into this category. In this setting, appropriate model systems may provide a means to support implication of specific genes. By high-resolution genotyping of 262 Htx subjects and 991 controls, we identify a twofold excess of subjects with rare genic copy number variations in Htx (14.5% vs. 7.4%, P = 1.5 × 10−4). Although 7 of 45 Htx copy number variations were large chromosomal abnormalities, 38 smaller copy number variations altered a total of 61 genes, 22 of which had Xenopus orthologs. In situ hybridization identified 7 of these 22 genes with expression in the ciliated LR organizer (gastrocoel roof plate), a marked enrichment compared with 40 of 845 previously studied genes (sevenfold enrichment, P < 10−6). Morpholino knockdown in Xenopus of Htx candidates demonstrated that five (NEK2, ROCK2, TGFBR2, GALNT11, and NUP188) strongly disrupted both morphological LR development and expression of pitx2, a molecular marker of LR patterning. These effects were specific, because 0 of 13 control genes from rare Htx or control copy number variations produced significant LR abnormalities (P = 0.001). These findings identify genes not previously implicated in LR patterning. PMID:21282601

Rare copy number variations in congenital heart disease patients identify unique genes in left-right patterning.

PubMed

Fakhro, Khalid A; Choi, Murim; Ware, Stephanie M; Belmont, John W; Towbin, Jeffrey A; Lifton, Richard P; Khokha, Mustafa K; Brueckner, Martina

2011-02-15

Dominant human genetic diseases that impair reproductive fitness and have high locus heterogeneity constitute a problem for gene discovery because the usual criterion of finding more mutations in specific genes than expected by chance may require extremely large populations. Heterotaxy (Htx), a congenital heart disease resulting from abnormalities in left-right (LR) body patterning, has features suggesting that many cases fall into this category. In this setting, appropriate model systems may provide a means to support implication of specific genes. By high-resolution genotyping of 262 Htx subjects and 991 controls, we identify a twofold excess of subjects with rare genic copy number variations in Htx (14.5% vs. 7.4%, P = 1.5 × 10(-4)). Although 7 of 45 Htx copy number variations were large chromosomal abnormalities, 38 smaller copy number variations altered a total of 61 genes, 22 of which had Xenopus orthologs. In situ hybridization identified 7 of these 22 genes with expression in the ciliated LR organizer (gastrocoel roof plate), a marked enrichment compared with 40 of 845 previously studied genes (sevenfold enrichment, P < 10(-6)). Morpholino knockdown in Xenopus of Htx candidates demonstrated that five (NEK2, ROCK2, TGFBR2, GALNT11, and NUP188) strongly disrupted both morphological LR development and expression of pitx2, a molecular marker of LR patterning. These effects were specific, because 0 of 13 control genes from rare Htx or control copy number variations produced significant LR abnormalities (P = 0.001). These findings identify genes not previously implicated in LR patterning.

Large-scale integrative network-based analysis identifies common pathways disrupted by copy number alterations across cancers

PubMed Central

2013-01-01

Background Many large-scale studies analyzed high-throughput genomic data to identify altered pathways essential to the development and progression of specific types of cancer. However, no previous study has been extended to provide a comprehensive analysis of pathways disrupted by copy number alterations across different human cancers. Towards this goal, we propose a network-based method to integrate copy number alteration data with human protein-protein interaction networks and pathway databases to identify pathways that are commonly disrupted in many different types of cancer. Results We applied our approach to a data set of 2,172 cancer patients across 16 different types of cancers, and discovered a set of commonly disrupted pathways, which are likely essential for tumor formation in majority of the cancers. We also identified pathways that are only disrupted in specific cancer types, providing molecular markers for different human cancers. Analysis with independent microarray gene expression datasets confirms that the commonly disrupted pathways can be used to identify patient subgroups with significantly different survival outcomes. We also provide a network view of disrupted pathways to explain how copy number alterations affect pathways that regulate cell growth, cycle, and differentiation for tumorigenesis. Conclusions In this work, we demonstrated that the network-based integrative analysis can help to identify pathways disrupted by copy number alterations across 16 types of human cancers, which are not readily identifiable by conventional overrepresentation-based and other pathway-based methods. All the results and source code are available at http://compbio.cs.umn.edu/NetPathID/. PMID:23822816

ALK amplification and protein expression predict inferior prognosis in neuroblastomas.

PubMed

Wang, Miao; Zhou, Chunju; Sun, Qinnuan; Cai, Rongqin; Li, Yong; Wang, Daye; Gong, Liping

2013-10-01

ALK gene has been identified as a major neuroblastoma (NBL) predisposition gene. But ALK gene copy number and protein expression in ganglioneuroblastoma (GNBL) and ganglioneuroma (GN) are poorly described in the literature. Furthermore, there are controversies on the correlation between ALK protein expression and clinical outcome in NBL. We evaluated MYCN/ALK gene copy number by fluorescence in situ hybridization (FISH) and detected ALK protein expression by immunohistochemistry (IHC) in 188 NBL, 52 GNBL and 6 GN samples and analyzed their association with clinical outcome of the patients. Although ALK gene copy number increase is a recurrent genetic aberration of neuroblastic tumors (NTs) (39.1%, 96/246), ALK amplification was only present in three NBLs (1.2%, 3/246). The frequency of ALK positivity in NBL (50.5%, 51/101) was significantly higher than in GNBL (22.6%, 7/31) and in GN (0.0%, 0/4) (P<0.05). In addition, ALK positivity also significantly correlates with MYCN/ALK gene copy number increases (P<0.05). Kaplan-Meier survival analysis indicated that MYCN/ALK amplification is correlated with decreased overall survival in NBL. A better prognosis trend was observed in patients with MYCN/ALK gain tumors compared with those with MYCN/ALK normal tumors. Furthermore, ALK positivity significantly correlated with inferior survival in NBL (P=0.044). ALK positivity in NTs correlated with advanced tumor types and MYCN/ALK gene copy number increases. ALK positivity predicts inferior prognosis in NBL and IHC is a simplified strategy to screen ALK positivity in clinical practice. Copyright © 2013 Elsevier Inc. All rights reserved.

Distinct responses in ammonia-oxidizing archaea and bacteria after addition of biosolids to an agricultural soil.

PubMed

Kelly, John J; Policht, Katherine; Grancharova, Tanya; Hundal, Lakhwinder S

2011-09-01

The recently discovered ammonia-oxidizing archaea (AOA) have been suggested as contributors to the first step of nitrification in terrestrial ecosystems, a role that was previously assigned exclusively to ammonia-oxidizing bacteria (AOB). The current study assessed the effects of agricultural management, specifically amendment of soil with biosolids or synthetic fertilizer, on nitrification rates and copy numbers of archaeal and bacterial ammonia monooxygenase (amoA) genes. Anaerobically digested biosolids or synthetic fertilizer was applied annually for three consecutive years to field plots used for corn production. Biosolids were applied at two loading rates, a typical agronomic rate (27 Mg hectare(-1) year(-1)) and double the agronomic rate (54 Mg hectare(-1) year(-1)), while synthetic fertilizer was applied at an agronomic rate typical for the region (291 kg N hectare(-1) year(-1)). Both biosolids amendments and synthetic fertilizer increased soil N and corn yield, but only the biosolids amendments resulted in significant increases in nitrification rates and increases in the copy numbers of archaeal and bacterial amoA genes. In addition, only archaeal amoA gene copy numbers increased in response to biosolids applied at the typical agronomic rate and showed a significant correlation with nitrification rates. Finally, copy numbers of archaeal amoA genes were significantly higher than copy numbers of bacterial amoA genes for all treatments. These results implicate AOA as being primarily responsible for the increased nitrification observed in an agricultural soil amended with biosolids. These results also support the hypothesis that physiological differences between AOA and AOB may enable them to occupy distinct ecological niches.

