Molecular evolution of psbA gene in ferns: unraveling selective pressure and co-evolutionary pattern

PubMed Central

2012-01-01

Background The photosynthetic oxygen-evolving photo system II (PS II) produces almost the entire oxygen in the atmosphere. This unique biochemical system comprises a functional core complex that is encoded by psbA and other genes. Unraveling the evolutionary dynamics of this gene is of particular interest owing to its direct role in oxygen production. psbA underwent gene duplication in leptosporangiates, in which both copies have been preserved since. Because gene duplication is often followed by the non-fictionalization of one of the copies and its subsequent erosion, preservation of both psbA copies pinpoint functional or regulatory specialization events. The aim of this study was to investigate the molecular evolution of psbA among fern lineages. Results We sequenced psbA , which encodes D1 protein in the core complex of PSII, in 20 species representing 8 orders of extant ferns; then we searched for selection and convolution signatures in psbA across the 11 fern orders. Collectively, our results indicate that: (1) selective constraints among D1 protein relaxed after the duplication in 4 leptosporangiate orders; (2) a handful positively selected codons were detected within species of single copy psbA, but none in duplicated ones; (3) a few sites among D1 protein were involved in co-evolution process which may intimate significant functional/structural communications between them. Conclusions The strong competition between ferns and angiosperms for light may have been the main cause for a continuous fixation of adaptive amino acid changes in psbA , in particular after its duplication. Alternatively, a single psbA copy may have undergone bursts of adaptive changes at the molecular level to overcome angiosperms competition. The strong signature of positive Darwinian selection in a major part of D1 protein is testament to this. At the same time, species own two psbA copies hardly have positive selection signals among the D1 protein coding sequences. In this study, eleven co-evolving sites have been detected via different molecules, which may be more important than others. PMID:22899792

Topological Constraints on Transvection between White Genes within the Transposing Element Te35b in Drosophila Melanogaster

PubMed Central

Gubb, D.; Roote, J.; Trenear, J.; Coulson, D.; Ashburner, M.

1997-01-01

The transposable element TE35B carries two copies of the white (w) gene at 35B1.2 on the second chromosome. These w genes are suppressed in a zeste-1 (z(1)) mutant background in a synapsis-dependent manner. Single-copy derivatives of the original TE35B stock give red eyes when heterozygous, but zeste eyes when homozygous. TE35B derivatives carrying single, double or triple copies of w were crossed to generate flies carrying from two to five ectopic w genes. Within this range, z(1)-mediated suppression is insensitive to copynumber and does not distinguish between w genes that are in cis or in trans. Suppression does not require the juxtaposition of even numbers of w genes, but is extremely sensitive to chromosomal topology. When arranged in a tight cluster, in triple-copy TE derivatives, w genes are nonsuppressible. Breakpoints falling within TE35B and separating two functional w genes act as partial suppressors of z(1). Similarly, breakpoints immediately proximal or distal to both w genes give partial suppression. This transvection-dependent downregulation of w genes may result from mis-activation of the X-chromosome dosage compensation mechanism. PMID:9215897

Papaya (Carica papaya L.).

PubMed

Zhu, Yun J; Fitch, Maureen M M; Moore, Paul H

2006-01-01

Transgenic papaya plants were initially obtained using particle bombardment, a method having poor efficiency in producing intact, single-copy insertion of transgenes. Single-copy gene insertion was improved using Agrobacterium tumefaciens. With progress being made in genome sequencing and gene discovery, there is a need for more efficient methods of transformation in order to study the function of these genes. We describe a protocol for Agrobacterium-mediated transformation using carborundum-wounded papaya embryogenic calli. This method should lead to high-throughput transformation, which on average produced at least one plant that was positive in polymerase chain reaction (PCR), histochemical staining, or by Southern blot hybridization from 10 to 20% of the callus clusters that had been co-cultivated with Agrobacterium. Plants regenerated from the callus clusters in 9 to 13 mo.

Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

PubMed

Clark, Iain C; Melnyk, Ryan A; Youngblut, Matthew D; Carlson, Hans K; Iavarone, Anthony T; Coates, John D

2015-05-19

Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. The ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism. Copyright © 2015 Clark et al.

Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome

PubMed Central

Opazo, Juan C.; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F.

2015-01-01

Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about ancestral functions of vertebrate globins. PMID:25743544

Target sequencing and CRISPR/Cas editing reveal simultaneous loss of UTX and UTY in urothelial bladder cancer.

PubMed

Ahn, Jinwoo; Kim, Kwang Hyun; Park, Sanghui; Ahn, Young-Ho; Kim, Ha Young; Yoon, Hana; Lee, Ji Hyun; Bang, Duhee; Lee, Dong Hyeon

2016-09-27

UTX is a histone demethylase gene located on the X chromosome and is a frequently mutated gene in urothelial bladder cancer (UBC). UTY is a paralog of UTX located on the Y chromosome. We performed target capture sequencing on 128 genes in 40 non-metastatic UBC patients. UTX was the most frequently mutated gene (30%, 12/40). Of the genetic alterations identified, 75% were truncating mutations. UTY copy number loss was detected in 8 male patients (22.8%, 8/35). Of the 9 male patients with UTX mutations, 6 also had copy number loss (66.7%). To evaluate the functional roles of UTX and UTY in tumor progression, we designed UTX and UTY single knockout and UTX-UTY double knockout experiments using a CRISPR/Cas9 lentiviral system, and compared the proliferative capacities of two UBC cell lines in vitro. Single UTX or UTY knockout increased cell proliferation as compared to UTX-UTY wild-type cells. UTX-UTY double knockout cells exhibited greater proliferation than single knockout cells. These findings suggest both UTX and UTY function as dose-dependent suppressors of UBC development. While UTX escapes X chromosome inactivation in females, UTY may function as a male homologue of UTX, which could compensate for dosage imbalances.

Coherent-state information concentration and purification in atomic memory

NASA Astrophysics Data System (ADS)

Herec, Jiří; Filip, Radim

2006-12-01

We propose a feasible method of coherent-state information concentration and purification utilizing quantum memory. The method allows us to optimally concentrate and purify information carried by many noisy copies of an unknown coherent state (randomly distributed in time) to a single copy. Thus nonclassical resources and operations can be saved, if we compare information processing with many noisy copies and a single copy with concentrated and purified information.

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.

Single-Cell Analysis Reveals Early Manifestation of Cancerous Phenotype in Pre-Malignant Esophageal Cells

PubMed Central

Wang, Jiangxin; Shi, Xu; Johnson, Roger H.; Kelbauskas, Laimonas; Zhang, Weiwen; Meldrum, Deirdre R.

2013-01-01

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE. PMID:24116039

77 FR 38632 - Findings of Research Misconduct

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-28

... counts of nigrostriatal neurons in brains of several mice and rats by copying a single data file from a... Used Herbicide, Atrazine: Altered Function and Loss of Neurons in Brain Monamine Systems.'' Environ... 2004 and 2006; Falsifying a bar graph representing brain proteasomal activity, by selectively altering...

Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

PubMed Central

2010-01-01

Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should consider and, when possible take advantage of, the implications of polyploidy. PMID:20977772

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

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 .

Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

PubMed

Rodriguez-Fernandez, Imilce A; Dell'Angelica, Esteban C

2015-01-01

The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

Mate-choice copying in single and coupled women: the influence of mate acceptance and mate rejection decisions of other women.

PubMed

Deng, Yan; Zheng, Yong

2015-01-26

Studies of humans and non-human animals indicate that females tend to change the likelihood of choosing a potential mate based on the decisions of other females; this is known as mate-choice copying. In a sample of both single and coupled women, we examined the influence of other women's (model) mate-choice decisions, including mate acceptance and mate rejection, on participants' attractiveness ratings of men (target) and willingness of mate selection. We also examined whether different types of relationships between the target men and the model women affected mate-choice copying. We found that both the single and coupled women showed mate-choice copying, but their response patterns differed. The significant effects for single women were dependent on a decrease in attractiveness ratings when they perceived the models' mate rejection. However, the significant findings for coupled women relied on an increase in attractiveness ratings when they observed the models' mate acceptance. Furthermore, the relationship status between the target men and the model women affected the magnitude of mate-choice copying effects for the single women. Specifically, they showed less mate-choice copying when the targets and models were in a committed romantic relationship than when in a temporary relationship.

Development of single-copy nuclear intron markers for species-level phylogenetics: Case study with Paullinieae (Sapindaceae).

PubMed

Chery, Joyce G; Sass, Chodon; Specht, Chelsea D

2017-09-01

We developed a bioinformatic pipeline that leverages a publicly available genome and published transcriptomes to design primers in conserved coding sequences flanking targeted introns of single-copy nuclear loci. Paullinieae (Sapindaceae) is used to demonstrate the pipeline. Transcriptome reads phylogenetically closer to the lineage of interest are aligned to the closest genome. Single-nucleotide polymorphisms are called, generating a "pseudoreference" closer to the lineage of interest. Several filters are applied to meet the criteria of single-copy nuclear loci with introns of a desired size. Primers are designed in conserved coding sequences flanking introns. Using this pipeline, we developed nine single-copy nuclear intron markers for Paullinieae. This pipeline is highly flexible and can be used for any group with available genomic and transcriptomic resources. This pipeline led to the development of nine variable markers for phylogenetic study without generating sequence data de novo.

The complete chloroplast genome sequence of Chikusichloa aquatica (Poaceae: Oryzeae).

PubMed

Zhang, Jie; Zhang, Dan; Shi, Chao; Gao, Ju; Gao, Li-Zhi

2016-07-01

The complete chloroplast sequence of the Chikusichloa aquatica was determined in this study. The genome consists of 136 563 bp containing a pair of inverted repeats (IRs) of 20 837 bp, which was separated by a large single-copy region and a small single-copy region of 82 315 bp and 33 411 bp, respectively. The C. aquatica cp genome encodes 111 functional genes (71 protein-coding genes, four rRNA genes, and 36 tRNA genes): 92 are unique, while 19 are duplicated in the IR regions. The genic regions account for 58.9% of whole cp genome, and the GC content of the plastome is 39.0%. A phylogenomic analysis showed that C. aquatica is closely related to Rhynchoryza subulata that belongs to the tribe Oryzeae.

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.

Cellular, Molecular and Functional Characterisation of YAC Transgenic Mouse Models of Friedreich Ataxia

PubMed Central

Anjomani Virmouni, Sara; Sandi, Chiranjeevi; Al-Mahdawi, Sahar; Pook, Mark A.

2014-01-01

Background Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene. We have previously established and performed preliminary characterisation of several human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing GAA repeat expansions, Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats). Methodology/Principal Findings We now report extended cellular, molecular and functional characterisation of these FXN YAC transgenic mouse models. FXN transgene copy number analysis of the FRDA mice demonstrated that the YG22R and Y47R lines each have a single copy of the FXN transgene while the YG8R line has two copies. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We identified significant functional deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8R and YG22R FRDA mice compared to Y47R and wild-type control mice. We also confirmed increased somatic GAA repeat instability in the cerebellum and brain of YG22R and YG8R mice, together with significantly reduced levels of FXN mRNA and protein in the brain and liver of YG8R and YG22R compared to Y47R. Conclusions/Significance Together these studies provide a detailed characterisation of our GAA repeat expansion-based YAC transgenic FRDA mouse models that will help investigations of FRDA disease mechanisms and therapy. PMID:25198290

Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

ScienceCinema

Sczyrba, Alex

2018-02-13

DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

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

Sczyrba, Alex

2011-10-13

DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

A Century of Copyright in the Library of Congress.

ERIC Educational Resources Information Center

Cole, John Y.

Before 1870, copyright registration and deposit were functions of several government departments. After the first Federal copyright law was passed in 1790, copyright registration was performed by the clerk in the U.S. district court for the district where the applicant resided, and single copies of copyright deposits were sent to different…

Detection of the free living amoeba Naegleria fowleri by using conventional and real-time PCR based on a single copy DNA sequence.

PubMed

Régoudis, Estelle; Pélandakis, Michel

2016-02-01

The amoeba-flagellate Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM). This thermophilic species occurs worldwide and tends to proliferate in warm aquatic environment. The PAM cases remain rare but this infection is mostly fatal. Here, we describe a single copy region which has been cloned and sequenced, and was used for both conventional and real-time PCR. Targeting a single-copy DNA sequence allows to directly quantify the N. fowleri cells. The real-time PCR results give a detection limit of 1 copy per reaction with high reproducibility without the need of a Taqman probe. This procedure is of interest as compared to other procedures which are mostly based on the detection of multi-copy DNA associated with a Taqman probe. Copyright © 2015 Elsevier Inc. All rights reserved.

A Likelihood-Based Framework for Association Analysis of Allele-Specific Copy Numbers.

PubMed

Hu, Y J; Lin, D Y; Sun, W; Zeng, D

2014-10-01

Copy number variants (CNVs) and single nucleotide polymorphisms (SNPs) co-exist throughout the human genome and jointly contribute to phenotypic variations. Thus, it is desirable to consider both types of variants, as characterized by allele-specific copy numbers (ASCNs), in association studies of complex human diseases. Current SNP genotyping technologies capture the CNV and SNP information simultaneously via fluorescent intensity measurements. The common practice of calling ASCNs from the intensity measurements and then using the ASCN calls in downstream association analysis has important limitations. First, the association tests are prone to false-positive findings when differential measurement errors between cases and controls arise from differences in DNA quality or handling. Second, the uncertainties in the ASCN calls are ignored. We present a general framework for the integrated analysis of CNVs and SNPs, including the analysis of total copy numbers as a special case. Our approach combines the ASCN calling and the association analysis into a single step while allowing for differential measurement errors. We construct likelihood functions that properly account for case-control sampling and measurement errors. We establish the asymptotic properties of the maximum likelihood estimators and develop EM algorithms to implement the corresponding inference procedures. The advantages of the proposed methods over the existing ones are demonstrated through realistic simulation studies and an application to a genome-wide association study of schizophrenia. Extensions to next-generation sequencing data are discussed.

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

Structure-Based Systematic Isolation of Conditional-Lethal Mutations in the Single Yeast Calmodulin Gene

PubMed Central

Ohya, Y.; Botstein, D.

1994-01-01

Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe -> Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium, 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl(2) in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks. PMID:7896089

Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome.

PubMed

Opazo, Juan C; Lee, Alison P; Hoffmann, Federico G; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F

2015-07-01

Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about ancestral functions of vertebrate globins. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms[OPEN

PubMed Central

Li, Zhen; Van de Peer, Yves; De Smet, Riet

2016-01-01

Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of “gene duplicability” is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes. PMID:26744215

Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms.

PubMed

Li, Zhen; Defoort, Jonas; Tasdighian, Setareh; Maere, Steven; Van de Peer, Yves; De Smet, Riet

2016-02-01

Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of "gene duplicability" is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes. © 2016 American Society of Plant Biologists. All rights reserved.

Quantitative and Functional Requirements for Bioluminescent Cancer Models.

PubMed

Feys, Lynn; Descamps, Benedicte; Vanhove, Christian; Vermeulen, Stefan; Vandesompele, J O; Vanderheyden, Katrien; Messens, Kathy; Bracke, Marc; De Wever, Olivier

2016-01-01

Bioluminescent cancer models are widely used but detailed quantification of the luciferase signal and functional comparison with a non-transfected control cell line are generally lacking. In the present study, we provide quantitative and functional tests for luciferase-transfected cells. We quantified the luciferase expression in BLM and HCT8/E11 transfected cancer cells, and examined the effect of long-term luciferin exposure. The present study also investigated functional differences between parental and transfected cancer cells. Our results showed that quantification of different single-cell-derived populations are superior with droplet digital polymerase chain reaction. Quantification of luciferase protein level and luciferase bioluminescent activity is only useful when there is a significant difference in copy number. Continuous exposure of cell cultures to luciferin leads to inhibitory effects on mitochondrial activity, cell growth and bioluminescence. These inhibitory effects correlate with luciferase copy number. Cell culture and mouse xenograft assays showed no significant functional differences between luciferase-transfected and parental cells. Luciferase-transfected cells should be validated by quantitative and functional assays before starting large-scale experiments. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Unambiguous discrimination between linearly dependent equidistant states with multiple copies

NASA Astrophysics Data System (ADS)

Zhang, Wen-Hai; Ren, Gang

2018-07-01

Linearly independent quantum states can be unambiguously discriminated, but linearly dependent ones cannot. For linearly dependent quantum states, however, if C copies of the single states are available, then they may form linearly independent states, and can be unambiguously discriminated. We consider unambiguous discrimination among N = D + 1 linearly dependent states given that C copies are available and that the single copies span a D-dimensional space with equal inner products. The maximum unambiguous discrimination probability is derived for all C with equal a priori probabilities. For this classification of the linearly dependent equidistant states, our result shows that if C is even then adding a further copy fails to increase the maximum discrimination probability.

Low-cost soft-copy display accuracy in the detection of pulmonary nodules by single-exposure dual-energy subtraction: comparison with hard-copy viewing.

PubMed

Kido, S; Kuriyama, K; Hosomi, N; Inoue, E; Kuroda, C; Horai, T

2000-02-01

This study endeavored to clarify the usefulness of single-exposure dual-energy subtraction computed radiography (CR) of the chest and the ability of soft-copy images to detect low-contrast simulated pulmonary nodules. Conventional and bone-subtracted CR images of 25 chest phantom image sets with a low-contrast nylon nodule and 25 without a nodule were interpreted by 12 observers (6 radiologists, 6 chest physicians) who rated each on a continuous confidence scale and marked the position of the nodule if one was present. Hard-copy images were 7 x 7-inch laser-printed CR films, and soft-copy images were displayed on a 21-inch noninterlaced color CRT monitor with an optimized dynamic range. Soft-copy images were adjusted to the same size as hard-copy images and were viewed under darkened illumination in the reading room. No significant differences were found between hard- and soft-copy images. In conclusion, the soft-copy images were found to be useful in detecting low-contrast simulated pulmonary nodules.

Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

PubMed

Cheung, Andrew K

2015-07-01

The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3). Copyright © 2015. Published by Elsevier Inc.

Ascorbate peroxidase-related (APx-R) is not a duplicable gene.

PubMed

Dunand, Christophe; Mathé, Catherine; Lazzarotto, Fernanda; Margis, Rogério; Margis-Pinheiro, Marcia

2011-12-01

Phylogenetic, genomic and functional analyses have allowed the identification of a new class of putative heme peroxidases, so called APx-R (APx-Related). These new class, mainly present in the green lineage (including green algae and land plants), can also be detected in other unicellular chloroplastic organisms. Except for recent polyploid organisms, only single-copy of APx-R gene was detected in each genome, suggesting that the majority of the APx-R extra-copies were lost after chromosomal or segmental duplications. In a similar way, most APx-R co-expressed genes in Arabidopsis genome do not have conserved extra-copies after chromosomal duplications and are predicted to be localized in organelles, as are the APx-R. The member of this gene network can be considered as unique gene, well conserved through the evolution due to a strong negative selection pressure and a low evolution rate. © 2011 Landes Bioscience

Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7

PubMed Central

Curtis, Helen J.; Wood, Matthew J.A.

2017-01-01

Abstract We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach. PMID:28575281

Binomial distribution for quantification of protein subunits in biological Nanoassemblies and functional nanomachines

PubMed Central

Fang, Huaming; Zhang, Peng; Huang, Lisa P.; Zhao, Zhengyi; Pi, Fengmei; Montemagno, Carlo; Guo, Peixuan

2014-01-01

Living systems produce ordered structures and nanomachines that inspire the development of biomimetic nanodevices such as chips, MEMS, actuators, sensors, sorters, and apparatuses for single-pore DNA sequencing, disease diagnosis, drug or therapeutic RNA delivery. Determination of the copy numbers of subunits that build these machines is challenging due to small size. Here we report a simple mathematical method to determine the stoichiometry, using phi29 DNA-packaging nanomotor as a model to elucidate the application of a formula ∑M=0Z(ZM)pZ−MqM, where p and q are the percentage of wild-type and inactive mutant in the empirical assay; M is the copy numbers of mutant and Z is the stoichiometry in question. Variable ratios of mutants and wild-type were mixed to inhibit motor function. Empirical data were plotted over the theoretical curves to determine the stoichiometry and the value of K, which is the number of mutant needed in each machine to block the function, all based on the condition that wild-type and mutant are equal in binding affinity. Both Z and K from 1–12 were investigated. The data precisely confirmed that phi29 motor contains six copies (Z) of the motor ATPase gp16, and K = 1. PMID:24650885

Large diversity of the piggyBac-like elements in the genome of Tribolium castaneum

PubMed Central

Wang, Jianjun; Du, Yuzhou; Wang, Suzhi; Brown, Sue; Park, Yoonseong

2011-01-01

The piggyBac transposable element, originally discovered in the cabbage looper, Trichoplusia ni, has been widely used in insect transgenesis including the red flour beetle Tribolium castaneum. We surveyed piggyBac-like (PLE) sequences in the genome of Tribolium castaneum by homology searches using as queries the diverse PLE sequences that have been described previously. The search yielded a total of 32 piggyBac-like elements (TcPLEs) which were classified into 14 distinct groups. Most of the TcPLEs contain defective functional motifs in that they are lacking inverted terminal repeats or have disrupted open reading frames. Only one single copy of TcPLE1 appears to be intact with imperfect 16 bp inverted terminal repeats flanking an open reading frame encoding a transposase of 571 amino acid residues. Many copies of TcPLEs were found to be inserted into or close to other transposon-like sequences. This large diversity of TcPLEs with generally low copy numbers suggests multiple invasions of the TcPLEs over a long evolutionary time without extensive multiplications or occurrence of rapid loss of TcPLEs copies. PMID:18342253

The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres.

PubMed

Zhang, Tao; Talbert, Paul B; Zhang, Wenli; Wu, Yufeng; Yang, Zujun; Henikoff, Jorja G; Henikoff, Steven; Jiang, Jiming

2013-12-10

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.

Network topologies and convergent aetiologies arising from deletions and duplications observed in individuals with autism.

PubMed

Noh, Hyun Ji; Ponting, Chris P; Boulding, Hannah C; Meader, Stephen; Betancur, Catalina; Buxbaum, Joseph D; Pinto, Dalila; Marshall, Christian R; Lionel, Anath C; Scherer, Stephen W; Webber, Caleb

2013-06-01

Autism Spectrum Disorders (ASD) are highly heritable and characterised by impairments in social interaction and communication, and restricted and repetitive behaviours. Considering four sets of de novo copy number variants (CNVs) identified in 181 individuals with autism and exploiting mouse functional genomics and known protein-protein interactions, we identified a large and significantly interconnected interaction network. This network contains 187 genes affected by CNVs drawn from 45% of the patients we considered and 22 genes previously implicated in ASD, of which 192 form a single interconnected cluster. On average, those patients with copy number changed genes from this network possess changes in 3 network genes, suggesting that epistasis mediated through the network is extensive. Correspondingly, genes that are highly connected within the network, and thus whose copy number change is predicted by the network to be more phenotypically consequential, are significantly enriched among patients that possess only a single ASD-associated network copy number changed gene (p = 0.002). Strikingly, deleted or disrupted genes from the network are significantly enriched in GO-annotated positive regulators (2.3-fold enrichment, corrected p = 2×10(-5)), whereas duplicated genes are significantly enriched in GO-annotated negative regulators (2.2-fold enrichment, corrected p = 0.005). The direction of copy change is highly informative in the context of the network, providing the means through which perturbations arising from distinct deletions or duplications can yield a common outcome. These findings reveal an extensive ASD-associated molecular network, whose topology indicates ASD-relevant mutational deleteriousness and that mechanistically details how convergent aetiologies can result extensively from CNVs affecting pathways causally implicated in ASD.

24 CFR 1000.548 - Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA...

Code of Federal Regulations, 2011 CFR

2011-04-01

... DEVELOPMENT NATIVE AMERICAN HOUSING ACTIVITIES Recipient Monitoring, Oversight and Accountability § 1000.548 Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA activities be... audit pursuant to the Single Audit Act relating to NAHASDA activities be submitted to HUD? 1000.548...

24 CFR 1000.548 - Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA...

Code of Federal Regulations, 2013 CFR

2013-04-01

... DEVELOPMENT NATIVE AMERICAN HOUSING ACTIVITIES Recipient Monitoring, Oversight and Accountability § 1000.548 Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA activities be... audit pursuant to the Single Audit Act relating to NAHASDA activities be submitted to HUD? 1000.548...

24 CFR 1000.548 - Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA...

Code of Federal Regulations, 2014 CFR

2014-04-01

... DEVELOPMENT NATIVE AMERICAN HOUSING ACTIVITIES Recipient Monitoring, Oversight and Accountability § 1000.548 Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA activities be... audit pursuant to the Single Audit Act relating to NAHASDA activities be submitted to HUD? 1000.548...

24 CFR 1000.548 - Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA...

Code of Federal Regulations, 2010 CFR

2010-04-01

... DEVELOPMENT NATIVE AMERICAN HOUSING ACTIVITIES Recipient Monitoring, Oversight and Accountability § 1000.548 Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA activities be... audit pursuant to the Single Audit Act relating to NAHASDA activities be submitted to HUD? 1000.548...

24 CFR 1000.548 - Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA...

Code of Federal Regulations, 2012 CFR

2012-04-01

... DEVELOPMENT NATIVE AMERICAN HOUSING ACTIVITIES Recipient Monitoring, Oversight and Accountability § 1000.548 Must a copy of the recipient's audit pursuant to the Single Audit Act relating to NAHASDA activities be... audit pursuant to the Single Audit Act relating to NAHASDA activities be submitted to HUD? 1000.548...

Organizational and visual memory deficits in schizophrenia and bipolar psychoses using the Rey-Osterrieth complex figure: effects of duration of illness.

PubMed

Seidman, Larry J; Lanca, Margaret; Kremen, William S; Faraone, Stephen V; Tsuang, Ming T

2003-10-01

Verbal declarative memory deficits in schizophrenia are well documented whereas visual declarative memory is less studied. Moreover, there are limited data on whether organizational and visual memory deficits are specific to schizophrenic psychoses. We compared visual memory and organizational function in patients with chronic schizophrenia (n=79) and chronic bipolar psychotic disorder (n=14), and in healthy controls (n=84) using the Rey-Osterrieth Complex Figure (ROCF), testing whether organizational impairments (i.e., executive dysfunctions) account for the visual memory deficit. Groups were comparable on age, handedness and expected intellectual ability (based on single word reading). Using analyses of covariance with sex, parental SES and ethnicity as co-variates, patients with schizophrenia were significantly more impaired than controls on copy accuracy, on recall accuracy, and on percent accuracy of recall. Patients with schizophrenia used a more detail-oriented style on copy and recall and had significantly worse recognition memory. After co-varying IQ, copy organization was also significantly different between the groups. Results for accuracy of copy and recall were not significantly attenuated when controlling for copy organization. Duration of illness was associated with visual memory. Bipolar patients performed at an intermediate level between controls and patients with schizophrenia. The data suggest that in schizophrenia, patients have a visual memory disorder characterized by both organizational processing impairments and retention difficulties, and that there is a decline in visual memory functions with duration of illness. Further research is required to determine whether similar mechanisms underlie the neurocognitive deficits in these psychotic disorders.

Single-site labeling of lysine in proteins through a metal-free multicomponent approach.

PubMed

Chilamari, Maheshwerreddy; Kalra, Neetu; Shukla, Sanjeev; Rai, Vishal

2018-06-15

We report a chemoselective and site-selective approach that distinguishes one Lys from its multiple copies, N-terminus, and other competitors. The phospha-Mannich protocol works with multiple proteins and installs probes without structural and functional perturbations. It delivers an antibody-drug conjugate with selective anti-proliferative activity towards HER2 expressing SKBR3 breast cancer cells.

Intra-isolate genome variation in arbuscular mycorrhizal fungi persists in the transcriptome.

PubMed

Boon, E; Zimmerman, E; Lang, B F; Hijri, M

2010-07-01

Arbuscular mycorrhizal fungi (AMF) are heterokaryotes with an unusual genetic makeup. Substantial genetic variation occurs among nuclei within a single mycelium or isolate. AMF reproduce through spores that contain varying fractions of this heterogeneous population of nuclei. It is not clear whether this genetic variation on the genome level actually contributes to the AMF phenotype. To investigate the extent to which polymorphisms in nuclear genes are transcribed, we analysed the intra-isolate genomic and cDNA sequence variation of two genes, the large subunit ribosomal RNA (LSU rDNA) of Glomus sp. DAOM-197198 (previously known as G. intraradices) and the POL1-like sequence (PLS) of Glomus etunicatum. For both genes, we find high sequence variation at the genome and transcriptome level. Reconstruction of LSU rDNA secondary structure shows that all variants are functional. Patterns of PLS sequence polymorphism indicate that there is one functional gene copy, PLS2, which is preferentially transcribed, and one gene copy, PLS1, which is a pseudogene. This is the first study that investigates AMF intra-isolate variation at the transcriptome level. In conclusion, it is possible that, in AMF, multiple nuclear genomes contribute to a single phenotype.

Homology-dependent Gene Silencing in Paramecium

PubMed Central

Ruiz, Françoise; Vayssié, Laurence; Klotz, Catherine; Sperling, Linda; Madeddu, Luisa

1998-01-01

Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant. PMID:9529389

Using Pinochle to motivate the restricted combinations with repetitions problem

NASA Astrophysics Data System (ADS)

Gorman, Patrick S.; Kunkel, Jeffrey D.; Vasko, Francis J.

2011-07-01

A standard example used in introductory combinatoric courses is to count the number of five-card poker hands possible from a straight deck of 52 distinct cards. A more interesting problem is to count the number of distinct hands possible from a Pinochle deck in which there are multiple, but obviously limited, copies of each type of card (two copies for single-deck, four for double deck). This problem is more interesting because our only concern is to count the number of distinguishable hands that can be dealt. In this note, under various scenarios, we will discuss two combinatoric techniques for counting these hands; namely, the inclusion-exclusion principle and generating functions. We will then show that these Pinochle examples motivate a general counting formula for what are called 'regular' combinations by Riordan. Finally, we prove the correctness of this formula using generating functions.

High-Resolution Mapping of Structural Mutations in Prostate Cancer with Single Nucleotide Polymorphism Arrays

DTIC Science & Technology

2006-11-01

study of the NCI60 panel of cancer cell lines [39]. More recently, amplifications of NOTCH3 were noted in ovarian tumors by an SNP array analysis...and the functional role of NOTCH3 was suggested by the ability to suppress cell proliferation by inhibiting NOTCH3 [40]. Allele-specific copy...Identified and functionally validated the oncogene MITF. 40 Park JT, Li M, Nakayama K, et al. Notch3 gene amplification in ovarian cancer. Cancer Res

Identifying uniformly mutated segments within repeats.

PubMed

Sahinalp, S Cenk; Eichler, Evan; Goldberg, Paul; Berenbrink, Petra; Friedetzky, Tom; Ergun, Funda

2004-12-01

Given a long string of characters from a constant size alphabet we present an algorithm to determine whether its characters have been generated by a single i.i.d. random source. More specifically, consider all possible n-coin models for generating a binary string S, where each bit of S is generated via an independent toss of one of the n coins in the model. The choice of which coin to toss is decided by a random walk on the set of coins where the probability of a coin change is much lower than the probability of using the same coin repeatedly. We present a procedure to evaluate the likelihood of a n-coin model for given S, subject a uniform prior distribution over the parameters of the model (that represent mutation rates and probabilities of copying events). In the absence of detailed prior knowledge of these parameters, the algorithm can be used to determine whether the a posteriori probability for n=1 is higher than for any other n>1. Our algorithm runs in time O(l4logl), where l is the length of S, through a dynamic programming approach which exploits the assumed convexity of the a posteriori probability for n. Our test can be used in the analysis of long alignments between pairs of genomic sequences in a number of ways. For example, functional regions in genome sequences exhibit much lower mutation rates than non-functional regions. Because our test provides means for determining variations in the mutation rate, it may be used to distinguish functional regions from non-functional ones. Another application is in determining whether two highly similar, thus evolutionarily related, genome segments are the result of a single copy event or of a complex series of copy events. This is particularly an issue in evolutionary studies of genome regions rich with repeat segments (especially tandemly repeated segments).

Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.

PubMed

Melo, Sônia C; Santos, Regineide X; Melgaço, Ana C; Pereira, Alanna C F; Pungartnik, Cristina; Brendel, Martin

2015-06-01

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet-C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene

PubMed Central

Melo, Sônia C.; Santos, Regineide X.; Melgaço, Ana C.; Pereira, Alanna C. F.; Pungartnik, Cristina; Brendel, Martin

2015-01-01

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae. PMID:26039235

Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1

PubMed Central

Clark, Iain C.; Melnyk, Ryan A.; Youngblut, Matthew D.; Carlson, Hans K.; Iavarone, Anthony T.

2015-01-01

ABSTRACT Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. PMID:25991681

Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene.

PubMed

Amit, Maayan; Sela, Noa; Keren, Hadas; Melamed, Ze'ev; Muler, Inna; Shomron, Noam; Izraeli, Shai; Ast, Gil

2007-11-29

Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes. Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells. The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains.

Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene

PubMed Central

Amit, Maayan; Sela, Noa; Keren, Hadas; Melamed, Ze'ev; Muler, Inna; Shomron, Noam; Izraeli, Shai; Ast, Gil

2007-01-01

Background Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes. Results Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells. Conclusion The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains. PMID:18047649

Single-cell copy number variation detection

PubMed Central

2011-01-01

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data. PMID:21854607

The Effectiveness of the Process of Socialisation to Behavioural Conformity Within the Public Schools Is Their Strength and also their Weakness: Some Observations

ERIC Educational Resources Information Center

Whitton, D. J.

1977-01-01

Analyzes the effectiveness of the process of socialization within the public schools (known as private schools in the United States) and seeks to provoke a reappraisal of their function in Australia. Available from: Australian College of Education, 916 Swanston Street, Carlton, Victoria 3053, Australia, $2.50 single copy. (Author/MLF)

Infantile spasms with periventricular nodular heterotopia, unbalanced chromosomal translocation 3p26.2 -10p15.1 and 6q22.31 duplication.

PubMed

Jones, Kevin; Weiss, Shelly K; Minassian, Berge

2016-07-01

Patients presenting with infantile spasms, dysmorphic features, and periventricular nodular heterotopia may benefit from genetic copy number variation microarray, or whole-exome sequencing to identify candidate genes. This will allow personalized diagnosis and prognostication and the eventual understanding of single and combined gene functions in brain health and disease.

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

EPA Science Inventory

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

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...

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.

High copy number of highly similar mariner-like transposons in planarian (Platyhelminthe): evidence for a trans-phyla horizontal transfer.

PubMed

Garcia-Fernàndez, J; Bayascas-Ramírez, J R; Marfany, G; Muñoz-Mármol, A M; Casali, A; Baguñà, J; Saló, E

1995-05-01

Several DNA sequences similar to the mariner element were isolated and characterized in the platyhelminthe Dugesia (Girardia) tigrina. They were 1,288 bp long, flanked by two 32 bp-inverted repeats, and contained a single 339 amino acid open-reading frame (ORF) encoding the transposase. The number of copies of this element is approximately 8,000 per haploid genome, constituting a member of the middle-repetitive DNA of Dugesia tigrina. Sequence analysis of several elements showed a high percentage of conservation between the different copies. Most of them presented an intact ORF and the standard signals of actively expressed genes, which suggests that some of them are or have recently been functional transposons. The high degree of similarity shared with other mariner elements from some arthropods, together with the fact that this element is undetectable in other planarian species, strongly suggests a case of horizontal transfer between these two distant phyla.

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.

Loss of function of 1-FEH IIb has more impact on post-harvest inulin degradation in Cichorium intybus than copy number variation of its close paralog 1-FEH IIa

PubMed Central

Dauchot, Nicolas; Raulier, Pierre; Maudoux, Olivier; Notté, Christine; Draye, Xavier; Van Cutsem, Pierre

2015-01-01

Key Message: The loss of mini-exon 2 in the 1-FEH IIb glycosyl-hydrolase results in a putative non-functional allele. This loss of function has a strong impact on the susceptibility to post-harvest inulin depolymerization. Significant variation of copy number was identified in its close paralog 1-FEH IIa, but no quantitative effect of copy number on carbohydrates-related phenotypes was detected. Inulin polyfructan is the second most abundant storage carbohydrate in flowering plants. After harvest, it is depolymerized by fructan exohydrolases (FEHs) as an adaptive response to end-season cold temperatures. In chicory, the intensity of this depolymerization differs between cultivars but also between individuals within a cultivar. Regarding this phenotypic variability, we recently identified statistically significant associations between inulin degradation and genetic polymorphisms located in three FEHs. We present here new results of a systematic analysis of copy number variation (CNV) in five key members of the chicory (Cichorium intybus) GH32 multigenic family, including three FEH genes and the two inulin biosynthesis genes: 1-SST and 1-FFT. qPCR analysis identified a significant variability of relative copy number only in the 1-FEH IIa gene. However, this CNV had no quantitative effect. Instead, cloning of the full length gDNA of a close paralogous sequence (1-FEH IIb) identified a 1028 bp deletion in lines less susceptible to post-harvest inulin depolymerization. This region comprises a 9 bp mini-exon containing one of the three conserved residues of the active site. This results in a putative non-functional 1-FEH IIb allele and an observed lower inulin depolymerization. Extensive genotyping confirmed that the loss of mini-exon 2 in 1-FEH IIb and the previously identified 47 bp duplication located in the 3′UTR of 1-FEH IIa belong to a single haplotype, both being statistically associated with reduced susceptibility to post-harvest inulin depolymerization. Emergence of these haplotypes is discussed. PMID:26157446

POTENTIAL FOR REDUCING STYRENE EXPOSURES FROM COPIED PAPER THROUGH USE OF LOW-EMITTING TONERS

EPA Science Inventory

The paper reports results of tests, conducted using 53-L chambers to determine styrene emission rates from freshly copied paper produced on a single photocopier using two toners manufactured for the copier having different styrene contents. Copied-paper styrene emissions with bot...

POTENTIAL FOR REDUCING INDOOR STYRENE EXPOSURE FROM COPIED PAPER THROUGH USE OF LOW-EMITTING TONERS

EPA Science Inventory

Tests were conducted, using 53-L dynamic chambers, to determine airborne styrene emission rates over time from freshly copied paper. Copies were produced on a single photocopier, using two toners manufactured for this copier but having different styrene contents. The resulting em...

Phylogeny of the cycads based on multiple single copy nuclear genes: congruence of concatenation and species tree inference methods

USDA-ARS?s Scientific Manuscript database

Despite a recent new classification, a stable tree of life for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study we apply five single copy nuclear genes (SCNGs) to the phylogeny of the order Cycadales. We specifically aim to evaluate seve...

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.

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.

High-resolution single-nucleotide polymorphism array-profiling in myeloproliferative neoplasms identifies novel genomic aberrations

PubMed Central

Stegelmann, Frank; Bullinger, Lars; Griesshammer, Martin; Holzmann, Karlheinz; Habdank, Marianne; Kuhn, Susanne; Maile, Carmen; Schauer, Stefanie; Döhner, Hartmut; Döhner, Konstanze

2010-01-01

Single-nucleotide polymorphism arrays allow for genome-wide profiling of copy-number alterations and copy-neutral runs of homozygosity at high resolution. To identify novel genetic lesions in myeloproliferative neoplasms, a large series of 151 clinically well characterized patients was analyzed in our study. Copy-number alterations were rare in essential thrombocythemia and polycythemia vera. In contrast, approximately one third of myelofibrosis patients exhibited small genomic losses (less than 5 Mb). In 2 secondary myelofibrosis cases the tumor suppressor gene NF1 in 17q11.2 was affected. Sequencing analyses revealed a mutation in the remaining NF1 allele of one patient. In terms of copy-neutral aberrations, no chromosomes other than 9p were recurrently affected. In conclusion, novel genomic aberrations were identified in our study, in particular in patients with myelofibrosis. Further analyses on single-gene level are necessary to uncover the mechanisms that are involved in the pathogenesis of myeloproliferative neoplasms. PMID:20015882

In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases

PubMed Central

Arand, Julia; Spieler, David; Karius, Tommy; Branco, Miguel R.; Meilinger, Daniela; Meissner, Alexander; Jenuwein, Thomas; Xu, Guoliang; Leonhardt, Heinrich; Wolf, Verena; Walter, Jörn

2012-01-01

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position–, cell type–, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs. PMID:22761581

Imaging Transcriptional Regulation of Eukaryotic mRNA Genes: Advances and Outlook.

PubMed

Yao, Jie

2017-01-06

Regulation of eukaryotic transcription in vivo occurs at distinct stages. Previous research has identified many active or repressive transcription factors (TFs) and core transcription components and studied their functions in vitro and in vivo. Nonetheless, how individual TFs act in concert to regulate mRNA gene expression in a single cell remains poorly understood. Direct observation of TF assembly and disassembly and various biochemical reactions during transcription of a single-copy gene in vivo is the ideal approach to study this problem. Research in this area requires developing novel techniques for single-cell transcription imaging and integrating imaging studies into understanding the molecular biology of transcription. In the past decade, advanced cell imaging has enabled unprecedented capabilities to visualize individual TF molecules, to track single transcription sites, and to detect individual mRNA in fixed and living cells. These studies have raised several novel insights on transcriptional regulation such as the "hit-and-run" model and transcription bursting that could not be obtained by in vitro biochemistry analysis. At this point, the key question is how to achieve deeper understandings or discover novel mechanisms of eukaryotic transcriptional regulation by imaging transcription in single cells. Meanwhile, further technical advancements are likely required for visualizing distinct kinetic steps of transcription on a single-copy gene in vivo. This review article summarizes recent progress in the field and describes the challenges and opportunities ahead. Copyright © 2016 Elsevier Ltd. All rights reserved.

A single exposure of sediment sulphate-reducing bacteria to oxytetracycline concentrations relevant to aquaculture enduringly disturbed their activity, abundance and community structure.

PubMed

Fernández, M L; Granados-Chinchilla, F; Rodríguez, C

2015-08-01

Although feed medicated with antibiotics is widely used in animal production to prevent and treat bacterial infections, the effect of these drugs on nontarget anaerobic bacteria is unknown. We aimed to clarify whether a single exposure of sulphate-reducing bacteria (SRB) from a tilapia pond to oxytetracycline (OTC) concentrations relevant to aquaculture impacts their function, abundance and community structure. To demonstrate changes in SO4(2-) content, SRB abundance, dsrB copy number and SRB diversity, sediment mesocosms were spiked with 5, 25, 50 and 75 mg OTC kg(-1) and examined for 30 days by means of ion chromatography, qPCR, cultivation and fluorescent in situ hybridization (FISH). On day 3, we measured higher SO4(2-) concentrations (ca. two-fold) and a reduction in dsrB copy numbers of approximately 50% in the treatments compared to the controls. After 30 days, a subtle yet measurable enrichment of bacteria from the order Desulfovibrionales occurred in mesocosms receiving ≥ 50 mg OTC kg(-1), notwithstanding that SRB counts decreased two orders of magnitude. OTC was dynamically and reversibly converted into 4-epioxytetracycline and other related compounds in a dose-dependent manner during the experiment. A single exposure to rather high OTC concentrations triggered functional and structural changes in a SRB community that manifested quickly and persisted for a month. This study improves our limited knowledge on the ecotoxicology of antibiotics in anaerobic environments. © 2015 The Society for Applied Microbiology.

Unified Engineering Software System

NASA Technical Reports Server (NTRS)

Purves, L. R.; Gordon, S.; Peltzman, A.; Dube, M.

1989-01-01

Collection of computer programs performs diverse functions in prototype engineering. NEXUS, NASA Engineering Extendible Unified Software system, is research set of computer programs designed to support full sequence of activities encountered in NASA engineering projects. Sequence spans preliminary design, design analysis, detailed design, manufacturing, assembly, and testing. Primarily addresses process of prototype engineering, task of getting single or small number of copies of product to work. Written in FORTRAN 77 and PROLOG.

Expression of Ornithine Decarboxylase Is Transiently Increased by Pollination, 2,4-Dichlorophenoxyacetic Acid, and Gibberellic Acid in Tomato Ovaries1

PubMed Central

Alabadí, David; Carbonell, Juan

1998-01-01

A cDNA encoding for a functional ornithine decarboxylase has been isolated from a cDNA library of carpels of tomato (Lycopersicon esculentum Mill.). Ornithine decarboxylase in tomato is represented by a single-copy gene that we show to be up-regulated during early fruit growth induced by 2,4-dichlorophenoxyacetic acid and gibberellic acid. PMID:9733552

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

Conservative site-specific and single-copy transgenesis in human LINE-1 elements

PubMed Central

Vijaya Chandra, Shree Harsha; Makhija, Harshyaa; Peter, Sabrina; Myint Wai, Cho Mar; Li, Jinming; Zhu, Jindong; Ren, Zhonglu; D'Alcontres, Martina Stagno; Siau, Jia Wei; Chee, Sharon; Ghadessy, Farid John; Dröge, Peter

2016-01-01

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. PMID:26673710

A versatile microsatellite instability reporter system in human cells.