Human enterovirus in the gastrocnemius of patients with peripheral arterial disease.

PubMed

Kim, Julian K S; Zhu, Zhen; Casale, George; Koutakis, Panagiotis; McComb, Rodney D; Swanson, Stanley; Thompson, Jonathan; Miserlis, Dimitrios; Johanning, Jason M; Haynatzki, Gleb; Pipinos, Iraklis I

2013-08-06

Peripheral arterial disease (PAD) is characterized by myofiber degeneration and loss of function in muscles of the lower limbs. Human enterovirus (HEV) infection has been implicated in the pathogenesis of a number of muscle diseases. However, its association with PAD has not been studied. In this study, we tested the hypothesis that infectious HEV is present in skeletal muscle of patients with PAD and is associated with severity of disease. Gastrocnemius biopsies from 37 patients with PAD and 14 controls were examined for the presence of HEV RNA, viral capsid protein, viral RNA copy number, and viral infectivity. HEV RNA was detected in 54% of the biopsies from patients with PAD but was not detected in muscle biopsies from control patients. This difference in prevalence among PAD and control patients was significant at P<0.001. Viral RNA copy numbers were increased significantly at the later stages of disease; Fontaine Stage IV (10(5.50) copies/mg muscle wet weight, at P<0.005) and Stage III (10(4.87) copies/mg, at P<0.010) compared to Stage II (10(2.50) copies/mg). Viral replication was confirmed by the presence of the negative-strand of viral RNA in all specimens positive for HEV RNA. Cultures of HeLa and human skeletal muscle cells treated with muscle homogenates showed HEV replication and the presence of HEV capsid protein. Our data identified infectious HEV in the gastrocnemius of PAD patients but not in controls. Viral copy number and prevalence of infection were higher in the later stages of disease. Our data point to the need for further studies to determine the contribution of HEV infection to the pathophysiology of PAD.

Genome-wide copy number variant analysis in Holstein cattle reveals variants associated with 10 production traits including residual feed intake and dry matter intake

USDA-ARS?s Scientific Manuscript database

Copy number variation (CNV) is an important type of genetic variation contributing to phenotypic differences among mammals and may serve as an alternative molecular marker to single nucleotide polymorphism (SNP) for genome-wide association study (GWAS). Recently, GWAS analysis using CNV has been app...

CHRONICITY OF DEPRESSION AND MOLECULAR MARKERS IN A LARGE SAMPLE OF HAN CHINESE WOMEN.

PubMed

Edwards, Alexis C; Aggen, Steven H; Cai, Na; Bigdeli, Tim B; Peterson, Roseann E; Docherty, Anna R; Webb, Bradley T; Bacanu, Silviu-Alin; Flint, Jonathan; Kendler, Kenneth S

2016-04-25

Major depressive disorder (MDD) has been associated with changes in mean telomere length and mitochondrial DNA (mtDNA) copy number. This study investigates if clinical features of MDD differentially impact these molecular markers. Data from a large, clinically ascertained sample of Han Chinese women with recurrent MDD were used to examine whether symptom presentation, severity, and comorbidity were related to salivary telomere length and/or mtDNA copy number (maximum N = 5,284 for both molecular and phenotypic data). Structural equation modeling revealed that duration of longest episode was positively associated with mtDNA copy number, while earlier age of onset of most severe episode and a history of dysthymia were associated with shorter telomeres. Other factors, such as symptom presentation, family history of depression, and other comorbid internalizing disorders, were not associated with these molecular markers. Chronicity of depressive symptoms is related to more pronounced telomere shortening and increased mtDNA copy number among individuals with a history of recurrent MDD. As these molecular markers have previously been implicated in physiological aging and morbidity, individuals who experience prolonged depressive symptoms are potentially at greater risk of adverse medical outcomes. © 2016 Wiley Periodicals, Inc.

Associations of mitochondrial haplogroups and mitochondrial DNA copy numbers with end-stage renal disease in a Han population.

PubMed

Zhang, Yuheng; Zhao, Ying; Wen, Shuzhen; Yan, Rengna; Yang, Qinglan; Chen, Huimei

2017-09-01

Mitochondrial DNA (mtDNA) is closely related to mitochondrion function, and variations have been suggested to be involved in pathogenesis of complex diseases. The present study sought to elucidate mitochondrial haplogroups and mtDNA copy number in end-stage renal disease (ESRD) in a Han population. First, the mitochondrial haplogroups of 37 ESRD patients were clustered into several haplogroups, and haplogroup A & D were taken as the candidate risk haplogroups for ESRD. Second, the frequencies of A and D were assessed in 344 ESRD patients and 438 healthy controls, respectively. Haplogroup D was found to be risk maker for ESRD in young subjects (<30 years) with an OR of 2.274. Finally, intracellular and cell-free mtDNA copy numbers were evaluated with quantitative-PCR. The ESRD patients exhibited greater cell-free mtDNA contents than the healthy controls but less intracellular mtDNA. Haplogroup D exhibited a further increase in cell-free mtDNA content and a decrease in intracellular mtDNA content among the ESRDs patients. Our findings suggest that mtNDA haplogroup D may contributes to pathogenesis of early-onset ESRD through alterations of mtDNA copy numbers.

Exploratory factor analysis of pathway copy number data with an application towards the integration with gene expression data.

PubMed

van Wieringen, Wessel N; van de Wiel, Mark A

2011-05-01

Realizing that genes often operate together, studies into the molecular biology of cancer shift focus from individual genes to pathways. In order to understand the regulatory mechanisms of a pathway, one must study its genes at all molecular levels. To facilitate such study at the genomic level, we developed exploratory factor analysis for the characterization of the variability of a pathway's copy number data. A latent variable model that describes the call probability data of a pathway is introduced and fitted with an EM algorithm. In two breast cancer data sets, it is shown that the first two latent variables of GO nodes, which inherit a clear interpretation from the call probabilities, are often related to the proportion of aberrations and a contrast of the probabilities of a loss and of a gain. Linking the latent variables to the node's gene expression data suggests that they capture the "global" effect of genomic aberrations on these transcript levels. In all, the proposed method provides an possibly insightful characterization of pathway copy number data, which may be fruitfully exploited to study the interaction between the pathway's DNA copy number aberrations and data from other molecular levels like gene expression.

Copy number analysis of NIPBL in a cohort of 510 patients reveals rare copy number variants and a mosaic deletion.

PubMed

Cheng, Yu-Wei; Tan, Christopher A; Minor, Agata; Arndt, Kelly; Wysinger, Latrice; Grange, Dorothy K; Kozel, Beth A; Robin, Nathaniel H; Waggoner, Darrel; Fitzpatrick, Carrie; Das, Soma; Del Gaudio, Daniela

2014-03-01

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder characterized by growth retardation, intellectual disability, upper limb abnormalities, hirsutism, and characteristic facial features. In this study we explored the occurrence of intragenic NIPBL copy number variations (CNVs) in a cohort of 510 NIPBL sequence-negative patients with suspected CdLS. Copy number analysis was performed by custom exon-targeted oligonucleotide array-comparative genomic hybridization and/or MLPA. Whole-genome SNP array was used to further characterize rearrangements extending beyond the NIPBL gene. We identified NIPBL CNVs in 13 patients (2.5%) including one intragenic duplication and a deletion in mosaic state. Breakpoint sequences in two patients provided further evidence of a microhomology-mediated replicative mechanism as a potential predominant contributor to CNVs in NIPBL. Patients for whom clinical information was available share classical CdLS features including craniofacial and limb defects. Our experience in studying the frequency of NIBPL CNVs in the largest series of patients to date widens the mutational spectrum of NIPBL and emphasizes the clinical utility of performing NIPBL deletion/duplication analysis in patients with CdLS.