PubMed

Koole, Wouter; Schäfer, Henning S; Agami, Reuven; van Haaften, Gijs; Tijsterman, Marcel

2013-09-01

Here, we report the investigation of microsatellite instability (MSI) in human cells with a newly developed reporter system based on fluorescence. We composed a vector into which microsatellites of different lengths and nucleotide composition can be introduced between a functional copy of the fluorescent protein mCherry and an out-of-frame copy of EGFP; in vivo frameshifting will lead to EGFP expression, which can be quantified by fluorescence activated cell sorting (FACS). Via targeted recombineering, single copy reporters were introduced in HEK293 and MCF-7 cells. We found predominantly -1 and +1 base pair frameshifts, the levels of which are kept in tune by mismatch repair. We show that tract length and composition greatly influences MSI. In contrast, a tracts' potential to form a G-quadruplex structure, its strand orientation or its transcriptional status is not affecting MSI. We further validated the functionality of the reporter system for screening microsatellite mutagenicity of compounds and for identifying modifiers of MSI: using a retroviral miRNA expression library, we identified miR-21, which targets MSH2, as a miRNA that induces MSI when overexpressed. Our data also provide proof of principle for the strategy of combining fluorescent reporters with next-generation sequencing technology to identify genetic factors in specific pathways.

36 CFR 1225.24 - When can an agency apply previously approved schedules to electronic records?

Code of Federal Regulations, 2010 CFR

2010-07-01

... may apply a previously approved schedule for hard copy records to electronic versions of the permanent records when the electronic records system replaces a single series of hard copy permanent records or the... have been previously scheduled as permanent in hard copy form, including special media records as...

Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110.

PubMed

Fomukong, N G; Tang, T H; al-Maamary, S; Ibrahim, W A; Ramayah, S; Yates, M; Zainuddin, Z F; Dale, J W

1994-12-01

DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

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

Characterization of the complete chloroplast genome of Platycarya strobilacea (Juglandaceae)

Treesearch

Jing Yan; Kai Han; Shuyun Zeng; Peng Zhao; Keith Woeste; Jianfang Li; Zhan-Lin Liu

2017-01-01

The whole chloroplast genome (cp genome) sequence of Platycarya strobilacea was characterized from Illumina pair-end sequencing data. The complete cp genome was 160,994 bp in length and contained a large single copy region (LSC) of 90,225 bp and a small single copy region (SSC) of 18,371 bp, which were separated by a pair of inverted repeat regions...

Nucleotide sequence of soybean chloroplast DNA regions which contain the psb A and trn H genes and cover the ends of the large single copy region and one end of the inverted repeats.

PubMed

Spielmann, A; Stutz, E

1983-10-25

The soybean chloroplast psb A gene (photosystem II thylakoid membrane protein of Mr 32 000, lysine-free) and the trn H gene (tRNAHisGUG), which both map in the large single copy region adjacent to one of the inverted repeat structures (IR1), have been sequenced including flanking regions. The psb A gene shows in its structural part 92% sequence homology with the corresponding genes of spinach and N. debneyi and contains also an open reading frame for 353 aminoacids. The aminoacid sequence of a potential primary translation product (calculated Mr, 38 904, no lysine) diverges from that of spinach and N. debneyi in only two positions in the C-terminal part. The trn H gene has the same polarity as the psb A gene and the coding region is located at the very end of the large single copy region. The deduced sequence of the soybean chloroplast tRNAHisGUG is identical with that of Zea mays chloroplasts. Both ends of the large single copy region were sequenced including a small segment of the adjacent IR1 and IR2.

Quetzal: a transposon of the Tc1 family in the mosquito Anopheles albimanus.

PubMed

Ke, Z; Grossman, G L; Cornel, A J; Collins, F H

1996-10-01

A member of the Tc1 family of transposable elements has been identified in the Central and South American mosquito Anopheles albimanus. The full-length Quetzal element is 1680 base pairs (bp) in length, possesses 236 bp inverted terminal repeats (ITRs), and has a single open reading frame (ORF) with the potential of encoding a 341-amino-acid (aa) protein that is similar to the transposases of other members of the Tc1 family, particularly elements described from three different Drosophila species. The approximately 10-12 copies per genome of Quetzal are found in the euchromatin of all three chromosomes of A. albimanus. One full-length clone, Que27, appears capable of encoding a complete transposase and may represent a functional copy of this element.

Construction of a restriction map and gene map of the lettuce chloroplast small single-copy region using Southern cross-hybridization.

PubMed

Mitchelson, K R

1996-01-01

The small single-copy region (SSCR) of the chloroplast genome of many higher plants typically contain ndh genes encoding proteins that share homology with subunits of the respiratory-chain reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase complex of mitochondria. A map of the lettuce chloroplast SSCR has been determined by Southern cross-hybridization, taking advantage of the high degree of homology between a tobacco small single-copy fragment and a corresponding lettuce chloroplast fragment. The gene order of the SSCR of lettuce and tobacco chloroplasts is similar. The cross-hybridization method can rapidly create a primary gene map of unknown chloroplast fragments, thus providing detailed information of the localization and arrangement of genes and conserved open reading frame regions.

A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2.

PubMed

Wiesenfahrt, Tobias; Duanmu, Jingjie; Snider, Frances; Moerman, Don; Au, Vinci; Li-Leger, Erica; Flibotte, Stephane; Parker, Dylan M; Marshall, Craig J; Nishimura, Erin Osborne; Mains, Paul E; McGhee, James D

2018-05-04

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p :: elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p :: elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82 , was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p :: elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen's design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10 -4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations. Copyright © 2018 Wiesenfahrt et al.

MicroRNA duplication accelerates the recruitment of new targets during vertebrate evolution

PubMed Central

Luo, Junjie; Wang, Yirong; Yuan, Jian

2018-01-01

The repertoire of miRNAs has considerably expanded during metazoan evolution, and duplication is an important mechanism for generating new functional miRNAs. However, relatively little is known about the functional divergence between paralogous miRNAs and the possible coevolution between duplicated miRNAs and the genomic contexts. By systematically examining small RNA expression profiles across various human tissues and interrogating the publicly available miRNA:mRNA pairing chimeras, we found that changes in expression patterns and targeting preferences are widespread for duplicated miRNAs in vertebrates. Both the empirical interactions and target predictions suggest that evolutionarily conserved homo-seed duplicated miRNAs pair with significantly higher numbers of target sites compared to the single-copy miRNAs. Our birth-and-death evolutionary analysis revealed that the new target sites of miRNAs experienced frequent gains and losses during function development. Our results suggest that a newly emerged target site has a higher probability to be functional and maintained by natural selection if it is paired to a seed shared by multiple paralogous miRNAs rather than being paired to a single-copy miRNA. We experimentally verified the divergence in target repression between two paralogous miRNAs by transfecting let-7a and let-7b mimics into kidney-derived cell lines of four mammalian species and measuring the resulting transcriptome alterations by extensive high-throughput sequencing. Our results also suggest that the gains and losses of let-7 target sites might be associated with the evolution of repressiveness of let-7 across mammalian species. PMID:29511046

75 FR 72832 - Neurological Devices Panel of the Medical Devices Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-26

..., rm. 1061, Rockville, MD 20852. Submit a single copy of electronic comments or a paper copy of any mailed comments, except that individuals may submit one paper copy. Comments are to be identified with... committee hot line/phone line to learn about possible modifications before coming to the meeting. Agenda: On...

Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

PubMed

Li, Chunmei; Yu, Zhilong; Fu, Yusi; Pang, Yuhong; Huang, Yanyi

2017-04-26

We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

The Role of Constitutional Copy Number Variants in Breast Cancer

PubMed Central

Walker, Logan C.; Wiggins, George A.R.; Pearson, John F.

2015-01-01

Constitutional copy number variants (CNVs) include inherited and de novo deviations from a diploid state at a defined genomic region. These variants contribute significantly to genetic variation and disease in humans, including breast cancer susceptibility. Identification of genetic risk factors for breast cancer in recent years has been dominated by the use of genome-wide technologies, such as single nucleotide polymorphism (SNP)-arrays, with a significant focus on single nucleotide variants. To date, these large datasets have been underutilised for generating genome-wide CNV profiles despite offering a massive resource for assessing the contribution of these structural variants to breast cancer risk. Technical challenges remain in determining the location and distribution of CNVs across the human genome due to the accuracy of computational prediction algorithms and resolution of the array data. Moreover, better methods are required for interpreting the functional effect of newly discovered CNVs. In this review, we explore current and future application of SNP array technology to assess rare and common CNVs in association with breast cancer risk in humans. PMID:27600231

Gross rearrangements within the 5'-untranslated region of the picornaviral genomes.

PubMed

Pilipenko, E V; Blinov, V M; Agol, V I

1990-06-11

An analysis of reported nucleotide sequences revealed several cases of gross rearrangements in the 5'-untranslated region (5-UTR) of picornaviral genomes. A large (greater than 100 nt) duplication was discovered in a downstream region of poliovirus 5-UTR involved in the translational control. Properties of the poliovirus mutants with large deletions [Kuge and Nomoto (1987) J. Virol. 61, 1478-1487] show that a single copy of the appropriate repeating unit is compatible with a wild type phenotype of the virus. In contrast to poliovirus and another enterovirus genomes, human rhinovirus RNAs contain only a single copy of this repeating unit. Another similarly large repeat was found in an upstream segment of the bovine enterovirus 5-UTR. A comparison of the primary and secondary structures of cardio- and aphthovirus 5-UTRs demonstrated the existence of a large (ca. 250 nucleotides) insertion/deletion in a region preceding the poly(C) tract. The two latter rearrangements appear to involve elements of the viral genome replication machinery. Possible origin as well as evolutionary and functional implications of these structural peculiarities are discussed.

A local duplication of the Melanocortin receptor 1 locus in Astyanax

PubMed Central

Gross, Joshua B.; Weagley, James; Stahl, Bethany A.; Ma, Li; Espinasa, Luis; McGaugh, Suzanne E.

2017-01-01

In this study, we report evidence of a novel duplication of Melanocortin receptor 1 (Mc1r) in the cavefish genome. This locus was discovered following the observation of excessive allelic diversity in a ~820 bp fragment of Mc1r amplified via degenerate PCR from a natural population of Astyanax aeneus fish from Guerrero, Mexico. The cavefish genome reveals the presence of two closely related Mc1r open reading frames separated by a 1.46 kb intergenic region. One open reading frame corresponds to the previously reported Mc1r receptor, and the other open reading frame (duplicate copy) is 975 bp in length, encoding a receptor of 325 amino acids. Sequence similarity analyses position both copies in the syntenic region of the single Mc1r locus in 16 representative craniate genomes spanning bony fish (including Astyanax) to mammals, suggesting we discovered tandem duplicates of this important gene. The two Mc1r copies share ~89% sequence similarity, and, within Astyanax, are more similar to one another compared to other melanocortin family members. Future studies will inform the precise functional significance of the duplicated Mc1r locus, and if this novel copy number variant may have adaptive significance for the Astyanax lineage. PMID:28738163

Single-molecule studies of multi-protein machines

NASA Astrophysics Data System (ADS)

van Oijen, Antoine

2010-03-01

Advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. In a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. Our group is developing the single-molecule tools that will make it possible to study biochemical pathways of arbitrary complexity at the single-molecule level. I will discuss results of single-molecule experiments on the replisome, the molecular machinery that is responsible for replication of DNA. We stretch individual DNA molecules and use their elastic properties to obtain dynamic information on the proteins that unwind the double helix and copy its genetic information. Furthermore, we visualize fluorescently labeled components of the replisome and thus obtain information on stochiometry and exchange kinetics. This simultaneous observation of catalytic activity and composition allows us to gain deeper insight into the structure-function relationship of the replisome.

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

Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells.

PubMed

Meyers, Robin M; Bryan, Jordan G; McFarland, James M; Weir, Barbara A; Sizemore, Ann E; Xu, Han; Dharia, Neekesh V; Montgomery, Phillip G; Cowley, Glenn S; Pantel, Sasha; Goodale, Amy; Lee, Yenarae; Ali, Levi D; Jiang, Guozhi; Lubonja, Rakela; Harrington, William F; Strickland, Matthew; Wu, Ting; Hawes, Derek C; Zhivich, Victor A; Wyatt, Meghan R; Kalani, Zohra; Chang, Jaime J; Okamoto, Michael; Stegmaier, Kimberly; Golub, Todd R; Boehm, Jesse S; Vazquez, Francisca; Root, David E; Hahn, William C; Tsherniak, Aviad

2017-12-01

The CRISPR-Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number-amplified regions. We developed CERES, a computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy number-specific effect. In our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this data set. We found that CERES decreased false-positive results and estimated sgRNA activity for both this data set and previously published screens performed with different sgRNA libraries. We further demonstrate the utility of this collection of screens, after CERES correction, for identifying cancer-type-specific vulnerabilities.

Computational correction of copy-number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells

PubMed Central

Meyers, Robin M.; Bryan, Jordan G.; McFarland, James M.; Weir, Barbara A.; Sizemore, Ann E.; Xu, Han; Dharia, Neekesh V.; Montgomery, Phillip G.; Cowley, Glenn S.; Pantel, Sasha; Goodale, Amy; Lee, Yenarae; Ali, Levi D.; Jiang, Guozhi; Lubonja, Rakela; Harrington, William F.; Strickland, Matthew; Wu, Ting; Hawes, Derek C.; Zhivich, Victor A.; Wyatt, Meghan R.; Kalani, Zohra; Chang, Jaime J.; Okamoto, Michael; Stegmaier, Kimberly; Golub, Todd R.; Boehm, Jesse S.; Vazquez, Francisca; Root, David E.; Hahn, William C.; Tsherniak, Aviad

2017-01-01

The CRISPR-Cas9 system has revolutionized gene editing both on single genes and in multiplexed loss-of-function screens, enabling precise genome-scale identification of genes essential to proliferation and survival of cancer cells1,2. However, previous studies reported that a gene-independent anti-proliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, leading to false positive results in copy number amplified regions3,4. We developed CERES, a computational method to estimate gene dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy-number-specific effect. As part of our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this dataset. We found that CERES reduced false positive results and estimated sgRNA activity for both this dataset and previously published screens performed with different sgRNA libraries. Here, we demonstrate the utility of this collection of screens, upon CERES correction, in revealing cancer-type-specific vulnerabilities. PMID:29083409

Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells.

PubMed

Tamminen, Manu V; Virta, Marko P J

2015-01-01

Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1), or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

Draft genomes of two blister beetles Hycleus cichorii and Hycleus phaleratus

PubMed Central

Wu, Yuan-Ming; Li, Jiang

2018-01-01

Abstract Background Commonly known as blister beetles or Spanish fly, there are more than 1500 species in the Meloidae family (Hexapoda: Coleoptera: Tenebrionoidea) that produce the potent defensive blistering agent cantharidin. Cantharidin and its derivatives have been used to treat cancers such as liver, stomach, lung, and esophageal cancers. Hycleus cichorii and Hycleus phaleratus are the most commercially important blister beetles in China due to their ability to biosynthesize this potent vesicant. However, there is a lack of genome reference, which has hindered development of studies on the biosynthesis of cantharidin and a better understanding of its biology and pharmacology. Results We report 2 draft genomes and quantified gene sets for the blister beetles H. cichorii and H. phaleratus, 2 complex genomes with >72% repeats and approximately 1% heterozygosity, using Illumina sequencing data. An integrated assembly pipeline was performed for assembly, and most of the coding regions were obtained. Benchmarking universal single-copy orthologs (BUSCO) assessment showed that our assembly obtained more than 98% of the Endopterygota universal single-copy orthologs. Comparison analysis showed that the completeness of coding genes in our assembly was comparable to other beetle genomes such as Dendroctonus ponderosae and Agrilus planipennis. Gene annotation yielded 13 813 and 13 725 protein-coding genes in H. cichorii and H. phaleratus, of which approximately 89% were functionally annotated. BUSCO assessment showed that approximately 86% and 84% of the Endopterygota universal single-copy orthologs were annotated completely in these 2 gene sets, whose completeness is comparable to that of D. ponderosae and A. planipennis. Conclusions Assembly of both blister beetle genomes provides a valuable resource for future biosynthesis of cantharidin and comparative genomic studies of blister beetles and other beetles. PMID:29444297

Draft genomes of two blister beetles Hycleus cichorii and Hycleus phaleratus.

PubMed

Wu, Yuan-Ming; Li, Jiang; Chen, Xiang-Sheng

2018-03-01

Commonly known as blister beetles or Spanish fly, there are more than 1500 species in the Meloidae family (Hexapoda: Coleoptera: Tenebrionoidea) that produce the potent defensive blistering agent cantharidin. Cantharidin and its derivatives have been used to treat cancers such as liver, stomach, lung, and esophageal cancers. Hycleus cichorii and Hycleus phaleratus are the most commercially important blister beetles in China due to their ability to biosynthesize this potent vesicant. However, there is a lack of genome reference, which has hindered development of studies on the biosynthesis of cantharidin and a better understanding of its biology and pharmacology. We report 2 draft genomes and quantified gene sets for the blister beetles H. cichorii and H. phaleratus, 2 complex genomes with >72% repeats and approximately 1% heterozygosity, using Illumina sequencing data. An integrated assembly pipeline was performed for assembly, and most of the coding regions were obtained. Benchmarking universal single-copy orthologs (BUSCO) assessment showed that our assembly obtained more than 98% of the Endopterygota universal single-copy orthologs. Comparison analysis showed that the completeness of coding genes in our assembly was comparable to other beetle genomes such as Dendroctonus ponderosae and Agrilus planipennis. Gene annotation yielded 13 813 and 13 725 protein-coding genes in H. cichorii and H. phaleratus, of which approximately 89% were functionally annotated. BUSCO assessment showed that approximately 86% and 84% of the Endopterygota universal single-copy orthologs were annotated completely in these 2 gene sets, whose completeness is comparable to that of D. ponderosae and A. planipennis. Assembly of both blister beetle genomes provides a valuable resource for future biosynthesis of cantharidin and comparative genomic studies of blister beetles and other beetles.

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.

Nucleotide sequence of soybean chloroplast DNA regions which contain the psb A and trn H genes and cover the ends of the large single copy region and one end of the inverted repeats.

PubMed Central

Spielmann, A; Stutz, E

1983-01-01

The soybean chloroplast psb A gene (photosystem II thylakoid membrane protein of Mr 32 000, lysine-free) and the trn H gene (tRNAHisGUG), which both map in the large single copy region adjacent to one of the inverted repeat structures (IR1), have been sequenced including flanking regions. The psb A gene shows in its structural part 92% sequence homology with the corresponding genes of spinach and N. debneyi and contains also an open reading frame for 353 aminoacids. The aminoacid sequence of a potential primary translation product (calculated Mr, 38 904, no lysine) diverges from that of spinach and N. debneyi in only two positions in the C-terminal part. The trn H gene has the same polarity as the psb A gene and the coding region is located at the very end of the large single copy region. The deduced sequence of the soybean chloroplast tRNAHisGUG is identical with that of Zea mays chloroplasts. Both ends of the large single copy region were sequenced including a small segment of the adjacent IR1 and IR2. PMID:6314279

Genes and gene expression: Localization, damage and control: A multilevel and inter-disciplinary study

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

Ts'o, P.O.P.

1990-09-01

The main objectives of this Program Project is to develop strategy and technology for the study of gene structure, organization and function in a multi-disciplinary, highly coordinated manner. In Project I, Molecular Cytology, the establishment of all instrumentation for the computerized microscopic imaging system (CMIS) has been completed with the software in place, including measurement of the third dimension (along the Z-axis). The technique is now at hand to measure single copy DNA in the nucleus, single copy mRNA in the cell, and finally, we are in the process of developing mathematical approaches for the analysis of the relative spatialmore » 3-D relationship among the chromosomes and the individual genes in the interphasal nucleus. Also, we have a sensitive and reliable method for measuring single-stranded DNA breaks which will be useful for the determination of damage to DNA caused by ionizing radiation. In Project II, the mapping of restriction fragments by 2-D enzymatic and electrophoretic analysis has been perfected for application. In Project III, a major finding is that the binding constant and effectiveness of antisense oligonucleotide analogues, Matagen, can be significantly improved by substituting 2{prime}-O-methylribos methylphosphonate backbones for the current 2{prime}-deoxyribomethylphosphonate backbones. 15 refs., 10 figs., 2 tabs.« less

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

PubMed

Zhang, Huifa; Jenkins, Gareth; Zou, Yuan; Zhu, Zhi; Yang, Chaoyong James

2012-04-17

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

PubMed

Möhlendick, Birte; Bartenhagen, Christoph; Behrens, Bianca; Honisch, Ellen; Raba, Katharina; Knoefel, Wolfram T; Stoecklein, Nikolas H

2013-01-01

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

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.

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

PubMed

Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

2016-04-19

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Multiple Genetic Backgrounds of the Amplified Plasmodium falciparum Multidrug Resistance (pfmdr1) Gene and Selective Sweep of 184F Mutation in Cambodia

PubMed Central

Vinayak, Sumiti; Alam, Md Tauqeer; Sem, Rithy; Shah, Naman K.; Susanti, Augustina I.; Lim, Pharath; Muth, Sinuon; Maguire, Jason D.; Rogers, William O.; Fandeur, Thierry; Barnwell, John W.; Escalante, Ananias A.; Wongsrichanalai, Chansuda; Ariey, Frederick; Meshnick, Steven R.; Udhayakumar, Venkatachalam

2011-01-01

Background The emergence of artesunate-mefloquine (AS+MQ)–resistant Plasmodium falciparum in the Thailand-Cambodia region is a major concern for malaria control. Studies indicate that copy number increase and key alleles in the pfmdr1 gene are associated with AS+MQ resistance. In the present study, we investigated evidence for a selective sweep around pfmdr1 because of the spread of adaptive mutation and/or multiple copies of this gene in the P. falciparum population in Cambodia. Methods We characterized 13 microsatellite loci flanking (± 99 kb) pfmdr1 in 93 single-clone P. falciparum infections, of which 31 had multiple copies and 62 had a single copy of the pfmdr1 gene. Results Genetic analysis revealed no difference in the mean (± standard deviation) expected heterozygosity (He) at loci around single (0.75 ± 0.03) and multiple (0.76 ± 0.04) copies of pfmdr1. Evidence of genetic hitchhiking with the selective sweep of certain haplotypes was seen around mutant (184F) pfmdr1 allele, irrespective of the copy number. There was an overall reduction of 28% in mean He (± SD) around mutant allele (0.56 ± 0.05), compared with wild-type allele (0.84 ± 0.02). Significant linkage disequilibrium was also observed between the loci flanking mutant pfmdr1 allele. Conclusion The 184F mutant allele is under selection, whereas amplification of pfmdr1 gene in this population occurs on multiple genetic backgrounds. PMID:20367478

Molecular adaptation in the world's deepest-living animal: Insights from transcriptome sequencing of the hadal amphipod Hirondellea gigas.

PubMed

Lan, Yi; Sun, Jin; Tian, Renmao; Bartlett, Douglas H; Li, Runsheng; Wong, Yue Him; Zhang, Weipeng; Qiu, Jian-Wen; Xu, Ting; He, Li-Sheng; Tabata, Harry G; Qian, Pei-Yuan

2017-07-01

The Challenger Deep in the Mariana Trench is the deepest point in the oceans of our planet. Understanding how animals adapt to this harsh environment characterized by high hydrostatic pressure, food limitation, dark and cold is of great scientific interest. Of the animals dwelling in the Challenger Deep, amphipods have been captured using baited traps. In this study, we sequenced the transcriptome of the amphipod Hirondellea gigas collected at a depth of 10,929 m from the East Pond of the Challenger Deep. Assembly of these sequences resulted in 133,041 contigs and 22,046 translated proteins. Functional annotation of these contigs was made using the go and kegg databases. Comparison of these translated proteins with those of four shallow-water amphipods revealed 10,731 gene families, of which 5659 were single-copy orthologs. Base substitution analysis on these single-copy orthologs showed that 62 genes are positively selected in H. gigas, including genes related to β-alanine biosynthesis, energy metabolism and genetic information processing. For multiple-copy orthologous genes, gene family expansion analysis revealed that cold-inducible proteins (i.e., transcription factors II A and transcription elongation factor 1) as well as zinc finger domains are expanded in H. gigas. Overall, our results indicate that genetic adaptation to the hadal environment by H. gigas may be mediated by both gene family expansion and amino acid substitutions of specific proteins. © 2017 John Wiley & Sons Ltd.

Application of next generation sequencing toward sensitive detection of enteric viruses isolated from celery samples as an example of produce.

PubMed

Yang, Zhihui; Mammel, Mark; Papafragkou, Efstathia; Hida, Kaoru; Elkins, Christopher A; Kulka, Michael

2017-11-16

Next generation sequencing (NGS) holds promise as a single application for both detection and sequence identification of foodborne viruses; however, technical challenges remain due to anticipated low quantities of virus in contaminated food. In this study, with a focus on data analysis using several bioinformatics tools, we applied NGS toward amplification-independent detection and identification of norovirus at low copy (<10 3 copies) or within multiple strains from produce. Celery samples were inoculated with human norovirus (stool suspension) either as a single norovirus strain, a mixture of strains (GII.4 and GII.6), or a mixture of different species (hepatitis A virus and norovirus). Viral RNA isolation and recovery was confirmed by RT-qPCR, and optimized for library generation and sequencing without amplification using the Illumina MiSeq platform. Extracts containing either a single virus or a two-virus mixture were analyzed using two different analytic approaches to achieve virus detection and identification. First an overall assessment of viral genome coverage for samples varying in copy numbers (1.1×10 3 to 1.7×10 7 ) and genomic content (single or multiple strains in various ratios) was completed by reference-guided mapping. Not unexpectedly, this targeted approach to identification was successful in correctly mapping reads, thus identifying each virus contained in the inoculums even at low copy (estimated at 12 copies). For the second (metagenomic) approach, samples were treated as "unknowns" for data analyses using (i) a sequence-based alignment with a local database, (ii) an "in-house" k-mer tool, (iii) a commercially available metagenomics bioinformatic analysis platform cosmosID, and (iv) an open-source program Kraken. Of the four metagenomics tools applied in this study, only the local database alignment and in-house k-mer tool were successful in detecting norovirus (as well as HAV) at low copy (down to <10 3 copies) and within a mixture of virus strains or species. The results of this investigation provide support for continued investigation into the development and integration of these analytical tools for identification and detection of foodborne viruses. Published by Elsevier B.V.

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

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

Making Aggressive Prostate Cancer Quiescent by Abrogating Cholesterol Esterification

DTIC Science & Technology

2017-10-01

and prostate cancer biology (Dr. T. Ratliff, co-PI). 15. SUBJECT TERMS- 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES...X. Liu, co-PI), and prostate cancer biology (Dr. T. Ratliff, co-PI). 2. KEYWORDS: Prostate cancer, lipid droplet, metabolism, cholesterol...presentations: 10 09-09-2016, “Lipid metabolism: from single cell biology to in vivo diagnosis”, Big Ten Cancer Research Consortium Summit, Indianapolis

Quantum jointly assisted cloning of an unknown three-dimensional equatorial state

NASA Astrophysics Data System (ADS)

Ma, Peng-Cheng; Chen, Gui-Bin; Li, Xiao-Wei; Zhan, You-Bang

2018-02-01

We present two schemes for perfectly cloning an unknown single-qutrit equatorial state with assistance from two and N state preparers, respectively. In the first scheme, the sender wishes to teleport an unknown single-qutrit equatorial state from two state preparers to a remote receiver, and then to create a perfect copy of the unknown state at her location. The scheme consists of two stages. The first stage of the scheme requires the usual teleportation. In the second stage, to help the sender realize the quantum cloning, two state preparers perform single-qutrit projective measurements on their own qutrits from the sender, then the sender can acquire a perfect copy of the unknown state. It is shown that, only if the two state preparers collaborate with each other, the sender can create a copy of the unknown state by means of some appropriate unitary operations. In the second scheme, we generalized the jointly assisted cloning in the first scheme to the case of N state prepares. In the present schemes, the total probability of success for assisted cloning of a perfect copy of the unknown state can reach 1.

nbCNV: a multi-constrained optimization model for discovering copy number variants in single-cell sequencing data.

PubMed

Zhang, Changsheng; Cai, Hongmin; Huang, Jingying; Song, Yan

2016-09-17

Variations in DNA copy number have an important contribution to the development of several diseases, including autism, schizophrenia and cancer. Single-cell sequencing technology allows the dissection of genomic heterogeneity at the single-cell level, thereby providing important evolutionary information about cancer cells. In contrast to traditional bulk sequencing, single-cell sequencing requires the amplification of the whole genome of a single cell to accumulate enough samples for sequencing. However, the amplification process inevitably introduces amplification bias, resulting in an over-dispersing portion of the sequencing data. Recent study has manifested that the over-dispersed portion of the single-cell sequencing data could be well modelled by negative binomial distributions. We developed a read-depth based method, nbCNV to detect the copy number variants (CNVs). The nbCNV method uses two constraints-sparsity and smoothness to fit the CNV patterns under the assumption that the read signals are negatively binomially distributed. The problem of CNV detection was formulated as a quadratic optimization problem, and was solved by an efficient numerical solution based on the classical alternating direction minimization method. Extensive experiments to compare nbCNV with existing benchmark models were conducted on both simulated data and empirical single-cell sequencing data. The results of those experiments demonstrate that nbCNV achieves superior performance and high robustness for the detection of CNVs in single-cell sequencing data.

Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR.

PubMed

Leng, Xuefei; Zhang, Wenhua; Wang, Chunming; Cui, Liang; Yang, Chaoyong James

2010-11-07

An agarose droplet method was developed for highly parallel and efficient single molecule emulsion PCR. The method capitalizes on the unique thermoresponsive sol-gel switching property of agarose for highly efficient DNA amplification and amplicon trapping. Uniform agarose solution droplets generated via a microfluidic chip serve as robust and inert nanolitre PCR reactors for single copy DNA molecule amplification. After PCR, agarose droplets are gelated to form agarose beads, trapping all amplicons in each reactor to maintain the monoclonality of each droplet. This method does not require cocapsulation of primer labeled microbeads, allows high throughput generation of uniform droplets and enables high PCR efficiency, making it a promising platform for many single copy genetic studies.

Genetic Structures of Copy Number Variants Revealed by Genotyping Single Sperm

PubMed Central

Luo, Minjie; Cui, Xiangfeng; Fredman, David; Brookes, Anthony J.; Azaro, Marco A.; Greenawalt, Danielle M.; Hu, Guohong; Wang, Hui-Yun; Tereshchenko, Irina V.; Lin, Yong; Shentu, Yue; Gao, Richeng; Shen, Li; Li, Honghua

2009-01-01

Background Copy number variants (CNVs) occupy a significant portion of the human genome and may have important roles in meiotic recombination, human genome evolution and gene expression. Many genetic diseases may be underlain by CNVs. However, because of the presence of their multiple copies, variability in copy numbers and the diploidy of the human genome, detailed genetic structure of CNVs cannot be readily studied by available techniques. Methodology/Principal Findings Single sperm samples were used as the primary subjects for the study so that CNV haplotypes in the sperm donors could be studied individually. Forty-eight CNVs characterized in a previous study were analyzed using a microarray-based high-throughput genotyping method after multiplex amplification. Seventeen single nucleotide polymorphisms (SNPs) were also included as controls. Two single-base variants, either allelic or paralogous, could be discriminated for all markers. Microarray data were used to resolve SNP alleles and CNV haplotypes, to quantitatively assess the numbers and compositions of the paralogous segments in each CNV haplotype. Conclusions/Significance This is the first study of the genetic structure of CNVs on a large scale. Resulting information may help understand evolution of the human genome, gain insight into many genetic processes, and discriminate between CNVs and SNPs. The highly sensitive high-throughput experimental system with haploid sperm samples as subjects may be used to facilitate detailed large-scale CNV analysis. PMID:19384415

48 CFR 52.223-13 - Acquisition of EPEAT®-Registered Imaging Equipment.

Code of Federal Regulations, 2014 CFR

2014-10-01

...) Facsimile machine (fax machine)—A commercially available imaging product whose primary functions are... available imaging product with a sole function of the production of hard copy duplicates from graphic hard... functionally integrated components, that performs two or more of the core functions of copying, printing...

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.

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

Phylotranscriptomic analysis uncovers a wealth of tissue inhibitor of metalloproteinases variants in echinoderms

PubMed Central

Clouse, Ronald M.; Linchangco, Gregorio V.; Kerr, Alexander M.; Reid, Robert W.; Janies, Daniel A.

2015-01-01

Tissue inhibitors of metalloproteinases (TIMPs) help regulate the extracellular matrix (ECM) in animals, mostly by inhibiting matrix metalloproteinases (MMPs). They are important activators of mutable collagenous tissue (MCT), which have been extensively studied in echinoderms, and the four TIMP copies in humans have been studied for their role in cancer. To understand the evolution of TIMPs, we combined 405 TIMPs from an echinoderm transcriptome dataset built from 41 specimens representing all five classes of echinoderms with variants from protostomes and chordates. We used multiple sequence alignment with various stringencies of alignment quality to cull highly divergent sequences and then conducted phylogenetic analyses using both nucleotide and amino acid sequences. Phylogenetic hypotheses consistently recovered TIMPs as diversifying in the ancestral deuterostome and these early lineages continuing to diversify in echinoderms. The four vertebrate TIMPs diversified from a single copy in the ancestral chordate, all other copies being lost. Consistent with greater MCT needs owing to body wall liquefaction, evisceration, autotomy and reproduction by fission, holothuroids had significantly more TIMPs and higher read depths per contig. Ten cysteine residues, an HPQ binding site and several other residues were conserved in at least 70% of all TIMPs. The conservation of binding sites and the placement of echinoderm TIMPs involved in MCT modification suggest that ECM regulation remains the primary function of TIMP genes, although within this role there are a large number of specialized copies. PMID:27017967

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.

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.

"The role of redundant information in cultural transmission and cultural stabilization": Correction to Acerbi and Tennie (2016).

PubMed

2016-05-01

Reports an error in "The role of redundant information in cultural transmission and cultural stabilization" by Alberto Acerbi and Claudio Tennie (Journal of Comparative Psychology, 2016[Feb], Vol 130[1], 62-70). In the article the copyright should have been "© 2016 The Author(s)". The author note also should have included the following license statement "This article has been published under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright for this article is retained by the author(s). Author(s) grant(s) the American Psychological Association the exclusive right to publish the article and identify itself as the original publisher." The online version of this article has been corrected. (The following abstract of the original article appeared in record 2016-07004-005.) Redundant copying has been proposed as a manner to achieve the high-fidelity necessary to pass on and preserve complex traits in human cultural transmission. There are at least 2 ways to define redundant copying. One refers to the possibility of copying repeatedly the same trait over time, and another to the ability to exploit multiple layers of information pointing to the same trait during a single copying event. Using an individual-based model, we explore how redundant copying (defined as in the latter way) helps to achieve successful transmission. The authors show that increasing redundant copying increases the likelihood of accurately transmitting a behavior more than either augmenting the number of copying occasions across time or boosting the general accuracy of social learning. They also investigate how different cost functions, deriving, for example, from the need to invest more energy in cognitive processing, impact the evolution of redundant copying. The authors show that populations converge either to high-fitness/high-costs states (with high redundant copying and complex culturally transmitted behaviors; resembling human culture) or to low-fitness/low-costs states (with low redundant copying and simple transmitted behaviors; resembling social learning forms typical of nonhuman animals). This outcome may help to explain why cumulative culture is rare in the animal kingdom. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

The maternal-effect, selfish genetic element Medea is associated with a composite Tc1 transposon.

PubMed

Lorenzen, Marcé D; Gnirke, Andreas; Margolis, Jonathan; Garnes, Jeffrey; Campbell, Margie; Stuart, Jeffrey J; Aggarwal, Rajat; Richards, Stephen; Park, Yoonseong; Beeman, Richard W

2008-07-22

Maternal-Effect Dominant Embryonic Arrest ("Medea") factors are selfish nuclear elements that combine maternal-lethal and zygotic-rescue activities to gain a postzygotic survival advantage. We show that Medea(1) activity in Tribolium castaneum is associated with a composite Tc1 transposon inserted just downstream of the neurotransmitter reuptake symporter bloated tubules (blot), whose Drosophila ortholog has both maternal and zygotic functions. The 21.5-kb insertion contains defective copies of elongation initiation factor-3, ATP synthase subunit C, and an RNaseD-related gene, as well as a potentially intact copy of a prokaryotic DUF1703 gene. Sequence comparisons suggest that the current distribution of Medea(1) reflects global emanation after a single transpositional event in recent evolutionary time. The Medea system in Tribolium represents an unusual type of intragenomic conflict and could provide a useful vehicle for driving desirable genes into populations.

The maternal-effect, selfish genetic element Medea is associated with a composite Tc1 transposon

PubMed Central

Lorenzen, Marcé D.; Gnirke, Andreas; Margolis, Jonathan; Garnes, Jeffrey; Campbell, Margie; Stuart, Jeffrey J.; Aggarwal, Rajat; Richards, Stephen; Park, Yoonseong; Beeman, Richard W.

2008-01-01

Maternal-Effect Dominant Embryonic Arrest (“Medea”) factors are selfish nuclear elements that combine maternal-lethal and zygotic-rescue activities to gain a postzygotic survival advantage. We show that Medea1 activity in Tribolium castaneum is associated with a composite Tc1 transposon inserted just downstream of the neurotransmitter reuptake symporter bloated tubules (blot), whose Drosophila ortholog has both maternal and zygotic functions. The 21.5-kb insertion contains defective copies of elongation initiation factor-3, ATP synthase subunit C, and an RNaseD-related gene, as well as a potentially intact copy of a prokaryotic DUF1703 gene. Sequence comparisons suggest that the current distribution of Medea1 reflects global emanation after a single transpositional event in recent evolutionary time. The Medea system in Tribolium represents an unusual type of intragenomic conflict and could provide a useful vehicle for driving desirable genes into populations. PMID:18621706

An Improved Binary Differential Evolution Algorithm to Infer Tumor Phylogenetic Trees.

PubMed

Liang, Ying; Liao, Bo; Zhu, Wen

2017-01-01

Tumourigenesis is a mutation accumulation process, which is likely to start with a mutated founder cell. The evolutionary nature of tumor development makes phylogenetic models suitable for inferring tumor evolution through genetic variation data. Copy number variation (CNV) is the major genetic marker of the genome with more genes, disease loci, and functional elements involved. Fluorescence in situ hybridization (FISH) accurately measures multiple gene copy number of hundreds of single cells. We propose an improved binary differential evolution algorithm, BDEP, to infer tumor phylogenetic tree based on FISH platform. The topology analysis of tumor progression tree shows that the pathway of tumor subcell expansion varies greatly during different stages of tumor formation. And the classification experiment shows that tree-based features are better than data-based features in distinguishing tumor. The constructed phylogenetic trees have great performance in characterizing tumor development process, which outperforms other similar algorithms.

Micro-Plasticity of Genomes As Illustrated by the Evolution of Glutathione Transferases in 12 Drosophila Species

PubMed Central

Saisawang, Chonticha; Ketterman, Albert J.

2014-01-01

Glutathione transferases (GST) are an ancient superfamily comprising a large number of paralogous proteins in a single organism. This multiplicity of GSTs has allowed the copies to diverge for neofunctionalization with proposed roles ranging from detoxication and oxidative stress response to involvement in signal transduction cascades. We performed a comparative genomic analysis using FlyBase annotations and Drosophila melanogaster GST sequences as templates to further annotate the GST orthologs in the 12 Drosophila sequenced genomes. We found that GST genes in the Drosophila subgenera have undergone repeated local duplications followed by transposition, inversion, and micro-rearrangements of these copies. The colinearity and orientations of the orthologous GST genes appear to be unique in many of the species which suggests that genomic rearrangement events have occurred multiple times during speciation. The high micro-plasticity of the genomes appears to have a functional contribution utilized for evolution of this gene family. PMID:25310450

Functional analysis of chloroplast early light inducible proteins (ELIPs)

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

Wetzel, Carolyn M

The objectives of this project were to characterize gene expression patterns of early light inducible protein (ELIP) genes in Arabidopsis thaliana and in Lycopersicon esculentum, to identify knock mutants of the 2 ELIP genes in Arabidopsis, and to characterize the effects of the knockouts. Expression in Arabidopsis was studied in response to thylakoid electron transport chain (PETC) capacity, where it was found that there is a signal for expression associated with reduction of the PETC. Expression in response to salt was also studied, with different responses of the two gene copies. Knockout lines for ELIP1 and ELIP2 have been identifiedmore » and are being characterized. In tomato, it was found that the single-copy ELIP gene is highly expressed in ripening fruit during the chloroplast-to-chromoplast transition. Studies of expression in tomato ripening mutants are ongoing.« less

The complete chloroplast genome sequence of Actinidia arguta using the PacBio RS II platform

PubMed Central

Lin, Miaomiao; Qi, Xiujuan; Chen, Jinyong; Sun, Leiming; Zhong, Yunpeng; Fang, Jinbao; Hu, Chungen

2018-01-01

Actinidia arguta is the most basal species in a phylogenetically and economically important genus in the family Actinidiaceae. To better understand the molecular basis of the Actinidia arguta chloroplast (cp), we sequenced the complete cp genome from A. arguta using Illumina and PacBio RS II sequencing technologies. The cp genome from A. arguta was 157,611 bp in length and composed of a pair of 24,232 bp inverted repeats (IRs) separated by a 20,463 bp small single copy region (SSC) and an 88,684 bp large single copy region (LSC). Overall, the cp genome contained 113 unique genes. The cp genomes from A. arguta and three other Actinidia species from GenBank were subjected to a comparative analysis. Indel mutation events and high frequencies of base substitution were identified, and the accD and ycf2 genes showed a high degree of variation within Actinidia. Forty-seven simple sequence repeats (SSRs) and 155 repetitive structures were identified, further demonstrating the rapid evolution in Actinidia. The cp genome analysis and the identification of variable loci provide vital information for understanding the evolution and function of the chloroplast and for characterizing Actinidia population genetics. PMID:29795601

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.

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

And then there were 12--distinguishing Van Leeuwenhoek microscopes from old or new copies.

PubMed

Robertson, Lesley A

2015-07-01

In the wake of announcements of the authentications of two previously unknown Van Leeuwenhoek microscopes in one month, this paper reviews the possibilities and potential pitfalls that might be involved in distinguishing 17th/18th century single-lensed microscopes from historical and modern copies. It is clear that a combination of characteristics must be considered, no single parameter will do. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Human Matrix Attachment Regions Are Necessary for the Establishment but Not the Maintenance of Transgene Insulation in Drosophila melanogaster

PubMed Central

Namciu, Stephanie J.; Fournier, R. E. K.

2004-01-01

Human matrix attachment regions (MARs) can insulate transgene expression from chromosomal position effects in Drosophila melanogaster. To gain insight into the mechanism(s) by which chromosomal insulation occurs, we studied the expression phenotypes of Drosophila transformants expressing mini-white transgenes in which MAR sequences from the human apoB gene were arranged in a variety of ways. In agreement with previous reports, we found that a single copy of the insulating element was not sufficient for position-independent transgene expression; rather, two copies were required. However, the arrangement of the two elements within the transgene was unimportant, since chromosomal insulation was equally apparent when both copies of the insulator were upstream of the mini-white reporter as when the transcription unit was flanked by insulator elements. Moreover, experiments in which apoB 3′ MAR sequences were removed from integrated transgenes in vivo by site-specific recombination demonstrated that MAR sequences were required for the establishment but not for the maintenance of chromosomal insulation. These observations are not compatible with the chromosomal loop model in its simplest form. Alternate mechanisms for MAR function in this system are proposed. PMID:15542833

Human neuronal changes in brain edema and increased intracranial pressure.