Drosophila mitochondrial transcription factor B1 modulates mitochondrial translation but not transcription or DNA copy number in Schneider cells.

PubMed

Matsushima, Yuichi; Adán, Cristina; Garesse, Rafael; Kaguni, Laurie S

2005-04-29

We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor (d-mtTF) B1. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB1 to 5% of its normal level in Schneider cells. In striking contrast with our previous study on d-mtTFB2, we found that RNAi knock-down of d-mtTFB1 does not change the abundance of specific mitochondrial RNA transcripts, nor does it affect the copy number of mitochondrial DNA. In a corollary manner, overexpression of d-mtTFB1 did not increase either the abundance of mitochondrial RNA transcripts or mitochondrial DNA copy number. Our data suggest that, unlike d-mtTFB2, d-mtTFB1 does not have a critical role in either transcription or regulation of the copy number of mitochondrial DNA. Instead, because we found that RNAi knockdown of d-mtTFB1 reduces mitochondrial protein synthesis, we propose that it serves its primary role in modulating translation. Our work represents the first study to document the role of mtTFB1 in vivo and establishes clearly functional differences between mtTFB1 and mtTFB2.

Effects of SULT1A1 Copy Number Variation on Estrogen Concentration and Tamoxifen-Associated Adverse Drug Reactions in Premenopausal Thai Breast Cancer Patients: A Preliminary Study.

PubMed

Charoenchokthavee, Wanaporn; Ayudhya, Duangchit Panomvana Na; Sriuranpong, Virote; Areepium, Nutthada

2016-01-01

Tamoxifen is a pharmacological estrogen inhibitor that binds to the estrogen receptor (ER) in breast cells. However, it shows an estrogenic effect in other organs, which causes adverse drug reactions (ADRs). The sulfotransferase 1A1 (SULT1A1) enzyme encoded by the SULT1A1 gene is involved in estrogen metabolism. Previous research has suggested that the SULT1A1 copy number is linked with the plasma estradiol (E2) concentration. Here, a total of 34 premenopausal breast cancer patients, selected from the Thai Tamoxifen (TTAM) Project, were screened for their SULT1A1 copy number, plasma E2 concentration and ADRs. The mean age was 44.3±11.1 years, and they were subtyped as ER+/ progesterone receptor (PR) + (28 patients), ER+/ PR- (5 patients) and ER-/PR- (1 patient). Three patients reported ADRs, which were irregular menstruation (2 patients) and vaginal discharge (1 patient). Most (33) patients had two SULT1A1 copies, with one patient having three copies. The median plasma E2 concentration was 1,575.6 (IQR 865.4) pg/ml. Patients with ADRs had significantly higher plasma E2 concentrations than those patients without ADRs (p = 0.014). The plasma E2 concentration was numerically higher in the patient with three SULT1A1 copies, but this lacked statistical significance.

CNV-seq, a new method to detect copy number variation using high-throughput sequencing.

PubMed

Xie, Chao; Tammi, Martti T

2009-03-06

DNA copy number variation (CNV) has been recognized as an important source of genetic variation. Array comparative genomic hybridization (aCGH) is commonly used for CNV detection, but the microarray platform has a number of inherent limitations. Here, we describe a method to detect copy number variation using shotgun sequencing, CNV-seq. The method is based on a robust statistical model that describes the complete analysis procedure and allows the computation of essential confidence values for detection of CNV. Our results show that the number of reads, not the length of the reads is the key factor determining the resolution of detection. This favors the next-generation sequencing methods that rapidly produce large amount of short reads. Simulation of various sequencing methods with coverage between 0.1x to 8x show overall specificity between 91.7 - 99.9%, and sensitivity between 72.2 - 96.5%. We also show the results for assessment of CNV between two individual human genomes.

Amy2B copy number variation reveals starch diet adaptations in ancient European dogs.

PubMed

Ollivier, Morgane; Tresset, Anne; Bastian, Fabiola; Lagoutte, Laetitia; Axelsson, Erik; Arendt, Maja-Louise; Bălăşescu, Adrian; Marshour, Marjan; Sablin, Mikhail V; Salanova, Laure; Vigne, Jean-Denis; Hitte, Christophe; Hänni, Catherine

2016-11-01

Extant dog and wolf DNA indicates that dog domestication was accompanied by the selection of a series of duplications on the Amy2B gene coding for pancreatic amylase. In this study, we used a palaeogenetic approach to investigate the timing and expansion of the Amy2B gene in the ancient dog populations of Western and Eastern Europe and Southwest Asia. Quantitative polymerase chain reaction was used to estimate the copy numbers of this gene for 13 ancient dog samples, dated to between 15 000 and 4000 years before present (cal. BP). This evidenced an increase of Amy2B copies in ancient dogs from as early as the 7th millennium cal. BP in Southeastern Europe. We found that the gene expansion was not fixed across all dogs within this early farming context, with ancient dogs bearing between 2 and 20 diploid copies of the gene. The results also suggested that selection for the increased Amy2B copy number started 7000 years cal. BP, at the latest. This expansion reflects a local adaptation that allowed dogs to thrive on a starch rich diet, especially within early farming societies, and suggests a biocultural coevolution of dog genes and human culture.

Amy2B copy number variation reveals starch diet adaptations in ancient European dogs

PubMed Central

Tresset, Anne; Bastian, Fabiola; Lagoutte, Laetitia; Arendt, Maja-Louise; Bălăşescu, Adrian; Marshour, Marjan; Sablin, Mikhail V.; Salanova, Laure; Vigne, Jean-Denis; Hitte, Christophe; Hänni, Catherine

2016-01-01

Extant dog and wolf DNA indicates that dog domestication was accompanied by the selection of a series of duplications on the Amy2B gene coding for pancreatic amylase. In this study, we used a palaeogenetic approach to investigate the timing and expansion of the Amy2B gene in the ancient dog populations of Western and Eastern Europe and Southwest Asia. Quantitative polymerase chain reaction was used to estimate the copy numbers of this gene for 13 ancient dog samples, dated to between 15 000 and 4000 years before present (cal. BP). This evidenced an increase of Amy2B copies in ancient dogs from as early as the 7th millennium cal. BP in Southeastern Europe. We found that the gene expansion was not fixed across all dogs within this early farming context, with ancient dogs bearing between 2 and 20 diploid copies of the gene. The results also suggested that selection for the increased Amy2B copy number started 7000 years cal. BP, at the latest. This expansion reflects a local adaptation that allowed dogs to thrive on a starch rich diet, especially within early farming societies, and suggests a biocultural coevolution of dog genes and human culture. PMID:28018628

Low D4Z4 copy number and gender difference in Korean patients with facioscapulohumeral muscular dystrophy type 1.

PubMed

Park, Hyung Jun; Hong, Ji-Man; Lee, Jung Hwan; Lee, Hyung Seok; Shin, Ha Young; Kim, Seung Min; Ki, Chang-Seok; Lee, Ji Hyun; Choi, Young-Chul

2015-11-01

The objective of this study was to investigate the clinical and genetic features of Korean patients with facioscapulohumeral muscular dystrophy type 1 (FSHD), and assessed the impact of molecular defects on phenotypic expression. We enrolled 104 FSHD patients from 87 unrelated Korean families with D4Z4 repeat array of less than 11 copies on 4q35. Sixty-one men and forty-three women were enrolled. Median D4Z4 copy number was 4 units and 99 (95%) Korean patients with FSHD carried 1-6 units. The median age at symptom onset was 13 [interquartile range: 8-17] years old. In 100 symptomatic patients, muscle weakness began in facial muscles in 58 patients, shoulder-girdle muscles in 37, and pelvic-girdle muscles in 5. Disease severity was significantly correlated with D4Z4 copy number. In addition, women were more severely affected than men even though there were no differences in age at examination or in D4Z4 copy number between the two genders. This gender difference among Korean patients was the opposite of analysis on individuals of European ancestry. In conclusion, the present study demonstrated the new diagnostic threshold for FSHD in Koreans based on the D4Z4 repeat array size distribution from 1 to 6 units and expanded the clinical spectrum. Copyright © 2015 Elsevier B.V. All rights reserved.