PubMed

Faragó, Nóra; Kocsis, Ágnes Katalin; Braskó, Csilla; Lovas, Sándor; Rózsa, Márton; Baka, Judith; Kovács, Balázs; Mikite, Katalin; Szemenyei, Viktor; Molnár, Gábor; Ozsvár, Attila; Oláh, Gáspár; Piszár, Ildikó; Zvara, Ágnes; Patócs, Attila; Barzó, Pál; Puskás, László G; Tamás, Gábor

2016-08-04

Functional and molecular changes associated with pathophysiological conditions are relatively easily detected based on tissue samples collected from patients. Population specific cellular responses to disease might remain undiscovered in samples taken from organs formed by a multitude of cell types. This is particularly apparent in the human cerebral cortex composed of a yet undefined number of neuron types with a potentially different involvement in disease processes. We combined cellular electrophysiology, anatomy and single cell digital PCR in human neurons identified in situ for the first time to assess mRNA expression and corresponding functional changes in response to edema and increased intracranial pressure. In single pyramidal cells, mRNA copy numbers of AQP1, AQP3, HMOX1, KCNN4, SCN3B and SOD2 increased, while CACNA1B, CRH decreased in edema. In addition, single pyramidal cells increased the copy number of AQP1, HTR5A and KCNS1 mRNAs in response to increased intracranial pressure. In contrast to pyramidal cells, AQP1, HMOX1and KCNN4 remained unchanged in single cell digital PCR performed on fast spiking cells in edema. Corroborating single cell digital PCR results, pharmacological and immunohistochemical results also suggested the presence of KCNN4 encoding the α-subunit of KCa3.1 channels in edema on pyramidal cells, but not on interneurons. We measured the frequency of spontaneous EPSPs on pyramidal cells in both pathophysiological conditions and on fast spiking interneurons in edema and found a significant decrease in each case, which was accompanied by an increase in input resistances on both cell types and by a drop in dendritic spine density on pyramidal cells consistent with a loss of excitatory synapses. Our results identify anatomical and/or physiological changes in human pyramidal and fast spiking cells in edema and increased intracranial pressure revealing cell type specific quantitative changes in gene expression. Some of the edema/increased intracranial pressure modulated and single human pyramidal cell verified gene products identified here might be considered as novel pharmacological targets in cell type specific neuroprotection.

Vocal copying of individually distinctive signature whistles in bottlenose dolphins

PubMed Central

King, Stephanie L.; Sayigh, Laela S.; Wells, Randall S.; Fellner, Wendi; Janik, Vincent M.

2013-01-01

Vocal learning is relatively common in birds but less so in mammals. Sexual selection and individual or group recognition have been identified as major forces in its evolution. While important in the development of vocal displays, vocal learning also allows signal copying in social interactions. Such copying can function in addressing or labelling selected conspecifics. Most examples of addressing in non-humans come from bird song, where matching occurs in an aggressive context. However, in other animals, addressing with learned signals is very much an affiliative signal. We studied the function of vocal copying in a mammal that shows vocal learning as well as complex cognitive and social behaviour, the bottlenose dolphin (Tursiops truncatus). Copying occurred almost exclusively between close associates such as mother–calf pairs and male alliances during separation and was not followed by aggression. All copies were clearly recognizable as such because copiers consistently modified some acoustic parameters of a signal when copying it. We found no evidence for the use of copying in aggression or deception. This use of vocal copying is similar to its use in human language, where the maintenance of social bonds appears to be more important than the immediate defence of resources. PMID:23427174

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

Hard copies for digital medical images: an overview

NASA Astrophysics Data System (ADS)

Blume, Hartwig R.; Muka, Edward

1995-04-01

This paper is a condensed version of an invited overview on the technology of film hard-copies used in radiology. Because the overview was given to an essentially nonmedical audience, the reliance on film hard-copies in radiology is outlined in greater detail. The overview is concerned with laser image recorders generating monochrome prints on silver-halide films. The basic components of laser image recorders are sketched. The paper concentrates on the physical parameters - characteristic function, dynamic range, digitization resolution, modulation transfer function, and noise power spectrum - which define image quality and information transfer capability of the printed image. A preliminary approach is presented to compare the printed image quality with noise in the acquired image as well as with the noise of state-of- the-art cathode-ray-tube display systems. High-performance laser-image- recorder/silver-halide-film/light-box systems are well capable of reproducing acquired radiologic information. Most recently development was begun toward a display function standard for soft-copy display systems to facilitate similarity of image presentation between different soft-copy displays as well as between soft- and hard-copy displays. The standard display function is based on perceptional linearization. The standard is briefly reviewed to encourage the printer industry to adopt it, too.

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

USDA-ARS?s Scientific Manuscript database

Technical abstract: 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 ...

Copy number variability in Parkinson's disease: assembling the puzzle through a systems biology approach.

PubMed

La Cognata, Valentina; Morello, Giovanna; D'Agata, Velia; Cavallaro, Sebastiano

2017-01-01

Parkinson's disease (PD), the second most common progressive neurodegenerative disorder of aging, was long believed to be a non-genetic sporadic origin syndrome. The proof that several genetic loci are responsible for rare Mendelian forms has represented a revolutionary breakthrough, enabling to reveal molecular mechanisms underlying this debilitating still incurable condition. While single nucleotide polymorphisms (SNPs) and small indels constitute the most commonly investigated DNA variations accounting for only a limited number of PD cases, larger genomic molecular rearrangements have emerged as significant PD-causing mutations, including submicroscopic Copy Number Variations (CNVs). CNVs constitute a prevalent source of genomic variations and substantially participate in each individual's genomic makeup and phenotypic outcome. However, the majority of genetic studies have focused their attention on single candidate-gene mutations or on common variants reaching a significant statistical level of acceptance. This gene-centric approach is insufficient to uncover the genetic background of polygenic multifactorial disorders like PD, and potentially masks rare individual CNVs that all together might contribute to disease development or progression. In this review, we will discuss literature and bioinformatic data describing the involvement of CNVs on PD pathobiology. We will analyze the most frequent copy number changes in familiar PD genes and provide a "systems biology" overview of rare individual rearrangements that could functionally act on commonly deregulated molecular pathways. Assessing the global genome-wide burden of CNVs in PD patients may reveal new disease-related molecular mechanisms, and open the window to a new possible genetic scenario in the unsolved PD puzzle.

Characterization of the Population of the Sulfur-Oxidizing Symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by Single-Cell Analyses▿ †

PubMed Central

Caro, Audrey; Gros, Olivier; Got, Patrice; De Wit, Rutger; Troussellier, Marc

2007-01-01

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 μm in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host. PMID:17259363

A widespread class of reverse transcriptase-related cellular genes.

PubMed

Gladyshev, Eugene A; Arkhipova, Irina R

2011-12-20

Reverse transcriptases (RTs) polymerize DNA on RNA templates. They fall into several structurally related but distinct classes and form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) synthesizing RNA on RNA templates. It is generally believed that most RT-like enzymes originate from retrotransposons or viruses and have no specific function in the host cell, with telomerases being the only notable exception. Here we report on the discovery and properties of a unique class of RT-related cellular genes collectively named rvt. We present evidence that rvts are not components of retrotransposons or viruses, but single-copy genes with a characteristic domain structure that may contain introns in evolutionarily conserved positions, occur in syntenic regions, and evolve under purifying selection. These genes can be found in all major taxonomic groups including protists, fungi, animals, plants, and even bacteria, although they exhibit patchy phylogenetic distribution in each kingdom. We also show that the RVT protein purified from one of its natural hosts, Neurospora crassa, exists in a multimeric form and has the ability to polymerize NTPs as well as dNTPs in vitro, with a strong preference for NTPs, using Mn(2+) as a cofactor. The existence of a previously unknown class of single-copy RT-related genes calls for reevaluation of the current views on evolution and functional roles of RNA-dependent polymerases in living cells.

Asymptotic structure of the Einstein-Maxwell theory on AdS3

NASA Astrophysics Data System (ADS)

Pérez, Alfredo; Riquelme, Miguel; Tempo, David; Troncoso, Ricardo

2016-02-01

The asymptotic structure of AdS spacetimes in the context of General Relativity coupled to the Maxwell field in three spacetime dimensions is analyzed. Although the fall-off of the fields is relaxed with respect to that of Brown and Henneaux, the variation of the canonical generators associated to the asymptotic Killing vectors can be shown to be finite once required to span the Lie derivative of the fields. The corresponding surface integrals then acquire explicit contributions from the electromagnetic field, and become well-defined provided they fulfill suitable integrability conditions, implying that the leading terms of the asymptotic form of the electromagnetic field are functionally related. Consequently, for a generic choice of boundary conditions, the asymptotic symmetries are broken down to {R}⊗ U(1)⊗ U(1) . Nonetheless, requiring compatibility of the boundary conditions with one of the asymptotic Virasoro symmetries, singles out the set to be characterized by an arbitrary function of a single variable, whose precise form depends on the choice of the chiral copy. Remarkably, requiring the asymptotic symmetries to contain the full conformal group selects a very special set of boundary conditions that is labeled by a unique constant parameter, so that the algebra of the canonical generators is given by the direct sum of two copies of the Virasoro algebra with the standard central extension and U (1). This special set of boundary conditions makes the energy spectrum of electrically charged rotating black holes to be well-behaved.

Microchambers with Solid-State Phosphorescent Sensor for Measuring Single Mitochondrial Respiration.

PubMed

Pham, Ted D; Wallace, Douglas C; Burke, Peter J

2016-07-09

It is now well established that, even within a single cell, multiple copies of the mitochondrial genome may be present (genetic heteroplasmy). It would be interesting to develop techniques to determine if and to what extent this genetic variation results in functional variation from one mitochondrion to the next (functional heteroplasmy). Measuring mitochondrial respiration can reveal the organelles' functional capacity for Adenosine triphosphate (ATP) production and determine mitochondrial damage that may arise from genetic or age related defects. However, available technologies require significant quantities of mitochondria. Here, we develop a technology to assay the respiration of a single mitochondrion. Our "micro-respirometer" consists of micron sized chambers etched out of borofloat glass substrates and coated with an oxygen sensitive phosphorescent dye Pt(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP) mixed with polystyrene. The chambers are sealed with a polydimethylsiloxane layer coated with oxygen impermeable Viton rubber to prevent diffusion of oxygen from the environment. As the mitochondria consume oxygen in the chamber, the phosphorescence signal increases, allowing direct determination of the respiration rate. Experiments with coupled vs. uncoupled mitochondria showed a substantial difference in respiration, confirming the validity of the microchambers as single mitochondrial respirometers. This demonstration could enable future high-throughput assays of mitochondrial respiration and benefit the study of mitochondrial functional heterogeneity, and its role in health and disease.

GRAbB: Selective Assembly of Genomic Regions, a New Niche for Genomic Research

PubMed Central

Zhang, Hao; van Diepeningen, Anne D.; van der Lee, Theo A. J.; Waalwijk, Cees; de Hoog, G. Sybren

2016-01-01

GRAbB (Genomic Region Assembly by Baiting) is a new program that is dedicated to assemble specific genomic regions from NGS data. This approach is especially useful when dealing with multi copy regions, such as mitochondrial genome and the rDNA repeat region, parts of the genome that are often neglected or poorly assembled, although they contain interesting information from phylogenetic or epidemiologic perspectives, but also single copy regions can be assembled. The program is capable of targeting multiple regions within a single run. Furthermore, GRAbB can be used to extract specific loci from NGS data, based on homology, like sequences that are used for barcoding. To make the assembly specific, a known part of the region, such as the sequence of a PCR amplicon or a homologous sequence from a related species must be specified. By assembling only the region of interest, the assembly process is computationally much less demanding and may lead to assemblies of better quality. In this study the different applications and functionalities of the program are demonstrated such as: exhaustive assembly (rDNA region and mitochondrial genome), extracting homologous regions or genes (IGS, RPB1, RPB2 and TEF1a), as well as extracting multiple regions within a single run. The program is also compared with MITObim, which is meant for the exhaustive assembly of a single target based on a similar query sequence. GRAbB is shown to be more efficient than MITObim in terms of speed, memory and disk usage. The other functionalities (handling multiple targets simultaneously and extracting homologous regions) of the new program are not matched by other programs. The program is available with explanatory documentation at https://github.com/b-brankovics/grabb. GRAbB has been tested on Ubuntu (12.04 and 14.04), Fedora (23), CentOS (7.1.1503) and Mac OS X (10.7). Furthermore, GRAbB is available as a docker repository: brankovics/grabb (https://hub.docker.com/r/brankovics/grabb/). PMID:27308864

GRAbB: Selective Assembly of Genomic Regions, a New Niche for Genomic Research.

PubMed

Brankovics, Balázs; Zhang, Hao; van Diepeningen, Anne D; van der Lee, Theo A J; Waalwijk, Cees; de Hoog, G Sybren

2016-06-01

GRAbB (Genomic Region Assembly by Baiting) is a new program that is dedicated to assemble specific genomic regions from NGS data. This approach is especially useful when dealing with multi copy regions, such as mitochondrial genome and the rDNA repeat region, parts of the genome that are often neglected or poorly assembled, although they contain interesting information from phylogenetic or epidemiologic perspectives, but also single copy regions can be assembled. The program is capable of targeting multiple regions within a single run. Furthermore, GRAbB can be used to extract specific loci from NGS data, based on homology, like sequences that are used for barcoding. To make the assembly specific, a known part of the region, such as the sequence of a PCR amplicon or a homologous sequence from a related species must be specified. By assembling only the region of interest, the assembly process is computationally much less demanding and may lead to assemblies of better quality. In this study the different applications and functionalities of the program are demonstrated such as: exhaustive assembly (rDNA region and mitochondrial genome), extracting homologous regions or genes (IGS, RPB1, RPB2 and TEF1a), as well as extracting multiple regions within a single run. The program is also compared with MITObim, which is meant for the exhaustive assembly of a single target based on a similar query sequence. GRAbB is shown to be more efficient than MITObim in terms of speed, memory and disk usage. The other functionalities (handling multiple targets simultaneously and extracting homologous regions) of the new program are not matched by other programs. The program is available with explanatory documentation at https://github.com/b-brankovics/grabb. GRAbB has been tested on Ubuntu (12.04 and 14.04), Fedora (23), CentOS (7.1.1503) and Mac OS X (10.7). Furthermore, GRAbB is available as a docker repository: brankovics/grabb (https://hub.docker.com/r/brankovics/grabb/).

The Complete Chloroplast Genome of Catha edulis: A Comparative Analysis of Genome Features with Related Species

PubMed Central

Tembrock, Luke R.; Zheng, Shaoyu; Wu, Zhiqiang

2018-01-01

Qat (Catha edulis, Celastraceae) is a woody evergreen species with great economic and cultural importance. It is cultivated for its stimulant alkaloids cathine and cathinone in East Africa and southwest Arabia. However, genome information, especially DNA sequence resources, for C. edulis are limited, hindering studies regarding interspecific and intraspecific relationships. Herein, the complete chloroplast (cp) genome of Catha edulis is reported. This genome is 157,960 bp in length with 37% GC content and is structurally arranged into two 26,577 bp inverted repeats and two single-copy areas. The size of the small single-copy and the large single-copy regions were 18,491 bp and 86,315 bp, respectively. The C. edulis cp genome consists of 129 coding genes including 37 transfer RNA (tRNA) genes, 8 ribosomal RNA (rRNA) genes, and 84 protein coding genes. For those genes, 112 are single copy genes and 17 genes are duplicated in two inverted regions with seven tRNAs, four rRNAs, and six protein coding genes. The phylogenetic relationships resolved from the cp genome of qat and 32 other species confirms the monophyly of Celastraceae. The cp genomes of C. edulis, Euonymus japonicus and seven Celastraceae species lack the rps16 intron, which indicates an intron loss took place among an ancestor of this family. The cp genome of C. edulis provides a highly valuable genetic resource for further phylogenomic research, barcoding and cp transformation in Celastraceae. PMID:29425128

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.

Protocols for Copying and Proofreading in Template-Assisted Polymerization

NASA Astrophysics Data System (ADS)

Pigolotti, Simone; Sartori, Pablo

2016-03-01

We discuss how information encoded in a template polymer can be stochastically copied into a copy polymer. We consider four different stochastic copy protocols of increasing complexity, inspired by building blocks of the mRNA translation pathway. In the first protocol, monomer incorporation occurs in a single stochastic transition. We then move to a more elaborate protocol in which an intermediate step can be used for error correction. Finally, we discuss the operating regimes of two kinetic proofreading protocols: one in which proofreading acts from the final copying step, and one in which it acts from an intermediate step. We review known results for these models and, in some cases, extend them to analyze all possible combinations of energetic and kinetic discrimination. We show that, in each of these protocols, only a limited number of these combinations leads to an improvement of the overall copying accuracy.

A second visual rhodopsin gene, rh1-2, is expressed in zebrafish photoreceptors and found in other ray-finned fishes.

PubMed

Morrow, James M; Lazic, Savo; Dixon Fox, Monica; Kuo, Claire; Schott, Ryan K; de A Gutierrez, Eduardo; Santini, Francesco; Tropepe, Vincent; Chang, Belinda S W

2017-01-15

Rhodopsin (rh1) is the visual pigment expressed in rod photoreceptors of vertebrates that is responsible for initiating the critical first step of dim-light vision. Rhodopsin is usually a single copy gene; however, we previously discovered a novel rhodopsin-like gene expressed in the zebrafish retina, rh1-2, which we identified as a functional photosensitive pigment that binds 11-cis retinal and activates in response to light. Here, we localized expression of rh1-2 in the zebrafish retina to a subset of peripheral photoreceptor cells, which indicates a partially overlapping expression pattern with rh1 We also expressed, purified and characterized Rh1-2, including investigation of the stability of the biologically active intermediate. Using fluorescence spectroscopy, we found the half-life of the rate of retinal release of Rh1-2 following photoactivation to be more similar to that of the visual pigment rhodopsin than to the non-visual pigment exo-rhodopsin (exorh), which releases retinal around 5 times faster. Phylogenetic and molecular evolutionary analyses show that rh1-2 has ancient origins within teleost fishes, is under similar selective pressure to rh1, and likely experienced a burst of positive selection following its duplication and divergence from rh1 These findings indicate that rh1-2 is another functional visual rhodopsin gene, which contradicts the prevailing notion that visual rhodopsin is primarily found as a single copy gene within ray-finned fishes. The reasons for retention of this duplicate gene, as well as possible functional consequences for the visual system, are discussed. © 2017. Published by The Company of Biologists Ltd.

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 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

The complete chloroplast genome sequence of Dendrobium officinale.

PubMed

Yang, Pei; Zhou, Hong; Qian, Jun; Xu, Haibin; Shao, Qingsong; Li, Yonghua; Yao, Hui

2016-01-01

The complete chloroplast sequence of Dendrobium officinale, an endangered and economically important traditional Chinese medicine, was reported and characterized. The genome size is 152,018 bp, with 37.5% GC content. A pair of inverted repeats (IRs) of 26,284 bp are separated by a large single-copy region (LSC, 84,944 bp) and a small single-copy region (SSC, 14,506 bp). The complete cp DNA contains 83 protein-coding genes, 39 tRNA genes and 8 rRNA genes. Fourteen genes contained one or two introns.

CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.

PubMed

Kuscu, Cem; Parlak, Mahmut; Tufan, Turan; Yang, Jiekun; Szlachta, Karol; Wei, Xiaolong; Mammadov, Rashad; Adli, Mazhar

2017-07-01

CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.

Development of early handwriting: Visual-motor control during letter copying.

PubMed

Maldarelli, Jennifer E; Kahrs, Björn A; Hunt, Sarah C; Lockman, Jeffrey J

2015-07-01

Despite the importance of handwriting for school readiness and early academic progress, prior research on the development of handwriting has focused primarily on the product rather than the process by which young children write letters. In contrast, in the present work, early handwriting is viewed as involving a suite of perceptual, motor, and cognitive abilities, which must work in unison if children are to write letters efficiently. To study such coordination, head-mounted eye-tracking technology was used to investigate the process of visual-motor coordination while kindergarten children (N = 23) and adults (N = 11) copied individual letters and strings of letters that differed in terms of their phonemic properties. Results indicated that kindergarten children were able to copy single letters efficiently, as did adults. When the cognitive demands of the task increased and children were presented with strings of letters, however, their ability to copy letters efficiently was compromised: Children frequently interrupted their writing midletter, whereas they did not do so on single letter trials. Yet, with increasing age, children became more efficient in copying letter strings, in part by using vision more prospectively when writing. Taken together, the results illustrate how the coordination of perceptual, motor, and cognitive processes contributes to advances in the development of letter writing skill. (c) 2015 APA, all rights reserved).

Development of Early Handwriting: Visual-Motor Control During Letter Copying

PubMed Central

Maldarelli, Jennifer E.; Kahrs, Björn A.; Hunt, Sarah C.; Lockman, Jeffrey J.

2015-01-01

Despite the importance of handwriting for school readiness and early academic progress, prior research on the development of handwriting has focused primarily on the product rather than the process by which young children write letters. In contrast, in the present work, early handwriting is viewed as involving a suite of perceptual, motor and cognitive abilities, which must work in unison if children are to write letters efficiently. To study such coordination, head-mounted eye-tracking technology was used to investigate the process of visual-motor coordination while kindergarten children (N=23) and adults (N=11) copied individual letters and strings of letters that differed in terms of their phonemic properties. Results indicated that kindergarten children were able to copy single letters efficiently, as did adults. When the cognitive demands of the task increased and children were presented with strings of letters, however, their ability to copy letters efficiently was compromised: children frequently interrupted their writing mid-letter, whereas they did not do so on single letter trials. Yet, with increasing age, children became more efficient in copying letter strings, in part by using vision more prospectively when writing. Taken together, the results illustrate how the coordination of perceptual, motor and cognitive processes contributes to advances in the development of letter writing skill. PMID:26029821

Simulation studies of the fidelity of biomolecular structure ensemble recreation

NASA Astrophysics Data System (ADS)

Lätzer, Joachim; Eastwood, Michael P.; Wolynes, Peter G.

2006-12-01

We examine the ability of Bayesian methods to recreate structural ensembles for partially folded molecules from averaged data. Specifically we test the ability of various algorithms to recreate different transition state ensembles for folding proteins using a multiple replica simulation algorithm using input from "gold standard" reference ensembles that were first generated with a Gō-like Hamiltonian having nonpairwise additive terms. A set of low resolution data, which function as the "experimental" ϕ values, were first constructed from this reference ensemble. The resulting ϕ values were then treated as one would treat laboratory experimental data and were used as input in the replica reconstruction algorithm. The resulting ensembles of structures obtained by the replica algorithm were compared to the gold standard reference ensemble, from which those "data" were, in fact, obtained. It is found that for a unimodal transition state ensemble with a low barrier, the multiple replica algorithm does recreate the reference ensemble fairly successfully when no experimental error is assumed. The Kolmogorov-Smirnov test as well as principal component analysis show that the overlap of the recovered and reference ensembles is significantly enhanced when multiple replicas are used. Reduction of the multiple replica ensembles by clustering successfully yields subensembles with close similarity to the reference ensembles. On the other hand, for a high barrier transition state with two distinct transition state ensembles, the single replica algorithm only samples a few structures of one of the reference ensemble basins. This is due to the fact that the ϕ values are intrinsically ensemble averaged quantities. The replica algorithm with multiple copies does sample both reference ensemble basins. In contrast to the single replica case, the multiple replicas are constrained to reproduce the average ϕ values, but allow fluctuations in ϕ for each individual copy. These fluctuations facilitate a more faithful sampling of the reference ensemble basins. Finally, we test how robustly the reconstruction algorithm can function by introducing errors in ϕ comparable in magnitude to those suggested by some authors. In this circumstance we observe that the chances of ensemble recovery with the replica algorithm are poor using a single replica, but are improved when multiple copies are used. A multimodal transition state ensemble, however, turns out to be more sensitive to large errors in ϕ (if appropriately gauged) and attempts at successful recreation of the reference ensemble with simple replica algorithms can fall short.