Fine mapping of copy number variations on two cattle genome assemblies using high density SNP array

USDA-ARS?s Scientific Manuscript database

Btau_4.0 and UMD3.1 are two distinct cattle reference genome assemblies. In our previous study using the low density BovineSNP50 array, we reported a copy number variation (CNV) analysis on Btau_4.0 with 521 animals of 21 cattle breeds, yielding 682 CNV regions with a total length of 139.8 megabases...

TCF21 hypermethylation in genetically quiescent clear cell sarcoma of the kidney | Office of Cancer Genomics

Cancer.gov

Clear Cell Sarcoma of the Kidney (CCSK) is a rare childhood tumor whose molecular pathogenesis remains poorly understood. We analyzed a discovery set of 13 CCSKs for changes in chromosome copy number, mutations, rearrangements, global gene expression and global DNA methylation. No recurrent segmental chromosomal copy number changes or somatic variants (single nucleotide or small insertion/deletion) were identified.

[Analysis of genomic copy number variations in two unrelated neonates with 8p deletion and duplication associated with congenital heart disease].

PubMed

Mei, Mei; Yang, Lin; Zhan, Guodong; Wang, Huijun; Ma, Duan; Zhou, Wenhao; Huang, Guoying

2014-06-01

To screen for genomic copy number variations (CNVs) in two unrelated neonates with multiple congenital abnormalities using Affymetrix SNP chip and try to find the critical region associated with congenital heart disease. Two neonates were tested for genomic copy number variations by using Cytogenetic SNP chip.Rare CNVs with potential clinical significance were selected of which deletion segments' size was larger than 50 kb and duplication segments' size was larger than 150 kb based on the analysis of ChAs software, without false positive CNVs and segments of normal population. The identified CNVs were compared with those of the cases in DECIPHER and ISCA databases. Eleven rare CNVs with size from 546.6-27 892 kb were identified in the 2 neonates. The deletion region and size of case 1 were 8p23.3-p23.1 (387 912-11 506 771 bp) and 11.1 Mb respectively, the duplication region and size of case 1 were 8p23.1-p11.1 (11 508 387-43 321 279 bp) and 31.8 Mb respectively. The deletion region and size of case 2 were 8p23.3-p23.1 (46 385-7 809 878 bp) and 7.8 Mb respectively, the duplication region and size of case 2 were 8p23.1-p11.21 (12 260 914-40 917 092 bp) and 28.7 Mb respectively. The comparison with Decipher and ISCA databases supported previous viewpoint that 8p23.1 had been associated with congenital heart disease and the region between 7 809 878-11 506 771 bp may play a role in the severe cardiac defects associated with 8p23.1 deletions. Case 1 had serious cardiac abnormalities whose GATA4 was located in the duplication segment and the copy number increased while SOX7 was located in the deletion segment and the copy number decreased. The region between 7 809 878-11 506 771 bp in 8p23.1 is associated with heart defects and copy number variants of SOX7 and GATA4 may result in congenital heart disease.

Discovery and analysis of an active long terminal repeat-retrotransposable element in Aspergillus oryzae.

PubMed

Jie Jin, Feng; Hara, Seiichi; Sato, Atsushi; Koyama, Yasuji

2014-01-01

Wild-type Aspergillus oryzae RIB40 contains two copies of the AO090005001597 gene. We previously constructed A. oryzae RIB40 strain, RKuAF8B, with multiple chromosomal deletions, in which the AO090005001597 copy number was found to be increased significantly. Sequence analysis indicated that AO090005001597 is part of a putative 6,000-bp retrotransposable element, flanked by two long terminal repeats (LTRs) of 669 bp, with characteristics of retroviruses and retrotransposons, and thus designated AoLTR (A. oryzae LTR-retrotransposable element). AoLTR comprised putative reverse transcriptase, RNase H, and integrase domains. The deduced amino acid sequence alignment of AoLTR showed 94% overall identity with AFLAV, an A. flavus Tf1/sushi retrotransposon. Quantitative real-time RT-PCR showed that AoLTR gene expression was significantly increased in the RKuAF8B, in accordance with the increased copy number. Inverse PCR indicated that the full-length retrotransposable element was randomly integrated into multiple genomic locations. However, no obvious phenotypic changes were associated with the increased AoLTR gene copy number.

[Molecular diagnosis of spinal muscular atrophy by multiplex ligation-dependent probe amplification].

PubMed

Zeng, Jian; Ke, Long-feng; Deng, Xiao-jun; Cai, Mei-ying; Tu, Xiang-dong; Lan, Feng-hua

2008-12-16

To investigate the effect of multiplex ligation-dependent probe amplification (MLPA) in molecular diagnosis of spinal muscular atrophy (SMA). Peripheral blood samples were collected from 13 SMA patients, 31 parents of SMA patients, 50 healthy individuals without family history of SMA, and 10 specimens of amniotic fluid from these families were collected too. Genomic DNA was analyzed by MLPA, conventional PCR-RFLP, and allele-specific PCR. In complete agreement with the results of conventional PCR-RFLP and allele-specific PCR, MLPA analysis showed that all of the 13 patients had homozygous deletion of the survival of motor neuron 1 (SMN1) gene, and there was significant difference between the SMA severity (type I to type III) and SMN2 copy number (P < 0.05). Of the 31 parents 29 (93.5%) had 1 copy of SMN1, 2 (6.5%) had 2 copies of SMN1. Of the 50 healthy individuals, 1 (2.0%) had 1 copy of SMN1, 48 (96.0%) had 2 copies of SMN1, and 1 (2.0%) had 3 copies. The SMN1 copy number of the parents was significantly higher than that of the healthy individuals (P < 0.01). Two of the 10 fetuses had homozygous deletion of SMN1. The MLPA technique has proved to be an accurate and reliable tool for the molecular diagnosis of SMA, both in patients and in healthy carriers.

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

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

Doller, Anke; Badawi, Amel; Schmid, Tobias

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuRmore » amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on different HuR activities. • Both cytoskeletal inhibitors disturbed the RNP-polysome HuR allocation in HepG2 cells. • Accordingly, both inhibitors reduced the stability and levels of HuR controlled mRNAs. • Functionally, important HuR-triggered cell functions were impaired by both compounds. • Targeting of HuR trafficking implies a useful approach for novel antitumor therapy.« less

Topoisomerase-1 and -2A gene copy numbers are elevated in mismatch repair-proficient colorectal cancers.

PubMed

Sønderstrup, Ida Marie Heeholm; Nygård, Sune Boris; Poulsen, Tim Svenstrup; Linnemann, Dorte; Stenvang, Jan; Nielsen, Hans Jørgen; Bartek, Jiri; Brünner, Nils; Nørgaard, Peter; Riis, Lene

2015-06-01

Topoisomerase 1 (TOP1) and 2A (TOP2A) are potential predictive biomarkers for irinotecan and anthracycline treatment, respectively, in colorectal cancer (CRC), and we have recently reported a high frequency of gene gain of the TOP1 and TOP2A genes in CRC. Furthermore, Mismatch Repair (MMR) subtypes of CRC have been associated with benefit from adjuvant chemotherapy of primary CRC. Given the involvement of the topoisomerase enzymes in DNA replication and repair, we raised the hypothesis that an association may exist between TOP gene copy numbers and MMR proficiency/deficiency in CRC. Test cohort: FISH analysis with an in-house TOP1/CEN20 probe mix and a commercially available TOP2A/CEN17 (Dako, Glostrup, Denmark) probe mix was performed on archival formalin fixed paraffin embedded (FFPE) tissue samples from 18 patients with proficient MMR (pMMR) CRC and 18 patients with deficient MMR (dMMR) CRC. TOP1 and TOP2A gene copy numbers and their ratios per nucleus were correlated with MMR status using the Mann-Whitney test. Validation cohort: FFPE samples from 154 patients with primary stage III CRC (originally included in the RANX05 study) were classified according to MMR status by immunohistochemical analysis using validated antibodies for MLH1, MLH2, MSH6 and PMS2, and information on TOP1, CEN20, TOP2A and CEN17 status was previously published for this cohort. The observed TOP1 gene copy numbers in the 36 CRC test cohort were significantly greater (p < 0.01) in the pMMR subgroup (mean: 3.84, SD: 2.03) than in the dMMR subgroup (mean: 1.50, SD: 0.12). Similarly, the TOP2A copy numbers were significantly greater (p < 0.01) in the pMMR subgroup (mean: 1.99, SD: 0.52) than in the dMMR subgroup (mean: 1.52, SD: 0.10). These findings were confirmed in the validation cohort, where in the pMMR subgroup 51% had ≥2 extra TOP1 copies per cell, while all tumors classified as dMMR had diploid TOP1 status and mean TOP2A copy numbers were 2.30 (SD: 1.36) and 1.80 (SD: 0.31) (p = 0.01) in the pMMR subgroup vs. dMMR subgroup, respectively. Our results show that TOP1 and TOP2A gene copy numbers are increased in the pMMR subgroup. We propose that this preference may reflect a selective pressure to gain and/or maintain the gained extra copies of topoisomerase genes whose products are required to cope with high replication stress present in the pMMR tumors, thereby providing a survival advantage selectively in pMMR tumors. Future studies should test this concept and explore potential differences between pMMR and dMMR tumors in response to Top1 and Top2 inhibitors. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Age Differences of Salivary Alpha-Amylase Levels of Basal and Acute Responses to Citric Acid Stimulation Between Chinese Children and Adults.

PubMed

Yang, Ze-Min; Chen, Long-Hui; Zhang, Min; Lin, Jing; Zhang, Jie; Chen, Wei-Wen; Yang, Xiao-Rong

2015-01-01

It remains unclear how salivary alpha-amylase (sAA) levels respond to mechanical stimuli in different age groups. In addition, the role played by the sAA gene (AMY1) copy number and protein expression (glycosylated and non-glycosylated) in sAA activity has also been rarely reported. In this study, we analyzed saliva samples collected before and after citric acid stimulation from 47 child and 47 adult Chinese subjects. We observed that adults had higher sAA activity and sAA glycosylated levels (glycosylated sAA amount/total sAA amount) in basal and stimulated saliva when compared with children, while no differences were found in total or glycosylated sAA amount between them. Interestingly, adults showed attenuated sAA activity levels increase over those of children after stimulation. Correlation analysis showed that total sAA amount, glycosylated sAA amount, and AMY1 copy number × total sAA amount were all positively correlated with sAA activity before and after stimulation in both groups. Interestingly, correlation r between sAA levels (glycosylated sAA amount and total sAA amount) and sAA activity decreased after stimulation in children, while adults showed an increase in correlation r. In addition, the correlation r between AMY1 copy number × total sAA amount and sAA activity was higher than that between AMY1 copy number, total sAA amount, and sAA activity, respectively. Taken together, our results suggest that total sAA amount, glycosylated sAA amount, and the positive interaction between AMY1 copy number and total sAA amount are crucial in influencing sAA activity before and after stimulation in children and adults.

Fusobacterium nucleatum as a prognostic marker of colorectal cancer in a Japanese population.

PubMed

Yamaoka, Yuko; Suehiro, Yutaka; Hashimoto, Shinichi; Hoshida, Tomomi; Fujimoto, Michiyo; Watanabe, Michiya; Imanaga, Daiki; Sakai, Kouhei; Matsumoto, Toshihiko; Nishioka, Mitsuaki; Takami, Taro; Suzuki, Nobuaki; Hazama, Shoichi; Nagano, Hiroaki; Sakaida, Isao; Yamasaki, Takahiro

2018-04-01

Accumulating evidence shows an overabundance of Fusobacterium nucleatum in colorectal tumor tissues. However, the correlation between the absolute copy number of F. nucleatum in colorectal cancer tissues and colorectal cancer progression is unclear from previous reports. Therefore, we performed a study to compare the abundance of F. nucleatum in colorectal tissues with clinicopathologic and molecular features of colorectal cancer. We collected 100 colorectal cancer tissues and 72 matched normal-appearing mucosal tissues. Absolute copy numbers of F. nucleatum were measured by droplet digital PCR. The detection rates of F. nucleatum were 63.9% (46/72) in normal-appearing mucosal tissues and 75.0% (75/100) in CRC tissue samples. The median copy number of F. nucleatum was 0.4/ng DNA in the normal-appearing colorectal mucosa in patients with colorectal cancer and 1.9/ng DNA in the colorectal cancer tissues (P = 0.0031). F. nucleatum copy numbers in stage IV colorectal cancer tissues were significantly higher than those in the normal-appearing mucosa in patients with colorectal cancer (P = 0.0016). The abundance of F. nucleatum in colorectal cancer tissues correlated with tumor size and KRAS mutation and was significantly associated with shorter overall survival times; this trend was notable in the patients with stage IV colorectal cancer. Focusing on normal-appearing mucosa in the patients with colorectal cancer, the F. nucleatum copy number was significantly higher in the patients with stage IV rather than stages I-III. These results suggest that determining F. nucleatum levels may help predict clinical outcomes in colorectal cancer patients. Further confirmatory studies using independent datasets are required to confirm our findings.

High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR.

PubMed

Miotke, Laura; Lau, Billy T; Rumma, Rowza T; Ji, Hanlee P

2014-03-04

In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene FLT3 and the common V600E point mutation in BRAF. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. As another novel feature, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as copy number changes from cancers even in the context of significant dilution with normal DNA. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay.

Expression of the enzymatically active legumain-like cysteine proteinase TvLEGU-1 of Trichomonas vaginalis in Pichia pastoris.

PubMed

Reséndiz-Cardiel, Gerardo; Arroyo, Rossana; Ortega-López, Jaime

2017-06-01

The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter. The active TvLEGU-1 recombinant protein (rTvLEGU-1) was secreted into the medium when tvlegu-1-opt was fused to the Aspergillus niger alpha-amylase signal peptide. The rTvLEGU-1 secretion was influenced by the gene copy number and induction temperature. Data indicate that increasing tvlegu-1-opt gene copy number was detrimental for heterologous expression of the enzymatically active TvLEGU-1. Indeed, expression of TvLEGU-1 had a greater impact on cell viability for those clones with 26 or 29 gene copy number, and cell lysis was observed when the induction was carried out at 30 °C. The enzyme activity in the medium was higher when the induction was carried out at 16 °C and in P. pastoris clones with lower gene copy number. The results presented here suggest that both copy number and induction temperature affect the rTvLEGU-1 expression in its native-like and active conformation. Copyright © 2017 Elsevier Inc. All rights reserved.

Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer

PubMed Central

Gao, Shiwu; Yang, Yingying; Wang, Chunfeng; Guo, Jinlong; Zhou, Dinggang; Wu, Qibin; Su, Yachun; Xu, Liping

2016-01-01

We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines. PMID:27093437

Age Differences of Salivary Alpha-Amylase Levels of Basal and Acute Responses to Citric Acid Stimulation Between Chinese Children and Adults

PubMed Central

Yang, Ze-Min; Chen, Long-Hui; Zhang, Min; Lin, Jing; Zhang, Jie; Chen, Wei-Wen; Yang, Xiao-Rong

2015-01-01

It remains unclear how salivary alpha-amylase (sAA) levels respond to mechanical stimuli in different age groups. In addition, the role played by the sAA gene (AMY1) copy number and protein expression (glycosylated and non-glycosylated) in sAA activity has also been rarely reported. In this study, we analyzed saliva samples collected before and after citric acid stimulation from 47 child and 47 adult Chinese subjects. We observed that adults had higher sAA activity and sAA glycosylated levels (glycosylated sAA amount/total sAA amount) in basal and stimulated saliva when compared with children, while no differences were found in total or glycosylated sAA amount between them. Interestingly, adults showed attenuated sAA activity levels increase over those of children after stimulation. Correlation analysis showed that total sAA amount, glycosylated sAA amount, and AMY1 copy number × total sAA amount were all positively correlated with sAA activity before and after stimulation in both groups. Interestingly, correlation r between sAA levels (glycosylated sAA amount and total sAA amount) and sAA activity decreased after stimulation in children, while adults showed an increase in correlation r. In addition, the correlation r between AMY1 copy number × total sAA amount and sAA activity was higher than that between AMY1 copy number, total sAA amount, and sAA activity, respectively. Taken together, our results suggest that total sAA amount, glycosylated sAA amount, and the positive interaction between AMY1 copy number and total sAA amount are crucial in influencing sAA activity before and after stimulation in children and adults. PMID:26635626

Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains.

PubMed

van den Broek, M; Bolat, I; Nijkamp, J F; Ramos, E; Luttik, M A H; Koopman, F; Geertman, J M; de Ridder, D; Pronk, J T; Daran, J-M

2015-09-01

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes. Copyright © 2015, van den Broek et al.

Amplification and up-regulation of MIR30D was associated with disease progression of cervical squamous cell carcinomas.

PubMed

Zhou, You; Hao, Yinghua; Li, Yuxia; Li, Ruizhen; Wu, Ruifang; Wang, Shubin; Fang, Zhengyu

2017-03-29

Cervical squamous cell carcinoma (CSCC) is the most frequent type among cervical cancers. Although the altered miRNA miR-30d expression and the amplified chromosome locus of MIR30D, 8q24, have been reported in somatic cancers, the definitive functional impact of such region especially in CSCC remains under-investigated. One hundred thirty-six cases of CSCC tissues and matched adjacent normal ovarian epithelial tissues were assessed in this study. FISH and qPCR were performed to detect the copy number and microRNA expression of MIR30D gene in the collected samples. In in-vitro study, proliferation of CSCC cells were analyzed using WST-1 assay and invasion abilities of CSCC cells were evaluated by transwell assay. In-vivo study using a model of nude mice bearing tumor was also performed. Copy number gains of MIR30D were detected in 22.8% (31 out of 136) of CSCC samples. Copy number of MIR30D was positively correlated with tumor progression. CSCCs with lymph node metastases (LNM) also showed more frequencies (36.4%) of MIR30D amplification than those without LNM (18.4%, p < 0.05). CSCCs with increased copy number of MIR30D also showed a positive correlation with miR-30d up-regulation. Inhibition of miR-30d in CSCC cells led to impaired tumor growth and migration. Copy number amplifications of MIR30D gene and enhanced expression of miR-30d were positively correlated with tumor progression in CSCCs, indicating miR-30d might play an oncomiric role in the progression of CSCC.

Complement component 4 copy number variation and CYP21A2 genotype associations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

PubMed

Chen, Wuyan; Xu, Zhi; Nishitani, Miki; Van Ryzin, Carol; McDonnell, Nazli B; Merke, Deborah P

2012-12-01

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of cortisol biosynthesis caused by CYP21A2 mutations. An increase in gene copy number variation (CNV) exists at the CYP21A2 locus. CNV of C4, a neighboring gene that encodes complement component 4, is associated with autoimmune disease susceptibility. In this study, we performed comprehensive genetic analysis of the RP-C4-CYP21-TNX (RCCX) region in 127 unrelated 21-OHD patients (100 classic, 27 nonclassic). C4 copy number was determined by Southern blot. C4 CNV and serum C4 levels were evaluated in relation to CYP21A2 mutations and relevant phenotypes. We found that the most common CYP21A2 mutation associated with the nonclassic form of CAH, V281L, was associated with high C4 copy number (p = 7.13 × 10(-16)). Large CYP21A2 deletion, a common mutation associated with the classic form of CAH, was associated with low C4 copy number (p = 1.61 × 10(-14)). Monomodular RCCX with a short C4 gene, a risk factor for autoimmune disease, was significantly less frequent in CAH patients compared to population estimates (2.8 vs. 10.6 %; p = 1.08 × 10(-4)). In conclusion, CAH patients have increased C4 CNV, with mutation-specific associations that may be protective for autoimmune disease. The study of CYP21A2 in relation to neighboring genes provides insight into the genetics of CNV hotspots, an important determinant of human health.

Characterization of genome-wide copy number aberrations in colonic mixed adenoneuroendocrine carcinoma and neuroendocrine carcinoma reveals recurrent amplification of PTGER4 and MYC genes.

PubMed

Sinha, Namita; Gaston, Daniel; Manders, Daniel; Goudie, Marissa; Matsuoka, Makoto; Xie, Tao; Huang, Weei-Yuarn

2018-03-01

Colonic mixed adenoneuroendocrine carcinoma (MANEC) is an aggressive neoplasm with worse prognosis compared with adenocarcinoma. To gain a better understanding of the molecular features of colonic MANEC, we characterized the genome-wide copy number aberrations of 14 MANECs and 5 neuroendocrine carcinomas using the OncoScan FFPE (Affymetrix, Santa Clara, CA) assay. Compared with 269 colonic adenocarcinomas, 19 of 42 chromosomal arms of MANEC exhibited a similar frequency of major aberrant events as adenocarcinomas, and 13 chromosomal arms exhibited a higher frequency of copy number gains. Among them, the most significant chromosomal arms were 5p (77% versus 13%, P = .000012) and 8q (85% versus 33%, P = .0018). The Genomic Identification of Significant Targets in Cancers algorithm identified 7 peaks that drive the tumorgenesis of MANEC. For all except 5p13.1, the peaks largely overlapped with those of adenocarcinoma. Two tumors exhibited MYC amplification localized in 8q24.21, and 2 tumors exhibited PTGER4 amplification localized in 5p13.1. A total of 8 tumors exhibited high copy number gain of PTGER4 and/or MYC. Whereas the frequency of MYC amplification was similar to adenocarcinoma (10.5% versus 4%, P = .2), the frequency of PTGER4 amplification was higher than adenocarcinoma (10.5% versus 0.3%, P = .01). Our study demonstrates similar, but also distinct, copy number aberrations in MANEC compared with adenocarcinoma and suggests an important role for the MYC pathway of colonic carcinoma with neuroendocrine differentiation. The discovery of recurrent PTGER4 amplification implies a potential of exploring targeting therapy to the prostaglandin synthesis pathways in a subset of these tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

Distinct Responses in Ammonia-Oxidizing Archaea and Bacteria after Addition of Biosolids to an Agricultural Soil▿

PubMed Central

Kelly, John J.; Policht, Katherine; Grancharova, Tanya; Hundal, Lakhwinder S.