Breakdown of Spatial Parallel Coding in Children's Drawing

ERIC Educational Resources Information Center

De Bruyn, Bart; Davis, Alyson

2005-01-01

When drawing real scenes or copying simple geometric figures young children are highly sensitive to parallel cues and use them effectively. However, this sensitivity can break down in surprisingly simple tasks such as copying a single line where robust directional errors occur despite the presence of parallel cues. Before we can conclude that this…

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...

17 CFR 240.15c2-12 - Municipal securities disclosure.

Code of Federal Regulations, 2010 CFR

2010-04-01

... business day, by first-class mail or other equally prompt means, to any potential customer, on request, a... and in sufficient time to accompany any confirmation that requests payment from any customer, copies... other equally prompt means, to any potential customer, on request, a single copy of the final official...

Parasites bearing a single copy of the multi-drug resistance gene (pfmdr-1) with wild-type SNPs predominate amongst Plasmodium falciparum isolates from Malawi.

PubMed

Nkhoma, Standwell; Nair, Shalini; Mukaka, Mavuto; Molyneux, Malcolm E; Ward, Stephen A; Anderson, Timothy J C

2009-07-01

We genotyped 160 P. falciparum infections from Malawi for pfmdr-1 copy number changes and SNPs associated with in vivo tolerance and poor in vitro sensitivity to the component drugs of Coartem. We also measured in vitro susceptibility of 49 of these isolates to a variety of drugs in clinical use or with a potential for use in Africa. All 160 infections carried a single copy of pfmdr-1 but 34% exhibited sequence variation at 4 of the 5 polymorphic sites in pfmdr-1. Isolates carrying 86-Asn and 184-Tyr pfmdr-1 alleles were significantly less sensitive (p<0.001) to mefloquine, lumefantrine, artemether and dihydroartemisinin compared with those bearing 86-Tyr and 184-Phe polymorphisms. This study provides baseline measures prior to policy change: continued surveillance for changes in baseline drug susceptibility, pfmdr-1 copy number and SNPs, and other putative Coartem resistance loci will be necessary to provide an early warning of emerging Coartem resistance in this setting.

The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid

PubMed Central

Tobiason, Deborah M; Seifert, H. Steven

2006-01-01

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts. PMID:16719561

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.

Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction

PubMed Central

Jalal, Hamid; Stephen, Hannah; Curran, Martin D.; Burton, Janet; Bradley, Michelle; Carne, Christopher

2006-01-01

A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload. PMID:16390971

High-Performance, Multi-Node File Copies and Checksums for Clustered File Systems

NASA Technical Reports Server (NTRS)

Kolano, Paul Z.; Ciotti, Robert B.

2012-01-01

Modern parallel file systems achieve high performance using a variety of techniques, such as striping files across multiple disks to increase aggregate I/O bandwidth and spreading disks across multiple servers to increase aggregate interconnect bandwidth. To achieve peak performance from such systems, it is typically necessary to utilize multiple concurrent readers/writers from multiple systems to overcome various singlesystem limitations, such as number of processors and network bandwidth. The standard cp and md5sum tools of GNU coreutils found on every modern Unix/Linux system, however, utilize a single execution thread on a single CPU core of a single system, and hence cannot take full advantage of the increased performance of clustered file systems. Mcp and msum are drop-in replacements for the standard cp and md5sum programs that utilize multiple types of parallelism and other optimizations to achieve maximum copy and checksum performance on clustered file systems. Multi-threading is used to ensure that nodes are kept as busy as possible. Read/write parallelism allows individual operations of a single copy to be overlapped using asynchronous I/O. Multinode cooperation allows different nodes to take part in the same copy/checksum. Split-file processing allows multiple threads to operate concurrently on the same file. Finally, hash trees allow inherently serial checksums to be performed in parallel. Mcp and msum provide significant performance improvements over standard cp and md5sum using multiple types of parallelism and other optimizations. The total speed-ups from all improvements are significant. Mcp improves cp performance over 27x, msum improves md5sum performance almost 19x, and the combination of mcp and msum improves verified copies via cp and md5sum by almost 22x. These improvements come in the form of drop-in replacements for cp and md5sum, so are easily used and are available for download as open source software at http://mutil.sourceforge.net.

The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex.

PubMed

Gribble, Kristin E; Mark Welch, David B

2012-08-01

Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP), a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. Isolates of the B. plicatilis species complex have 1-4 copies of mmr-b, each composed of 2-9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving rapidly, and novel alleles may be maintained and increase in frequency via asexual reproduction. Our analyses indicate that mate recognition, controlled by MMR-B, may drive reproductive isolation and allow saltational sympatric speciation within the B. plicatilis cryptic species complex, and that this process may be largely neutral.

COPI selectively drives maturation of the early Golgi

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

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

COPI selectively drives maturation of the early Golgi

DOE PAGES

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham; ...

2015-12-28

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

The Landscape of Somatic Chromosomal Copy Number Aberrations in GEM Models of Prostate Carcinoma

PubMed Central

Bianchi-Frias, Daniella; Hernandez, Susana A.; Coleman, Roger; Wu, Hong; Nelson, Peter S.

2015-01-01

Human prostate cancer (PCa) is known to harbor recurrent genomic aberrations consisting of chromosomal losses, gains, rearrangements and mutations that involve oncogenes and tumor suppressors. Genetically engineered mouse (GEM) models have been constructed to assess the causal role of these putative oncogenic events and provide molecular insight into disease pathogenesis. While GEM models generally initiate neoplasia by manipulating a single gene, expression profiles of GEM tumors typically comprise hundreds of transcript alterations. It is unclear whether these transcriptional changes represent the pleiotropic effects of single oncogenes, and/or cooperating genomic or epigenomic events. Therefore, it was determined if structural chromosomal alterations occur in GEM models of PCa and whether the changes are concordant with human carcinomas. Whole genome array-based comparative genomic hybridization (CGH) was used to identify somatic chromosomal copy number aberrations (SCNAs) in the widely used TRAMP, Hi-Myc, Pten-null and LADY GEM models. Interestingly, very few SCNAs were identified and the genomic architecture of Hi-Myc, Pten-null and LADY tumors were essentially identical to the germline. TRAMP neuroendocrine carcinomas contained SCNAs, which comprised three recurrent aberrations including a single copy loss of chromosome 19 (encoding Pten). In contrast, cell lines derived from the TRAMP, Hi-Myc, and Pten-null tumors were notable for numerous SCNAs that included copy gains of chromosome 15 (encoding Myc) and losses of chromosome 11 (encoding p53). PMID:25298407

Trial and error: how the unclonable human mitochondrial genome was cloned in yeast.

PubMed

Bigger, Brian W; Liao, Ai-Yin; Sergijenko, Ana; Coutelle, Charles

2011-11-01

Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene. We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination. Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated. This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.

Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora.

PubMed

Wu, Dongliang; Navet, Natasha; Liu, Yingchao; Uchida, Janice; Tian, Miaoying

2016-09-06

As an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility. In this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion. This system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.

Linking transcriptional and genetic tumor heterogeneity through allele analysis of single-cell RNA-seq data.

PubMed

Fan, Jean; Lee, Hae-Ock; Lee, Soohyun; Ryu, Da-Eun; Lee, Semin; Xue, Catherine; Kim, Seok Jin; Kim, Kihyun; Barkas, Nikolas; Park, Peter J; Park, Woong-Yang; Kharchenko, Peter V

2018-06-13

Characterization of intratumoral heterogeneity is critical to cancer therapy, as presence of phenotypically diverse cell populations commonly fuels relapse and resistance to treatment. Although genetic variation is a well-studied source of intratumoral heterogeneity, the functional impact of most genetic alterations remains unclear. Even less understood is the relative importance of other factors influencing heterogeneity, such as epigenetic state or tumor microenvironment. To investigate the relationship between genetic and transcriptional heterogeneity in a context of cancer progression, we devised a computational approach called HoneyBADGER to identify copy number variation and loss-of-heterozygosity in individual cells from single-cell RNA-sequencing data. By integrating allele and normalized expression information, HoneyBADGER is able to identify and infer the presence of subclone-specific alterations in individual cells and reconstruct underlying subclonal architecture. Examining several tumor types, we show that HoneyBADGER is effective at identifying deletion, amplifications, and copy-neutral loss-of-heterozygosity events, and is capable of robustly identifying subclonal focal alterations as small as 10 megabases. We further apply HoneyBADGER to analyze single cells from a progressive multiple myeloma patient to identify major genetic subclones that exhibit distinct transcriptional signatures relevant to cancer progression. Surprisingly, other prominent transcriptional subpopulations within these tumors did not line up with the genetic subclonal structure, and were likely driven by alternative, non-clonal mechanisms. These results highlight the need for integrative analysis to understand the molecular and phenotypic heterogeneity in cancer. Published by Cold Spring Harbor Laboratory Press.

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

PubMed

Ferrarini, Alberto; Forcato, Claudio; Buson, Genny; Tononi, Paola; Del Monaco, Valentina; Terracciano, Mario; Bolognesi, Chiara; Fontana, Francesca; Medoro, Gianni; Neves, Rui; Möhlendick, Birte; Rihawi, Karim; Ardizzoni, Andrea; Sumanasuriya, Semini; Flohr, Penny; Lambros, Maryou; de Bono, Johann; Stoecklein, Nikolas H; Manaresi, Nicolò

2018-01-01

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.

Models for loosely linked gene duplicates suggest lengthy persistence of both copies.

PubMed

O'Hely, Martin; Wockner, Leesa

2007-06-21

Consider the appearance of a duplicate copy of a gene at a locus linked loosely, if at all, to the locus at which the gene is usually found. If all copies of the gene are subject to non-functionalizing mutations, then two fates are possible: loss of functional copies at the duplicate locus (loss of duplicate expression), or loss of functional copies at the original locus (map change). This paper proposes a simple model to address the probability of map change, the time taken for a map change and/or loss of duplicate expression, and considers where in the spectrum between loss of duplicate expression and map change such a duplicate complex is likely to be found. The findings are: the probability of map change is always half the reciprocal of the population size N, the time for a map change to occur is order NlogN generations, and that there is a marked tendency for duplicates to remain near equi-frequency with the gene at the original locus for a large portion of that time. This is in excellent agreement with simulations.

Legal and technical means to secure the commercial distribution of copyrighted electronic images on the information superhighway

NASA Astrophysics Data System (ADS)

Laurie, Ronald S.

1995-04-01

The arrival of the information super highway has introduced a number of problems, both for the owners of copyrighted images and for those who would like to legitimately use those images. The digitization of all content, whether still image, animation, video, music, or text, has made copying infinitely easier and cheaper. Further, the instantaneous access to this wealth of digital copyrighted material by millions of potential users, and would-be pirates, has the potential to greatly proliferate the number of unauthorized copies being made. For most of this century, technology has facilitated the unauthorized, and thus uncompensated, use of copyrighted material. Thus, xerography, audio and video recording systems, and personal computers have all contributed to the problem. However, we are entering a new era, where encryption techniques will not only prevent unauthorized use of copyrighted material but facilitate the use of that material by those that are willing to pay a commercially reasonable fee for such use. The technology, referred to as 'information metering,' allows usage fees to be based on a variety of use parameters. Thus, the material can either be 'rented' for a single use or a fixed number of uses, or it can be 'purchased' for an unlimited number of future uses. One price can be charged for on-screen viewing, with a higher price for hard copy print-out. In the case of digital images, the browsing function can be free, while the price of the print-out function can be based on the resolution of the image.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms

PubMed Central

Abylkassimova, Nikara; Hugall, Andrew F.; O'Hara, Timothy D.; Elphick, Maurice R.

2017-01-01

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide ‘cocktails’ is functionally important, but with plasticity over long evolutionary time scales. PMID:28878039

Discrete gene replication events drive coupling between the cell cycle and circadian clocks

PubMed Central

Paijmans, Joris; Bosman, Mark; ten Wolde, Pieter Rein; Lubensky, David K.

2016-01-01

Many organisms possess both a cell cycle to control DNA replication and a circadian clock to anticipate changes between day and night. In some cases, these two rhythmic systems are known to be coupled by specific, cross-regulatory interactions. Here, we use mathematical modeling to show that, additionally, the cell cycle generically influences circadian clocks in a nonspecific fashion: The regular, discrete jumps in gene-copy number arising from DNA replication during the cell cycle cause a periodic driving of the circadian clock, which can dramatically alter its behavior and impair its function. A clock built on negative transcriptional feedback either phase-locks to the cell cycle, so that the clock period tracks the cell division time, or exhibits erratic behavior. We argue that the cyanobacterium Synechococcus elongatus has evolved two features that protect its clock from such disturbances, both of which are needed to fully insulate it from the cell cycle and give it its observed robustness: a phosphorylation-based protein modification oscillator, together with its accompanying push–pull read-out circuit that responds primarily to the ratios of different phosphoform concentrations, makes the clock less susceptible to perturbations in protein synthesis; the presence of multiple, asynchronously replicating copies of the same chromosome diminishes the effect of replicating any single copy of a gene. PMID:27035936

Discrete gene replication events drive coupling between the cell cycle and circadian clocks.

PubMed

Paijmans, Joris; Bosman, Mark; Ten Wolde, Pieter Rein; Lubensky, David K

2016-04-12

Many organisms possess both a cell cycle to control DNA replication and a circadian clock to anticipate changes between day and night. In some cases, these two rhythmic systems are known to be coupled by specific, cross-regulatory interactions. Here, we use mathematical modeling to show that, additionally, the cell cycle generically influences circadian clocks in a nonspecific fashion: The regular, discrete jumps in gene-copy number arising from DNA replication during the cell cycle cause a periodic driving of the circadian clock, which can dramatically alter its behavior and impair its function. A clock built on negative transcriptional feedback either phase-locks to the cell cycle, so that the clock period tracks the cell division time, or exhibits erratic behavior. We argue that the cyanobacterium Synechococcus elongatus has evolved two features that protect its clock from such disturbances, both of which are needed to fully insulate it from the cell cycle and give it its observed robustness: a phosphorylation-based protein modification oscillator, together with its accompanying push-pull read-out circuit that responds primarily to the ratios of different phosphoform concentrations, makes the clock less susceptible to perturbations in protein synthesis; the presence of multiple, asynchronously replicating copies of the same chromosome diminishes the effect of replicating any single copy of a gene.

A Comparison of Math Cover, Copy, Compare Intervention Procedures for Children with Autism Spectrum Disorder.

PubMed

Morton, Reeva C; Gadke, Daniel L

2018-03-01

Cover, Copy, Compare (CCC) and Copy, Cover, Compare (MCCC) procedures are effective interventions for improving math fluency. However, there is a gap in literature exploring the use of these interventions for children with autism spectrum disorders (ASD). The purpose of the current study was to compare the use of CCC and MCCC for children with ASD using a multi-component single-case experimental design. The results showed no notable difference between the interventions. Implications and limitations, particularly surrounding experimental control, are discussed in detail.

47,XXX male: A clinical and molecular study.

PubMed

Ogata, T; Matsuo, M; Muroya, K; Koyama, Y; Fukutani, K

2001-02-01

We report a 53-year-old Japanese male with a 47,XXX karyotype. His clinical features included hypoplastic scrotal testes (4 ml bilaterally), normally formed small penis (3.8 cm), relatively poor pubic hair development (Tanner stage 3), gynecomastia, age-appropriate male height (159.1 cm), and mental retardation (verbal IQ of 56). Serum testosterone was markedly reduced (0.6 nmol/L). A needle biopsy showed severe testicular degeneration. FISH analysis revealed complex mosaicism consisting of (1) 47,XXX cells with a single copy of SRY (n = 177), two copies of SRY (n = 3), and no SRY (n = 1); (2) 46,XX cells with a single copy of SRY (n = 9) and no SRY (n = 3); (3) 45,X cells with no SRY (n = 5); and (4) 48,XXXX cells with a single copy of SRY (n = 1) and two copies of SRY (n = 1). PCR analysis showed the presence of Yp portion with the breakpoint between DYS264 and AMELY. Microsatellite analysis demonstrated three alleles for DMD and AR. X-inactivation analysis for the methylation status of the AR gene showed random inactivation of the three X chromosomes. The results suggest that this 47,XXX male has resulted from abnormal X-Y interchange during paternal meiosis and X-X nondisjunction during maternal meiosis. Complex mosaicism may be due to the age-related increase in mitotic nondisjunction which is prone to occur in rapidly dividing lymphocytes and to the presence of two randomly inactivated X chromosomes which may behave asynchronously during mitosis, and clinical features of this male would primarily be explained by the genetic information on the SRY (+) der(X) chromosome and his advanced age. Copyright 2001 Wiley-Liss, Inc.

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.

Comparison of the protein-coding genomes of three deep-sea, sulfur-oxidising bacteria: "Candidatus Ruthia magnifica", "Candidatus Vesicomyosocius okutanii" and Thiomicrospira crunogena.

PubMed

McGill, Susan E; Barker, Daniel

2017-07-20

" Candidatus Ruthia magnifica", "Candidatus Vesicomyosocius okutanii" and Thiomicrospira crunogena are all sulfur-oxidising bacteria found in deep-sea vent environments. Recent research suggests that the two symbiotic organisms, "Candidatus R. magnifica" and "Candidatus V. okutanii", may share common ancestry with the autonomously living species T. crunogena. We used comparative genomics to examine the genome-wide protein-coding content of all three species to explore their similarities. In particular, we used the OrthoMCL algorithm to sort proteins into groups of putative orthologs on the basis of sequence similarity. The OrthoMCL inflation parameter was tuned using biological criteria. Using the tuned value, OrthoMCL delimited 1070 protein groups. 63.5% of these groups contained one protein from each species. Two groups contained duplicate protein copies from all three species. 123 groups were unique to T. crunogena and ten groups included multiple copies of T. crunogena proteins but only single copies from the other species. "Candidatus R. magnifica" had one unique group, and had multiple copies in one group where the other species had a single copy. There were no groups unique to "Candidatus V. okutanii", and no groups in which there were multiple "Candidatus V. okutanii" proteins but only single proteins from the other species. Results align with previous suggestions that all three species share a common ancestor. However this is not definitive evidence to make taxonomic conclusions and the possibility of horizontal gene transfer was not investigated. Methodologically, the tuning of the OrthoMCL inflation parameter using biological criteria provides further methods to refine the OrthoMCL procedure.

Reduction of IgE binding and nonpromotion of Aspergillus flavus fungal growth by simultaneously silencing Ara h 2 and Ara h 6 in peanut.

USDA-ARS?s Scientific Manuscript database

The most potent peanut allergens, Ara h 2 and 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies ...

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...

Oculomotor learning revisited: a model of reinforcement learning in the basal ganglia incorporating an efference copy of motor actions

PubMed Central

Fee, Michale S.

2012-01-01

In its simplest formulation, reinforcement learning is based on the idea that if an action taken in a particular context is followed by a favorable outcome, then, in the same context, the tendency to produce that action should be strengthened, or reinforced. While reinforcement learning forms the basis of many current theories of basal ganglia (BG) function, these models do not incorporate distinct computational roles for signals that convey context, and those that convey what action an animal takes. Recent experiments in the songbird suggest that vocal-related BG circuitry receives two functionally distinct excitatory inputs. One input is from a cortical region that carries context information about the current “time” in the motor sequence. The other is an efference copy of motor commands from a separate cortical brain region that generates vocal variability during learning. Based on these findings, I propose here a general model of vertebrate BG function that combines context information with a distinct motor efference copy signal. The signals are integrated by a learning rule in which efference copy inputs gate the potentiation of context inputs (but not efference copy inputs) onto medium spiny neurons in response to a rewarded action. The hypothesis is described in terms of a circuit that implements the learning of visually guided saccades. The model makes testable predictions about the anatomical and functional properties of hypothesized context and efference copy inputs to the striatum from both thalamic and cortical sources. PMID:22754501

Oculomotor learning revisited: a model of reinforcement learning in the basal ganglia incorporating an efference copy of motor actions.

PubMed

Fee, Michale S

2012-01-01

In its simplest formulation, reinforcement learning is based on the idea that if an action taken in a particular context is followed by a favorable outcome, then, in the same context, the tendency to produce that action should be strengthened, or reinforced. While reinforcement learning forms the basis of many current theories of basal ganglia (BG) function, these models do not incorporate distinct computational roles for signals that convey context, and those that convey what action an animal takes. Recent experiments in the songbird suggest that vocal-related BG circuitry receives two functionally distinct excitatory inputs. One input is from a cortical region that carries context information about the current "time" in the motor sequence. The other is an efference copy of motor commands from a separate cortical brain region that generates vocal variability during learning. Based on these findings, I propose here a general model of vertebrate BG function that combines context information with a distinct motor efference copy signal. The signals are integrated by a learning rule in which efference copy inputs gate the potentiation of context inputs (but not efference copy inputs) onto medium spiny neurons in response to a rewarded action. The hypothesis is described in terms of a circuit that implements the learning of visually guided saccades. The model makes testable predictions about the anatomical and functional properties of hypothesized context and efference copy inputs to the striatum from both thalamic and cortical sources.

21 CFR 1316.68 - Copies of petitions for judicial review.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Copies of petitions for judicial review. 1316.68 Section 1316.68 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE ADMINISTRATIVE FUNCTIONS, PRACTICES, AND PROCEDURES Administrative Hearings § 1316.68 Copies of petitions for judicial...

The developmental transcriptome atlas of the spoon worm Urechis unicinctus (Echiurida: Annelida).