2011-01-01

The recently discovered ammonia-oxidizing archaea (AOA) have been suggested as contributors to the first step of nitrification in terrestrial ecosystems, a role that was previously assigned exclusively to ammonia-oxidizing bacteria (AOB). The current study assessed the effects of agricultural management, specifically amendment of soil with biosolids or synthetic fertilizer, on nitrification rates and copy numbers of archaeal and bacterial ammonia monooxygenase (amoA) genes. Anaerobically digested biosolids or synthetic fertilizer was applied annually for three consecutive years to field plots used for corn production. Biosolids were applied at two loading rates, a typical agronomic rate (27 Mg hectare−1 year−1) and double the agronomic rate (54 Mg hectare−1 year−1), while synthetic fertilizer was applied at an agronomic rate typical for the region (291 kg N hectare−1 year−1). Both biosolids amendments and synthetic fertilizer increased soil N and corn yield, but only the biosolids amendments resulted in significant increases in nitrification rates and increases in the copy numbers of archaeal and bacterial amoA genes. In addition, only archaeal amoA gene copy numbers increased in response to biosolids applied at the typical agronomic rate and showed a significant correlation with nitrification rates. Finally, copy numbers of archaeal amoA genes were significantly higher than copy numbers of bacterial amoA genes for all treatments. These results implicate AOA as being primarily responsible for the increased nitrification observed in an agricultural soil amended with biosolids. These results also support the hypothesis that physiological differences between AOA and AOB may enable them to occupy distinct ecological niches. PMID:21803892

Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains

PubMed Central

van den Broek, M.; Bolat, I.; Nijkamp, J. F.; Ramos, E.; Luttik, M. A. H.; Koopman, F.; Geertman, J. M.; de Ridder, D.; Pronk, J. T.

2015-01-01

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes. PMID:26150454

Short interspersed CAN SINE elements as prognostic markers in canine mammary neoplasia.

PubMed

Gelaleti, Gabriela B; Granzotto, Adriana; Leonel, Camila; Jardim, Bruna V; Moschetta, Marina G; Carareto, Claudia M A; Zuccari, Debora Ap P C

2014-01-01

The genome of mammals is characterized by a large number of non-LTR retrotransposons, and among them, the CAN SINEs are characteristics of the canine species. Small amounts of DNA freely circulate in normal blood serum and high amounts are found in human patients with cancer, characterizing it as a candidate tumor-biomarker. The aim of this study was to estimate, through its absolute expression, the number of copies of CAN SINE sequences present in free circulating DNA of female dogs with mammary cancer, in order to correlate with the clinical and pathological characteristics and the follow-up period. The copy number of CAN SINE sequences was estimated by qPCR in 28 female dogs with mammary neoplasia. The univariate analysis showed an increased number of copies in female dogs with mammary tumor in female dogs >10 years old (p=0.02) and tumor time >18 months (p<0.05). The Kaplan-Meier test demonstrated a negative correlation between an increased number of copies and survival time (p=0.03). High amounts of CAN SINE fragments can be good markers for the detection of tumor DNA in blood and may characterize it as a marker of poor prognosis, being related to female dogs with shorter survival times. This estimate can be used as a prognostic marker in non-invasive breast cancer research and is useful in predicting tumor progression and patient monitoring.

Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in primary myelofibrosis.

PubMed

Salati, Simona; Zini, Roberta; Nuzzo, Simona; Guglielmelli, Paola; Pennucci, Valentina; Prudente, Zelia; Ruberti, Samantha; Rontauroli, Sebastiano; Norfo, Ruggiero; Bianchi, Elisa; Bogani, Costanza; Rotunno, Giada; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Salmoiraghi, Silvia; Pietra, Daniela; Ferrari, Sergio; Barosi, Giovanni; Rambaldi, Alessandro; Cazzola, Mario; Bicciato, Silvio; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella

2016-04-01

Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients. © 2015 UICC.

GEAR: genomic enrichment analysis of regional DNA copy number changes.

PubMed

Kim, Tae-Min; Jung, Yu-Chae; Rhyu, Mun-Gan; Jung, Myeong Ho; Chung, Yeun-Jun

2008-02-01

We developed an algorithm named GEAR (genomic enrichment analysis of regional DNA copy number changes) for functional interpretation of genome-wide DNA copy number changes identified by array-based comparative genomic hybridization. GEAR selects two types of chromosomal alterations with potential biological relevance, i.e. recurrent and phenotype-specific alterations. Then it performs functional enrichment analysis using a priori selected functional gene sets to identify primary and clinical genomic signatures. The genomic signatures identified by GEAR represent functionally coordinated genomic changes, which can provide clues on the underlying molecular mechanisms related to the phenotypes of interest. GEAR can help the identification of key molecular functions that are activated or repressed in the tumor genomes leading to the improved understanding on the tumor biology. GEAR software is available with online manual in the website, http://www.systemsbiology.co.kr/GEAR/.

NanoStringNormCNV: pre-processing of NanoString CNV data.

PubMed

Sendorek, Dorota H; Lalonde, Emilie; Yao, Cindy Q; Sabelnykova, Veronica Y; Bristow, Robert G; Boutros, Paul C

2018-03-15

The NanoString System is a well-established technology for measuring RNA and DNA abundance. Although it can estimate copy number variation, relatively few tools support analysis of these data. To address this gap, we created NanoStringNormCNV, an R package for pre-processing and copy number variant calling from NanoString data. This package implements algorithms for pre-processing, quality-control, normalization and copy number variation detection. A series of reporting and data visualization methods support exploratory analyses. To demonstrate its utility, we apply it to a new dataset of 96 genes profiled on 41 prostate tumour and 24 matched normal samples. NanoStringNormCNV is implemented in R and is freely available at http://labs.oicr.on.ca/boutros-lab/software/nanostringnormcnv. paul.boutros@oicr.on.ca. Supplementary data are available at Bioinformatics online.

Comparison of medical students' learning approaches between electronic and hard copy team-based learning.

PubMed

Sharaf, Fawzy; Alnohair, Sultan

2017-01-01

To compare the students' perception of team-based learning (TBL): The paper (hard copy) compared with the e-copy (electronic copy) in the family medicine course of the fifth year medical students, Qassim University College of Medicine. A cross-sectional study was conducted during the family medicine course in 2015-2016 to compare the hard copy and the e-copy TBL sessions. We used Google drive to distribute, collect and analyze the questionnaire. The results of the e-copy TBL are shown and displayed directly with each session to the students, which was not the same as practiced with hard copy. We used also SPSS (version 17 for Windows) for more statistical analysis. The total number of respondents of students in each was 96; a phase of TBL phase 1 (hard copy) and phase 2 (e-copy). Male were 64 (66.7%) and females 32 (33.3%). The first three knowledge questions showed no difference between the mean score between paper and e-copy TBL, but of the perception questions showed a significant difference between the paper and e-copy TBL. The results of the survey showed that the students prefer e-copy TBL as a course format, as it was an attraction for most of the students and making them even more successful in the key exam and e-copy TBL develop the skills needed to work productively in task-groups.

Small Molecule Recognition and Tools to Study Modulation of r(CGG)(exp) in Fragile X-Associated Tremor Ataxia Syndrome.