PubMed

Park, Chungoo; Han, Yong-Hee; Lee, Sung-Gwon; Ry, Kyoung-Bin; Oh, Jooseong; Kern, Elizabeth M A; Park, Joong-Ki; Cho, Sung-Jin

2018-03-01

Echiurida is one of the most intriguing major subgroups of annelida because, unlike most other annelids, echiurids lack metameric body segmentation as adults. For this reason, transcriptome analyses from various developmental stages of echiurid species can be of substantial value for understanding precise expression levels and the complex regulatory networks during early and larval development. A total of 914 million raw RNA-Seq reads were produced from 14 developmental stages of Urechis unicinctus and were de novo assembled into contigs spanning 63,928,225 bp with an N50 length of 2700 bp. The resulting comprehensive transcriptome database of the early developmental stages of U. unicinctus consists of 20,305 representative functional protein-coding transcripts. Approximately 66% of unigenes were assigned to superphylum-level taxa, including Lophotrochozoa (40%). The completeness of the transcriptome assembly was assessed using benchmarking universal single-copy orthologs; 75.7% of the single-copy orthologs were presented in our transcriptome database. We observed 3 distinct patterns of global transcriptome profiles from 14 developmental stages and identified 12,705 genes that showed dynamic regulation patterns during the differentiation and maturation of U. unicinctus cells. We present the first large-scale developmental transcriptome dataset of U. unicinctus and provide a general overview of the dynamics of global gene expression changes during its early developmental stages. The analysis of time-course gene expression data is a first step toward understanding the complex developmental gene regulatory networks in U. unicinctus and will furnish a valuable resource for analyzing the functions of gene repertoires in various developmental phases.

The complete chloroplast genome sequence of Hibiscus syriacus.

PubMed

Kwon, Hae-Yun; Kim, Joon-Hyeok; Kim, Sea-Hyun; Park, Ji-Min; Lee, Hyoshin

2016-09-01

The complete chloroplast genome sequence of Hibiscus syriacus L. is presented in this study. The genome is composed of 161 019 bp in length, with a typical circular structure containing a pair of inverted repeats of 25 745 bp of length separated by a large single-copy region and a small single-copy region of 89 698 bp and 19 831 bp of length, respectively. The overall GC content is 36.8%. One hundred and fourteen genes were annotated, including 81 protein-coding genes, 4 ribosomal RNA genes and 29 transfer RNA genes.

Single Parent and Homemaker Projects Resource Notebook (Working Copy).

ERIC Educational Resources Information Center

Ohio State Dept. of Education, Columbus. Div. of Vocational and Career Education.

This guide is intended to assist developers and providers of programs for single parents and homemakers. The first section discusses the special needs of single parents and homemakers, the provisions of the Carl D. Perkins Vocational Education Act of 1984, and Ohio's single parent and homemaker projects. Section 2 covers the the following aspects…

Determining Complementary Properties with Quantum Clones.

PubMed

Thekkadath, G S; Saaltink, R Y; Giner, L; Lundeen, J S

2017-08-04

In a classical world, simultaneous measurements of complementary properties (e.g., position and momentum) give a system's state. In quantum mechanics, measurement-induced disturbance is largest for complementary properties and, hence, limits the precision with which such properties can be determined simultaneously. It is tempting to try to sidestep this disturbance by copying the system and measuring each complementary property on a separate copy. However, perfect copying is physically impossible in quantum mechanics. Here, we investigate using the closest quantum analog to this copying strategy, optimal cloning. The coherent portion of the generated clones' state corresponds to "twins" of the input system. Like perfect copies, both twins faithfully reproduce the properties of the input system. Unlike perfect copies, the twins are entangled. As such, a measurement on both twins is equivalent to a simultaneous measurement on the input system. For complementary observables, this joint measurement gives the system's state, just as in the classical case. We demonstrate this experimentally using polarized single photons.

Determining Complementary Properties with Quantum Clones

NASA Astrophysics Data System (ADS)

Thekkadath, G. S.; Saaltink, R. Y.; Giner, L.; Lundeen, J. S.

2017-08-01

In a classical world, simultaneous measurements of complementary properties (e.g., position and momentum) give a system's state. In quantum mechanics, measurement-induced disturbance is largest for complementary properties and, hence, limits the precision with which such properties can be determined simultaneously. It is tempting to try to sidestep this disturbance by copying the system and measuring each complementary property on a separate copy. However, perfect copying is physically impossible in quantum mechanics. Here, we investigate using the closest quantum analog to this copying strategy, optimal cloning. The coherent portion of the generated clones' state corresponds to "twins" of the input system. Like perfect copies, both twins faithfully reproduce the properties of the input system. Unlike perfect copies, the twins are entangled. As such, a measurement on both twins is equivalent to a simultaneous measurement on the input system. For complementary observables, this joint measurement gives the system's state, just as in the classical case. We demonstrate this experimentally using polarized single photons.

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.

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

Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer.

PubMed

Bailey, Swneke D; Desai, Kinjal; Kron, Ken J; Mazrooei, Parisa; Sinnott-Armstrong, Nicholas A; Treloar, Aislinn E; Dowar, Mark; Thu, Kelsie L; Cescon, David W; Silvester, Jennifer; Yang, S Y Cindy; Wu, Xue; Pezo, Rossanna C; Haibe-Kains, Benjamin; Mak, Tak W; Bedard, Philippe L; Pugh, Trevor J; Sallari, Richard C; Lupien, Mathieu

2016-10-01

Sustained expression of the estrogen receptor-α (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program. Somatic copy number alterations involving the ESR1 gene occur in approximately 1% of ESR1-positive breast cancers, suggesting that other mechanisms underlie the persistent expression of ESR1. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating ESR1 in 7% of ESR1-positive breast cancers. These mutations regulate ESR1 expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk-associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.

The comparative chloroplast genomic analysis of photosynthetic orchids and developing DNA markers to distinguish Phalaenopsis orchids.

PubMed

Jheng, Cheng-Fong; Chen, Tien-Chih; Lin, Jhong-Yi; Chen, Ting-Chieh; Wu, Wen-Luan; Chang, Ching-Chun

2012-07-01

The chloroplast genome of Phalaenopsis equestris was determined and compared to those of Phalaenopsis aphrodite and Oncidium Gower Ramsey in Orchidaceae. The chloroplast genome of P. equestris is 148,959 bp, and a pair of inverted repeats (25,846 bp) separates the genome into large single-copy (85,967 bp) and small single-copy (11,300 bp) regions. The genome encodes 109 genes, including 4 rRNA, 30 tRNA and 75 protein-coding genes, but loses four ndh genes (ndhA, E, F and H) and seven other ndh genes are pseudogenes. The rate of inter-species variation between the two moth orchids was 0.74% (1107 sites) for single nucleotide substitution and 0.24% for insertions (161 sites; 1388 bp) and deletions (189 sites; 1393 bp). The IR regions have a lower rate of nucleotide substitution (3.5-5.8-fold) and indels (4.3-7.1-fold) than single-copy regions. The intergenic spacers are the most divergent, and based on the length variation of the three intergenic spacers, 11 native Phalaenopsis orchids could be successfully distinguished. The coding genes, IR junction and RNA editing sites are relatively more conserved between the two moth orchids than between those of Phalaenopsis and Oncidium spp. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA Lengths

PubMed Central

Gül, O. Tolga; Pugliese, Kaitlin M.; Choi, Yongki; Sims, Patrick C.; Pan, Deng; Rajapakse, Arith J.; Weiss, Gregory A.; Collins, Philip G.

2016-01-01

As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein’s activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF’s base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures. PMID:27348011

Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA Lengths.

PubMed

Gül, O Tolga; Pugliese, Kaitlin M; Choi, Yongki; Sims, Patrick C; Pan, Deng; Rajapakse, Arith J; Weiss, Gregory A; Collins, Philip G

2016-06-24

As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein's activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF's base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures.

Rare copy number variants in patients with congenital conotruncal heart defects.

PubMed

Xie, Hongbo M; Werner, Petra; Stambolian, Dwight; Bailey-Wilson, Joan E; Hakonarson, Hakon; White, Peter S; Taylor, Deanne M; Goldmuntz, Elizabeth

2017-03-01

Previous studies using different cardiac phenotypes, technologies and designs suggest a burden of large, rare or de novo copy number variants (CNVs) in subjects with congenital heart defects. We sought to identify disease-related CNVs, candidate genes, and functional pathways in a large number of cases with conotruncal and related defects that carried no known genetic syndrome. Cases and control samples were divided into two cohorts and genotyped to assess each subject's CNV content. Analyses were performed to ascertain differences in overall CNV prevalence and to identify enrichment of specific genes and functional pathways in conotruncal cases relative to healthy controls. Only findings present in both cohorts are presented. From 973 total conotruncal cases, a burden of rare CNVs was detected in both cohorts. Candidate genes from rare CNVs found in both cohorts were identified based on their association with cardiac development or disease, and/or their reported disruption in published studies. Functional and pathway analyses revealed significant enrichment of terms involved in either heart or early embryonic development. Our study tested one of the largest cohorts specifically with cardiac conotruncal and related defects. These results confirm and extend previous findings that CNVs contribute to disease risk for congenital heart defects in general and conotruncal defects in particular. As disease heterogeneity renders identification of single recurrent genes or loci difficult, functional pathway and gene regulation network analyses appear to be more informative. Birth Defects Research 109:271-295, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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/.

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

COPI selectively drives maturation of the early Golgi

PubMed Central

Papanikou, Effrosyni; Day, Kasey J; Austin, Jotham; Glick, Benjamin S

2015-01-01

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins. DOI: http://dx.doi.org/10.7554/eLife.13232.001 PMID:26709839

Copy-Editing: The Cambridge Handbook.

ERIC Educational Resources Information Center

Butcher, Judith

This handbook is designed as a reference manual for copy editors who prepare typescript for printing. It deals with the following topics: the copy editor's function; the work to be done at each stage in the production process; some difficult points of spelling, capitalization, and other features collectively known as "house style"; the parts of a…

Specific functions of the Rep and Rep' proteins of porcine circovirus during copy-release and rolling-circle DNA replication

USDA-ARS?s Scientific Manuscript database

The roles of two porcine circovirus replication initiator proteins, Rep and Rep', in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replica...

Polymorphisms of CCL3L1/CCR5 genes and recurrence of hepatitis B in liver transplant recipients.

PubMed

Li, Hong; Xie, Hai-Yang; Zhou, Lin; Wang, Wei-Lin; Liang, Ting-Bo; Zhang, Min; Zheng, Shu-Sen

2011-12-01

The genetic diversity of chemokines and chemokine receptors has been associated with the outcome of hepatitis B virus infection. The aim of this study was to evaluate whether the copy number variation in the CCL3L1 gene and the polymorphisms of CCR5Δ32 and CCR5-2459A→G (rs1799987) are associated with recurrent hepatitis B in liver transplantation for hepatitis B virus infection-related end-stage liver disease. A total of 185 transplant recipients were enrolled in this study. The genomic DNA was extracted from whole blood, the copy number of the CCL3L1 gene was determined by a quantitative real-time PCR based assay, CCR5Δ32 was detected by a sizing PCR method, and a single-nucleotide polymorphism in CCR5-2459 was detected by restriction fragment length polymorphism PCR. No CCR5Δ32 mutation was detected in any of the individuals from China. Neither copy number variation nor polymorphism in CCR5-2459 was associated with post-transplant re-infection with hepatitis B virus. However, patients with fewer copies (<4) of the CCL3L1 gene compared with the population median in combination with the CCR5G allele had a significantly higher risk for recurrent hepatitis B (odds ratio=1.93, 95% CI: 1.00-3.69; P=0.047). Patients possessing the compound decreased functional genotype of both CCL3L1 and CCR5 genes might be more likely to have recurrence of hepatitis B after transplantation.

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

Identification and qualification of 500 nuclear, single-copy, orthologous genes for the Eupulmonata (Gastropoda) using transcriptome sequencing and exon capture.

PubMed

Teasdale, Luisa C; Köhler, Frank; Murray, Kevin D; O'Hara, Tim; Moussalli, Adnan

2016-09-01

The qualification of orthology is a significant challenge when developing large, multiloci phylogenetic data sets from assembled transcripts. Transcriptome assemblies have various attributes, such as fragmentation, frameshifts and mis-indexing, which pose problems to automated methods of orthology assessment. Here, we identify a set of orthologous single-copy genes from transcriptome assemblies for the land snails and slugs (Eupulmonata) using a thorough approach to orthology determination involving manual alignment curation, gene tree assessment and sequencing from genomic DNA. We qualified the orthology of 500 nuclear, protein-coding genes from the transcriptome assemblies of 21 eupulmonate species to produce the most complete phylogenetic data matrix for a major molluscan lineage to date, both in terms of taxon and character completeness. Exon capture targeting 490 of the 500 genes (those with at least one exon >120 bp) from 22 species of Australian Camaenidae successfully captured sequences of 2825 exons (representing all targeted genes), with only a 3.7% reduction in the data matrix due to the presence of putative paralogs or pseudogenes. The automated pipeline Agalma retrieved the majority of the manually qualified 500 single-copy gene set and identified a further 375 putative single-copy genes, although it failed to account for fragmented transcripts resulting in lower data matrix completeness when considering the original 500 genes. This could potentially explain the minor inconsistencies we observed in the supported topologies for the 21 eupulmonate species between the manually curated and 'Agalma-equivalent' data set (sharing 458 genes). Overall, our study confirms the utility of the 500 gene set to resolve phylogenetic relationships at a range of evolutionary depths and highlights the importance of addressing fragmentation at the homolog alignment stage for probe design. © 2016 John Wiley & Sons Ltd.

Comparative Chloroplast Genomes of Photosynthetic Orchids: Insights into Evolution of the Orchidaceae and Development of Molecular Markers for Phylogenetic Applications

PubMed Central

Niu, Zhi-Tao; Liu, Wei; Xue, Qing-Yun; Ding, Xiao-Yu

2014-01-01

The orchid family Orchidaceae is one of the largest angiosperm families, including many species of important economic value. While chloroplast genomes are very informative for systematics and species identification, there is very limited information available on chloroplast genomes in the Orchidaceae. Here, we report the complete chloroplast genomes of the medicinal plant Dendrobium officinale and the ornamental orchid Cypripedium macranthos, demonstrating their gene content and order and potential RNA editing sites. The chloroplast genomes of the above two species and five known photosynthetic orchids showed similarities in structure as well as gene order and content, but differences in the organization of the inverted repeat/small single-copy junction and ndh genes. The organization of the inverted repeat/small single-copy junctions in the chloroplast genomes of these orchids was classified into four types; we propose that inverted repeats flanking the small single-copy region underwent expansion or contraction among Orchidaceae. The AT-rich regions of the ycf1 gene in orchids could be linked to the recombination of inverted repeat/small single-copy junctions. Relative species in orchids displayed similar patterns of variation in ndh gene contents. Furthermore, fifteen highly divergent protein-coding genes were identified, which are useful for phylogenetic analyses in orchids. To test the efficiency of these genes serving as markers in phylogenetic analyses, coding regions of four genes (accD, ccsA, matK, and ycf1) were used as a case study to construct phylogenetic trees in the subfamily Epidendroideae. High support was obtained for placement of previously unlocated subtribes Collabiinae and Dendrobiinae in the subfamily Epidendroideae. Our findings expand understanding of the diversity of orchid chloroplast genomes and provide a reference for study of the molecular systematics of this family. PMID:24911363

Comparative chloroplast genomes of photosynthetic orchids: insights into evolution of the Orchidaceae and development of molecular markers for phylogenetic applications.

PubMed

Luo, Jing; Hou, Bei-Wei; Niu, Zhi-Tao; Liu, Wei; Xue, Qing-Yun; Ding, Xiao-Yu

2014-01-01

The orchid family Orchidaceae is one of the largest angiosperm families, including many species of important economic value. While chloroplast genomes are very informative for systematics and species identification, there is very limited information available on chloroplast genomes in the Orchidaceae. Here, we report the complete chloroplast genomes of the medicinal plant Dendrobium officinale and the ornamental orchid Cypripedium macranthos, demonstrating their gene content and order and potential RNA editing sites. The chloroplast genomes of the above two species and five known photosynthetic orchids showed similarities in structure as well as gene order and content, but differences in the organization of the inverted repeat/small single-copy junction and ndh genes. The organization of the inverted repeat/small single-copy junctions in the chloroplast genomes of these orchids was classified into four types; we propose that inverted repeats flanking the small single-copy region underwent expansion or contraction among Orchidaceae. The AT-rich regions of the ycf1 gene in orchids could be linked to the recombination of inverted repeat/small single-copy junctions. Relative species in orchids displayed similar patterns of variation in ndh gene contents. Furthermore, fifteen highly divergent protein-coding genes were identified, which are useful for phylogenetic analyses in orchids. To test the efficiency of these genes serving as markers in phylogenetic analyses, coding regions of four genes (accD, ccsA, matK, and ycf1) were used as a case study to construct phylogenetic trees in the subfamily Epidendroideae. High support was obtained for placement of previously unlocated subtribes Collabiinae and Dendrobiinae in the subfamily Epidendroideae. Our findings expand understanding of the diversity of orchid chloroplast genomes and provide a reference for study of the molecular systematics of this family.

Linking soil bacterial biodiversity and soil carbon stability.

PubMed

Mau, Rebecca L; Liu, Cindy M; Aziz, Maliha; Schwartz, Egbert; Dijkstra, Paul; Marks, Jane C; Price, Lance B; Keim, Paul; Hungate, Bruce A

2015-06-01

Native soil carbon (C) can be lost in response to fresh C inputs, a phenomenon observed for decades yet still not understood. Using dual-stable isotope probing, we show that changes in the diversity and composition of two functional bacterial groups occur with this 'priming' effect. A single-substrate pulse suppressed native soil C loss and reduced bacterial diversity, whereas repeated substrate pulses stimulated native soil C loss and increased diversity. Increased diversity after repeated C amendments contrasts with resource competition theory, and may be explained by increased predation as evidenced by a decrease in bacterial 16S rRNA gene copies. Our results suggest that biodiversity and composition of the soil microbial community change in concert with its functioning, with consequences for native soil C stability.

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...

One-to-one quantum dot-labeled single long DNA probes.

PubMed

He, Shibin; Huang, Bi-Hai; Tan, Junjun; Luo, Qing-Ying; Lin, Yi; Li, Jun; Hu, Yong; Zhang, Lu; Yan, Shihan; Zhang, Qi; Pang, Dai-Wen; Li, Lijia

2011-08-01

Quantum dots (QDs) have been received most attention due to their unique properties. Constructing QDs conjugated with certain number of biomolecules is considered as one of the most important research goals in nanobiotechnology. In this study, we report polymerase chain reaction (PCR) amplification of primer oligonucleotides bound to QDs, termed as QD-based PCR. Characterization of QD-based PCR products by gel electrophoresis and atomic force microscopy showed that QD-labeled long DNA strands were synthesized and only a single long DNA strand was conjugated with a QD. The QD-based PCR products still kept fluorescence properties. Moreover, the one-to-one QD-labeled long DNA conjugates as probes could detect a single-copy gene on maize chromosomes by fluorescence in situ hybridization. Labeling a single QD to a single long DNA will make detection of small single-copy DNA fragments, quantitative detection and single molecule imaging come true by nanotechnology, and it will promote medical diagnosis and basic biological research as well as nano-material fabrication. Copyright © 2011 Elsevier Ltd. All rights reserved.

Evaluation of targeted exome sequencing for 28 protein-based blood group systems, including the homologous gene systems, for blood group genotyping.

PubMed

Schoeman, Elizna M; Lopez, Genghis H; McGowan, Eunike C; Millard, Glenda M; O'Brien, Helen; Roulis, Eileen V; Liew, Yew-Wah; Martin, Jacqueline R; McGrath, Kelli A; Powley, Tanya; Flower, Robert L; Hyland, Catherine A

2017-04-01

Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting. © 2017 AABB.

The complete chloroplast genome of Sinopodophyllum hexandrum Ying (Berberidaceae).

PubMed

Meng, Lihua; Liu, Ruijuan; Chen, Jianbing; Ding, Chenxu

2017-05-01

The complete nucleotide sequence of the Sinopodophyllum hexandrum Ying chloroplast genome (cpDNA) was determined based on next-generation sequencing technologies in this study. The genome was 157 203 bp in length, containing a pair of inverted repeat (IRa and IRb) regions of 25 960 bp, which were separated by a large single-copy (LSC) region of 87 065 bp and a small single-copy (SSC) region of 18 218 bp, respectively. The cpDNA contained 148 genes, including 96 protein-coding genes, 8 ribosomal RNA genes, and 44 tRNA genes. In these genes, eight harbored a single intron, and two (ycf3 and clpP) contained a couple of introns. The cpDNA AT content of S. hexandrum cpDNA is 61.5%.

Evolution of RLSB, a nuclear-encoded S1 domain RNA binding protein associated with post-transcriptional regulation of plastid-encoded rbcL mRNA in vascular plants.

PubMed

Yerramsetty, Pradeep; Stata, Matt; Siford, Rebecca; Sage, Tammy L; Sage, Rowan F; Wong, Gane Ka-Shu; Albert, Victor A; Berry, James O

2016-06-29

RLSB, an S-1 domain RNA binding protein of Arabidopsis, selectively binds rbcL mRNA and co-localizes with Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within chloroplasts of C3 and C4 plants. Previous studies using both Arabidopsis (C3) and maize (C4) suggest RLSB homologs are post-transcriptional regulators of plastid-encoded rbcL mRNA. While RLSB accumulates in all Arabidopsis leaf chlorenchyma cells, in C4 leaves RLSB-like proteins accumulate only within Rubisco-containing bundle sheath chloroplasts of Kranz-type species, and only within central compartment chloroplasts in the single cell C4 plant Bienertia. Our recent evidence implicates this mRNA binding protein as a primary determinant of rbcL expression, cellular localization/compartmentalization, and photosynthetic function in all multicellular green plants. This study addresses the hypothesis that RLSB is a highly conserved Rubisco regulatory factor that occurs in the chloroplasts all higher plants. Phylogenetic analysis has identified RLSB orthologs and paralogs in all major plant groups, from ancient liverworts to recent angiosperms. RLSB homologs were also identified in algae of the division Charophyta, a lineage closely related to land plants. RLSB-like sequences were not identified in any other algae, suggesting that it may be specific to the evolutionary line leading to land plants. The RLSB family occurs in single copy across most angiosperms, although a few species with two copies were identified, seemingly randomly distributed throughout the various taxa, although perhaps correlating in some cases with known ancient whole genome duplications. Monocots of the order Poales (Poaceae and Cyperaceae) were found to contain two copies, designated here as RLSB-a and RLSB-b, with only RLSB-a implicated in the regulation of rbcL across the maize developmental gradient. Analysis of microsynteny in angiosperms revealed high levels of conservation across eudicot species and for both paralogs in grasses, highlighting the possible importance of maintaining this gene and its surrounding genomic regions. Findings presented here indicate that the RLSB family originated as a unique gene in land plant evolution, perhaps in the common ancestor of charophytes and higher plants. Purifying selection has maintained this as a highly conserved single- or two-copy gene across most extant species, with several conserved gene duplications. Together with previous findings, this study suggests that RLSB has been sustained as an important regulatory protein throughout the course of land plant evolution. While only RLSB-a has been directly implicated in rbcL regulation in maize, RLSB-b could have an overlapping function in the co-regulation of rbcL, or may have diverged as a regulator of one or more other plastid-encoded mRNAs. This analysis confirms that RLSB is an important and unique photosynthetic regulatory protein that has been continuously expressed in land plants as they emerged and diversified from their ancient common ancestor.