PubMed

Yang, Wang-Yong; He, Fang; Strack, Rita L; Oh, Seok Yoon; Frazer, Michelle; Jaffrey, Samie R; Todd, Peter K; Disney, Matthew D

2016-09-16

RNA transcripts containing expanded nucleotide repeats cause many incurable diseases via various mechanisms. One such disorder, fragile X-associated tremor ataxia syndrome (FXTAS), is caused by a noncoding r(CGG) repeat expansion (r(CGG)(exp)) that (i) sequesters proteins involved in RNA metabolism in nuclear foci, causing dysregulation of alternative pre-mRNA splicing, and (ii) undergoes repeat associated non-ATG translation (RANT), which produces toxic homopolymeric proteins without using a start codon. Here, we describe the design of two small molecules that inhibit both modes of toxicity and the implementation of various tools to study perturbation of these cellular events. Competitive Chemical Cross Linking and Isolation by Pull Down (C-Chem-CLIP) established that compounds bind r(CGG)(exp) and defined small molecule occupancy of r(CGG)(exp) in cells, the first approach to do so. Using an RNA GFP mimic, r(CGG)(exp)-Spinach2, we observe that our optimal designed compound binds r(CGG)(exp) and affects RNA localization by disrupting preformed RNA foci. These events correlate with an improvement of pre-mRNA splicing defects caused by RNA gain of function. In addition, the compounds reduced levels of toxic homopolymeric proteins formed via RANT. Polysome profiling studies showed that small molecules decreased loading of polysomes onto r(CGG)(exp), explaining decreased translation.

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism.

PubMed

Li, Ruixi; Sun, Ruobai; Hicks, Glenn R; Raikhel, Natasha V

2015-01-06

The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red staining suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.

Integrative analysis of copy number and gene expression in breast cancer using formalin-fixed paraffin-embedded core biopsy tissue: a feasibility study.

PubMed

Iddawela, Mahesh; Rueda, Oscar; Eremin, Jenny; Eremin, Oleg; Cowley, Jed; Earl, Helena M; Caldas, Carlos

2017-07-11

An absence of reliable molecular markers has hampered individualised breast cancer treatments, and a major limitation for translational research is the lack of fresh tissue. There are, however, abundant banks of formalin-fixed paraffin-embedded (FFPE) tissue. This study evaluated two platforms available for the analysis of DNA copy number and gene expression using FFPE samples. The cDNA-mediated annealing, selection, extension, and ligation assay (DASL™) has been developed for gene expression analysis and the Molecular Inversion Probes assay (Oncoscan™), were used for copy number analysis using FFPE tissues. Gene expression and copy number were evaluated in core-biopsy samples from patients with breast cancer undergoing neoadjuvant chemotherapy (NAC). Forty-three core-biopsies were evaluated and characteristic copy number changes in breast cancers, gains in 1q, 8q, 11q, 17q and 20q and losses in 6q, 8p, 13q and 16q, were confirmed. Regions that frequently exhibited gains in tumours showing a pathological complete response (pCR) to NAC were 1q (55%), 8q (40%) and 17q (40%), whereas 11q11 (37%) gain was the most frequent change in non-pCR tumours. Gains associated with poor survival were 11q13 (62%), 8q24 (54%) and 20q (47%). Gene expression assessed by DASL correlated with immunohistochemistry (IHC) analysis for oestrogen receptor (ER) [area under the curve (AUC) = 0.95], progesterone receptor (PR)(AUC = 0.90) and human epidermal growth factor type-2 receptor (HER-2) (AUC = 0.96). Differential expression analysis between ER+ and ER- cancers identified over-expression of TTF1, LAF-4 and C-MYB (p ≤ 0.05), and between pCR vs non-pCRs, over-expression of CXCL9, AREG, B-MYB and under-expression of ABCG2. This study was an integrative analysis of copy number and gene expression using FFPE core biopsies and showed that molecular marker data from FFPE tissues were consistent with those in previous studies using fresh-frozen samples. FFPE tissue can provide reliable information and will be a useful tool in molecular marker studies. Trial registration number ISRCTN09184069 and registered retrospectively on 02/06/2010.

Genomic Basis of Prostate Cancer Health Disparity Among African-American Men

DTIC Science & Technology

2013-07-01

amplification and deletion) in a limited repertoire of genes Is highly predictive of prostate cancer metastasis. This signature is present in primary...laboratory has demonstrated that the specific genes within metastatic prostate cancers have been altered by amplification (increase in the copy number...or deletion (decrease in the copy number) (3). These genes appeared to have been selected by the advantages that they conveyed to tumors, such as

Global Characterization of Protein-Altering Mutations in Prostate Cancer

DTIC Science & Technology

2012-08-01

observed, and assess prevalence; (3) Perform integrative analyses of somatic mutation with gene expression and copy number change data collected on the...v) completed CGH assays on 200 prostate cancers; (vi) initiated the integrated analyses of gene expression, copy number and mutation in prostate...histories of individual mutations within the progression of the cancer in which it was observed, and to assess the prevalence of candidate cancer genes

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

Treesearch

Yong-Su Jin; Thomas W. Jeffries

2003-01-01

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes(XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into...

Center of Excellence for Individuation of Therapy for Breast Cancer

DTIC Science & Technology

2012-03-01

Sledge, B. Leyland-Jones (2011) Gene copy number and expression of TYMP and TYMS are predictive of outcome in breast cancer patients treated with... Gene copy number and expression of TYMP and TYMS are predictive of outcome in breast cancer patients treated with capecitabine. R. Audet, C...determine if a specific gene expression signature could be used as predictive marker for treatment outcome . Results summary for Cohort A: doxorubicin

BACOM2.0 facilitates absolute normalization and quantification of somatic copy number alterations in heterogeneous tumor

NASA Astrophysics Data System (ADS)

Fu, Yi; Yu, Guoqiang; Levine, Douglas A.; Wang, Niya; Shih, Ie-Ming; Zhang, Zhen; Clarke, Robert; Wang, Yue

2015-09-01

Most published copy number datasets on solid tumors were obtained from specimens comprised of mixed cell populations, for which the varying tumor-stroma proportions are unknown or unreported. The inability to correct for signal mixing represents a major limitation on the use of these datasets for subsequent analyses, such as discerning deletion types or detecting driver aberrations. We describe the BACOM2.0 method with enhanced accuracy and functionality to normalize copy number signals, detect deletion types, estimate tumor purity, quantify true copy numbers, and calculate average-ploidy value. While BACOM has been validated and used with promising results, subsequent BACOM analysis of the TCGA ovarian cancer dataset found that the estimated average tumor purity was lower than expected. In this report, we first show that this lowered estimate of tumor purity is the combined result of imprecise signal normalization and parameter estimation. Then, we describe effective allele-specific absolute normalization and quantification methods that can enhance BACOM applications in many biological contexts while in the presence of various confounders. Finally, we discuss the advantages of BACOM in relation to alternative approaches. Here we detail this revised computational approach, BACOM2.0, and validate its performance in real and simulated datasets.

Genome-wide copy number variation (CNV) in patients with autoimmune Addison's disease

PubMed Central

2011-01-01

Background Addison's disease (AD) is caused by an autoimmune destruction of the adrenal cortex. The pathogenesis is multi-factorial, involving genetic components and hitherto unknown environmental factors. The aim of the present study was to investigate if gene dosage in the form of copy number variation (CNV) could add to the repertoire of genetic susceptibility to autoimmune AD. Methods A genome-wide study using the Affymetrix GeneChip® Genome-Wide Human SNP Array 6.0 was conducted in 26 patients with AD. CNVs in selected genes were further investigated in a larger material of patients with autoimmune AD (n = 352) and healthy controls (n = 353) by duplex Taqman real-time polymerase chain reaction assays. Results We found that low copy number of UGT2B28 was significantly more frequent in AD patients compared to controls; conversely high copy number of ADAM3A was associated with AD. Conclusions We have identified two novel CNV associations to ADAM3A and UGT2B28 in AD. The mechanism by which this susceptibility is conferred is at present unclear, but may involve steroid inactivation (UGT2B28) and T cell maturation (ADAM3A). Characterization of these proteins may unravel novel information on the pathogenesis of autoimmunity. PMID:21851588