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.

The complete chloroplast genome sequence of the medicinal plant Andrographis paniculata.

PubMed

Ding, Ping; Shao, Yanhua; Li, Qian; Gao, Junli; Zhang, Runjing; Lai, Xiaoping; Wang, Deqin; Zhang, Huiye

2016-07-01

The complete chloroplast genome of Andrographis paniculata, an important medicinal plant with great economic value, has been studied in this article. The genome size is 150,249 bp in length, with 38.3% GC content. A pair of inverted repeats (IRs, 25,300 bp) are separated by a large single copy region (LSC, 82,459 bp) and a small single-copy region (SSC, 17,190 bp). The chloroplast genome contains 114 unique genes, 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes. In these genes, 15 genes contained 1 intron and 3 genes comprised of 2 introns.

The complete chloroplast genome sequence of Dianthus superbus var. longicalycinus.

PubMed

Gurusamy, Raman; Lee, Do-Hyung; Park, SeonJoo

2016-05-01

The complete chloroplast genome (cpDNA) sequence of Dianthus superbus var. longicalycinus is an economically important traditional Chinese medicine was reported and characterized. The cpDNA of Dianthus superbus var. longicalycinus is 149,539 bp, with 36.3% GC content. A pair of inverted repeats (IRs) of 24,803 bp is separated by a large single-copy region (LSC, 82,805 bp) and a small single-copy region (SSC, 17,128 bp). It encodes 85 protein-coding genes, 36 tRNA genes and 8 rRNA genes. Of 129 individual genes, 13 genes encoded one intron and three genes have two introns.

The complete chloroplast genome sequence of Dendrobium nobile.

PubMed

Yan, Wenjin; Niu, Zhitao; Zhu, Shuying; Ye, Meirong; Ding, Xiaoyu

2016-11-01

The complete chloroplast (cp) genome sequence of Dendrobium nobile, an endangered and traditional Chinese medicine with important economic value, is presented in this article. The total genome size is 150,793 bp, containing a large single copy (LSC) region (84,939 bp) and a small single copy region (SSC) (13,310 bp) which were separated by two inverted repeat (IRs) regions (26,272 bp). The overall GC contents of the plastid genome were 38.8%. In total, 130 unique genes were annotated and they were consisted of 76 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Fourteen genes contained one or two introns.

The complete chloroplast genome sequence of Curcuma flaviflora (Curcuma).

PubMed

Zhang, Yan; Deng, Jiabin; Li, Yangyi; Gao, Gang; Ding, Chunbang; Zhang, Li; Zhou, Yonghong; Yang, Ruiwu

2016-09-01

The complete chloroplast (cp) genome of Curcuma flaviflora, a medicinal plant in Southeast Asia, was sequenced. The genome size was 160 478 bp in length, with 36.3% GC content. A pair of inverted repeats (IRs) of 26 946 bp were separated by a large single copy (LSC) of 88 008 bp and a small single copy (SSC) of 18 578 bp, respectively. The cp genome contained 132 annotated genes, including 79 protein coding genes, 30 tRNA genes, and four rRNA genes. And 19 of these genes were duplicated in inverted repeat regions.

Development of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae.

PubMed

Liu, Hailan; Guo, Xiaoqin; Wu, Jiasheng; Chen, Guo-Bo; Ying, Yeqing

2013-03-01

KEY MESSAGE : We develop a set of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae. Being evolutionary conserved, single-copy orthologous (COSII) genes are particularly useful in comparative mapping and phylogenetic investigation among species. In this study, we identified 2,684 COSII genes based on five sequenced Poaceae genomes including rice, maize, sorghum, foxtail millet, and brachypodium, and then developed 1,072 COSII markers whose transferability and polymorphism among five bamboo species were further evaluated with 46 pairs of randomly selected primers. 91.3 % of the 46 primers obtained clear amplification in at least one bamboo species, and 65.2 % of them produced polymorphism in more than one species. We also used 42 of them to construct the phylogeny for the five bamboo species, and it might reflect more precise evolutionary relationship than the one based on the vegetative morphology. The results indicated a promising prospect of applying these markers to the investigation of genetic diversity and the classification of Poaceae. To ease and facilitate access of the information of common interest to readers, a web-based database of the COSII markers is provided ( http://www.sicau.edu.cn/web/yms/PCOSWeb/PCOS.html ).

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

PubMed Central

Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

2016-01-01

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

Exon skipping of AGAMOUS homolog PrseAG in developing double flowers of Prunus lannesiana (Rosaceae).

PubMed

Liu, Zhixiong; Zhang, Dandan; Liu, Di; Li, Fenglan; Lu, Hai

2013-02-01

KEY MESSAGE : Two transcript isoforms of AGAMOUS homologs, from single and double flower Prunus lannesiana, respectively, showed different functions. The Arabidopsis floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity. Loss of AG function results in homeotic conversions of stamens into petals and formation of double flowers. In order to present a molecular dissection of a double-flower cultivar in Prunus lannesiana (Rosaceae), we isolated and identified a single-copy gene, AG homolog from two genetically cognate P. lannesiana bearing single and double flowers, respectively. Sequence analysis revealed that the AG homolog, prseag-1, from double flowers showed a 170-bp exon skipping as compared to PrseAG (Prunus serrulata AGAMOUS) from the single flowers. Genomic DNA sequence revealed that abnormal splicing resulted in mutant prseag-1 protein with the C-terminal AG motifs I and II deletions. In addition, protein sequence alignment and phylogenetic analyses revealed that the PrseAG was grouped into the euAG lineage. A semi-quantitative PCR analysis showed that the expression of PrseAG was restricted to reproductive organs of stamens and carpels in single flowers of P. lannesiana 'speciosa', while the prseag-1 mRNA was highly transcribed throughout the petals, stamens, and carpels in double flowers from 'Albo-rosea'. The transgenic Arabidopsis containing 35S::PrseAG displayed extremely early flowering, bigger stamens and carpels and homeotic conversion of petals into staminoid organs, but ectopic expression of prseag-1 could not mimic the phenotypic ectopic expression of PrseAG in Arabidopsis. In general, this study provides evidences to show that double flower 'Albo-rosea' is a putative C functional ag mutant in P. lannesiana.

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.

Surface code implementation of block code state distillation.

PubMed

Fowler, Austin G; Devitt, Simon J; Jones, Cody

2013-01-01

State distillation is the process of taking a number of imperfect copies of a particular quantum state and producing fewer better copies. Until recently, the lowest overhead method of distilling states produced a single improved [formula: see text] state given 15 input copies. New block code state distillation methods can produce k improved [formula: see text] states given 3k + 8 input copies, potentially significantly reducing the overhead associated with state distillation. We construct an explicit surface code implementation of block code state distillation and quantitatively compare the overhead of this approach to the old. We find that, using the best available techniques, for parameters of practical interest, block code state distillation does not always lead to lower overhead, and, when it does, the overhead reduction is typically less than a factor of three.

Surface code implementation of block code state distillation

PubMed Central

Fowler, Austin G.; Devitt, Simon J.; Jones, Cody

2013-01-01

State distillation is the process of taking a number of imperfect copies of a particular quantum state and producing fewer better copies. Until recently, the lowest overhead method of distilling states produced a single improved |A〉 state given 15 input copies. New block code state distillation methods can produce k improved |A〉 states given 3k + 8 input copies, potentially significantly reducing the overhead associated with state distillation. We construct an explicit surface code implementation of block code state distillation and quantitatively compare the overhead of this approach to the old. We find that, using the best available techniques, for parameters of practical interest, block code state distillation does not always lead to lower overhead, and, when it does, the overhead reduction is typically less than a factor of three. PMID:23736868

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.

The Shadow of Hate: A History of Intolerance in America. Student Text ("Us and Them") and Teacher's Guide.

ERIC Educational Resources Information Center

Carnes, Jim; Roberson, Houston

"The Shadow of Hate" resource kit provides a videotape program (40 minutes), 20 copies of a 128-page student text ("Us and Them"), and a 32-page teacher's guide. This document consists of single copies of the two printed components of this kit. The resource traces the history of racial, religious, and social intolerance in the…

Copy number variation in metabolic phenotypes.

PubMed

Lanktree, M; Hegele, R A

2008-01-01

Despite successes in identifying genetic contributors to common metabolic phenotypes, only part of the heritable component of these traits has thus far been explained. Copy number variation (CNV) is likely to be responsible for some of the unexplained variation. As observed with single nucleotide changes, it is probable that both rare and common CNVs will contribute to susceptibility to metabolic disease. For instance, CNVs in the LDLR gene underlie a substantial portion of disease in patients with heterozygous familial hypercholesterolemia. As well, a common CNV in LPA encoding apolipoprotein(a) is the primary determinant of plasma lipoprotein(a) concentrations, a risk factor for atherosclerosis. Recent efforts to map CNVs in control populations have defined their size, frequency and distribution. Many of the identified CNVs overlap genes with important functions in metabolic pathways. The overlap of CNVs that were found in control datasets with functional candidate genes or genes with previous evidence of association with metabolic syndrome presents an important subset for future CNV association studies. Finally, we describe an approach to search for CNVs in a rare high-penetrance metabolic disorder, namely lipodystrophy. As methods to identify CNVs increase in precision and accuracy, the prospect of identifying their role in both rare Mendelian and common complex metabolic phenotypes will become a reality. Copyright 2009 S. Karger AG, Basel.

A Study of Multifunctional Document Centers that Are Accessible to People Who Are Visually Impaired

ERIC Educational Resources Information Center

Huffman, Lee A.; Uslan, Mark M.; Burton, Darren M.; Eghtesadi, Caesar

2009-01-01

The capabilities of modern photocopy machines have advanced beyond the simple duplication of documents. In addition to the standard functions of copying, collating, and stapling, such machines can be a part of telecommunication networks and provide printing, scanning, faxing, and e-mailing functions. No longer just copy machines, these devices are…

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.

PubMed

Aguirre, Andrew J; Meyers, Robin M; Weir, Barbara A; Vazquez, Francisca; Zhang, Cheng-Zhong; Ben-David, Uri; Cook, April; Ha, Gavin; Harrington, William F; Doshi, Mihir B; Kost-Alimova, Maria; Gill, Stanley; Xu, Han; Ali, Levi D; Jiang, Guozhi; Pantel, Sasha; Lee, Yenarae; Goodale, Amy; Cherniack, Andrew D; Oh, Coyin; Kryukov, Gregory; Cowley, Glenn S; Garraway, Levi A; Stegmaier, Kimberly; Roberts, Charles W; Golub, Todd R; Meyerson, Matthew; Root, David E; Tsherniak, Aviad; Hahn, William C

2016-08-01

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. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803. 2016 American Association for Cancer Research.

Single-cell sequencing deciphers a convergent evolution of copy number alterations from primary to circulating tumor cells.

PubMed

Gao, Yan; Ni, Xiaohui; Guo, Hua; Su, Zhe; Ba, Yi; Tong, Zhongsheng; Guo, Zhi; Yao, Xin; Chen, Xixi; Yin, Jian; Yan, Zhao; Guo, Lin; Liu, Ying; Bai, Fan; Xie, X Sunney; Zhang, Ning

2017-08-01

Copy number alteration (CNA) is a major contributor to genome instability, a hallmark of cancer. Here, we studied genomic alterations in single primary tumor cells and circulating tumor cells (CTCs) from the same patient. Single-nucleotide variants (SNVs) in single cells from both samples occurred sporadically, whereas CNAs among primary tumor cells emerged accumulatively rather than abruptly, converging toward the CNA in CTCs. Focal CNAs affecting the MYC gene and the PTEN gene were observed only in a minor portion of primary tumor cells but were present in all CTCs, suggesting a strong selection toward metastasis. Single-cell structural variant (SV) analyses revealed a two-step mechanism, a complex rearrangement followed by gene amplification, for the simultaneous formation of anomalous CNAs in multiple chromosome regions. Integrative CNA analyses of 97 CTCs from 23 patients confirmed the convergence of CNAs and revealed single, concurrent, and mutually exclusive CNAs that could be the driving events in cancer metastasis. © 2017 Gao et al.; Published by Cold Spring Harbor Laboratory Press.

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

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

Cui, Yi; Hu, Dehong; Markillie, Lye Meng

Quantitative gene expression analysis in intact single cells can be achieved using single molecule- based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple FISH sub-probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific binding of sub-probes and tissue autofluorescence, limiting its accuracy. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on blinking frequencies of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses blinking frequency patterns, emitted frommore » a transcript bound to multiple sub-probes, which are distinct from blinking patterns emitted from partial or nonspecifically bound sub-probes and autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2, and their ratio (Nkx2-2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct blinking frequency patterns, laying the foundation for reliable single-cell transcriptomics.« less

Long-Read Single Molecule Sequencing to Resolve Tandem Gene Copies: The Mst77Y Region on the Drosophila melanogaster Y Chromosome

PubMed Central

Krsticevic, Flavia J.; Schrago, Carlos G.; Carvalho, A. Bernardo

2015-01-01

The autosomal gene Mst77F of Drosophila melanogaster is essential for male fertility. In 2010, Krsticevic et al. (Genetics 184: 295−307) found 18 Y-linked copies of Mst77F (“Mst77Y”), which collectively account for 20% of the functional Mst77F-like mRNA. The Mst77Y genes were severely misassembled in the then-available genome assembly and were identified by cloning and sequencing polymerase chain reaction products. The genomic structure of the Mst77Y region and the possible existence of additional copies remained unknown. The recent publication of two long-read assemblies of D. melanogaster prompted us to reinvestigate this challenging region of the Y chromosome. We found that the Illumina Synthetic Long Reads assembly failed in the Mst77Y region, most likely because of its tandem duplication structure. The PacBio MHAP assembly of the Mst77Y region seems to be very accurate, as revealed by comparisons with the previously found Mst77Y genes, a bacterial artificial chromosome sequence, and Illumina reads of the same strain. We found that the Mst77Y region spans 96 kb and originated from a 3.4-kb transposition from chromosome 3L to the Y chromosome, followed by tandem duplications inside the Y chromosome and invasion of transposable elements, which account for 48% of its length. Twelve of the 18 Mst77Y genes found in 2010 were confirmed in the PacBio assembly, the remaining six being polymerase chain reaction−induced artifacts. There are several identical copies of some Mst77Y genes, coincidentally bringing the total copy number to 18. Besides providing a detailed picture of the Mst77Y region, our results highlight the utility of PacBio technology in assembling difficult genomic regions such as tandemly repeated genes. PMID:25858959

Dwarfism

MedlinePlus

... normal copy to his or her own children. Turner syndrome Turner syndrome, a condition that affects only girls and women, ... X chromosome from each parent. A girl with Turner syndrome has only one fully functioning copy of the ...

HU content and dynamics in Escherichia coli during the cell cycle and at different growth rates.

PubMed

Abebe, Anteneh Hailu; Aranovich, Alexander; Fishov, Itzhak

2017-10-16

DNA-binding proteins play an important role in maintaining bacterial chromosome structure and functions. Heat-unstable (HU) histone-like protein is one of the most abundant of these proteins and participates in all major chromosome-related activities. Owing to its low sequence specificity, HU fusions with fluorescent proteins were used for general staining of the nucleoid, aiming to reveal its morphology and dynamics. We have exploited a single chromosomal copy of hupA-egfp fusion under the native promoter and used quantitative microscopy imaging to investigate the amount and dynamics of HUα in Escherichia coli cells. We found that in steady-state growing populations the cellular HUα content is proportional to the cell size, whereas its concentration is size independent. Single-cell live microscopy imaging confirmed that the amount of HUα exponentially increases during the cell cycle, but its concentration is maintained constant. This supports the existence of an auto-regulatory mechanism underlying the HUα cellular level, in addition to reflecting the gene copy number. Both the HUα amount and concentration strongly increase with the cell growth rate in different culture media. Unexpectedly, the HU/DNA stoichiometry also remarkably increases with the growth rate. This last finding may be attributed to a higher requirement for maintaining the chromosome structure in nucleoids with higher complexity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack)

PubMed Central

2012-01-01

Background While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Results Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. Conclusions The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants. PMID:23006412

Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack).

PubMed

Hensel, Goetz; Oleszczuk, Sylwia; Daghma, Diaa Eldin S; Zimny, Janusz; Melzer, Michael; Kumlehn, Jochen

2012-09-25

While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants.

The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex

PubMed Central

2012-01-01

Background Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP), a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. Results Isolates of the B. plicatilis species complex have 1–4 copies of mmr-b, each composed of 2–9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. Conclusions Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving rapidly, and novel alleles may be maintained and increase in frequency via asexual reproduction. Our analyses indicate that mate recognition, controlled by MMR-B, may drive reproductive isolation and allow saltational sympatric speciation within the B. plicatilis cryptic species complex, and that this process may be largely neutral. PMID:22852831

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.

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.

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

Jimenez, O.; Departamento de Fisica, Facultad de Ciencias Basicas, Universidad de Antofagasta, Casilla 170, Antofagasta; Bergou, J.

We study the probabilistic cloning of three symmetric states. These states are defined by a single complex quantity, the inner product among them. We show that three different probabilistic cloning machines are necessary to optimally clone all possible families of three symmetric states. We also show that the optimal cloning probability of generating M copies out of one original can be cast as the quotient between the success probability of unambiguously discriminating one and M copies of symmetric states.

The puroindoline b-2 variants are expressed at low levels relative to the puroindoline D1 genes in wheat seeds

USDA-ARS?s Scientific Manuscript database

Grain hardness in wheat is largely controlled by the Hardness locus. This locus contains the Puroindoline a and b genes, which were thought to exist as single copy genes on chromosome 5D. In fact, four additional copies of Pinb have been reported, termed Pinb-2v-1 – Pinb-2v4, which map to the grou...

CFTR Genotype and Maximal Exercise Capacity in Cystic Fibrosis: A Cross-sectional Study.

PubMed

Radtke, Thomas; Hebestreit, Helge; Gallati, Sabina; Schneiderman, Jane E; Braun, Julia; Stevens, Daniel; Hulzebos, Erik Hj; Takken, Tim; Boas, Steven R; Urquhart, Don S; Lands, Larry C; Tejero, Sergio; Sovtic, Aleksandar; Dwyer, Tiffany; Petrovic, Milos; Harris, Ryan A; Karila, Chantal; Savi, Daniela; Usemann, Jakob; Mei-Zahav, Meir; Hatziagorou, Elpis; Ratjen, Felix; Kriemler, Susi

2018-02-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human skeletal muscle cells. Variations of CFTR dysfunction among patients with cystic fibrosis may be an important determinant of maximal exercise capacity in cystic fibrosis. Previous studies on the relationship between CFTR genotype and maximal exercise capacity are scarce and contradictory. This study was designed to explore factors influencing maximal exercise capacity, expressed as peak oxygen uptake (V.O2peak), with a specific focus on CFTR genotype in children and adults with cystic fibrosis. In an international, multicenter, cross-sectional study, we collected data on CFTR genotype and cardiopulmonary exercise tests in patients with cystic fibrosis who were ages 8 years and older. CFTR mutations were classified into functional classes I–V. The final analysis included 726 patients (45% females; age range, 8–61 yr; forced expiratory volume in 1 s, 16 to 123% predicted) from 17 cystic fibrosis centers in North America, Europe, Australia, and Asia, all of whom had both valid maximal cardiopulmonary exercise tests and complete CFTR genotype data. Overall, patients exhibited exercise intolerance (V.O2peak, 77.3 ± 19.1% predicted), but values were comparable among different CFTR classes. We did not detect an association between CFTR genotype functional classes I–III and either V.O2peak (percent predicted) (adjusted β = −0.95; 95% CI, −4.18 to 2.29; P = 0.57) or maximum work rate (Wattmax) (adjusted β = −1.38; 95% CI, −5.04 to 2.27; P = 0.46) compared with classes IV–V. Those with at least one copy of a F508del-CFTR mutation and one copy of a class V mutation had a significantly lower V.O2peak (β = −8.24%; 95% CI, −14.53 to −2.99; P = 0.003) and lower Wattmax (adjusted β = −7.59%; 95% CI, −14.21 to −0.95; P = 0.025) than those with two copies of a class II mutation. On the basis of linear regression analysis adjusted for relevant confounders, lung function and body mass index were associated with V.O2peak. CFTR functional genotype class was not associated with maximal exercise capacity in patients with cystic fibrosis overall, but those with at least one copy of a F508del-CFTR mutation and a single class V mutation had lower maximal exercise capacity.

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.

Differential allelic expression of IL13 and CSF2 genes associated with asthma.

PubMed

Burkhardt, Jana; Kirsten, Holger; Wolfram, Grit; Quente, Elfi; Ahnert, Peter

2012-07-01

An important area of genetic research is the identification of functional mechanisms in polymorphisms associated with diseases. A highly relevant functional mechanism is the influence of polymorphisms on gene expression levels (differential allelic expression, DAE). The coding single nucleotide polymorphisms (SNPs) CSF2(rs25882) and IL13(rs20541) have been associated with asthma. In this work, we investigated whether the mRNA expression levels of CSF2 or IL13 were correlated with these SNPs. Samples were analyzed by mass spectrometry-based quantification of gene expression. Both SNPs influenced gene expression levels (CSF2(rs25882): p(overall) = 0.008 and p(DAE samples) = 0.00006; IL13(rs20541): p(overall) = 0.059 and p(DAE samples) = 0.036). For CSF2, the expression level was increased by 27.4% (95% CI: 18.5%-35.4%) in samples with significant DAE in the presence of one copy of risk variant CSF2(rs25882-T). The average expression level of IL13 was increased by 29.8% (95% CI: 3.1%-63.4%) in samples with significant DAE in the presence of one copy of risk variant IL13(rs20541-A). Enhanced expression of CSF2 could stimulate macrophages and neutrophils during inflammation and may be related to the etiology of asthma. For IL-13, higher expression could enhance the functional activity of the asthma-associated isoform. Overall, the analysis of DAE provides an efficient approach for identifying possible functional mechanisms that link disease-associated variants with altered gene expression levels.

Defining a Structural and Kinetic Rationale for Paralogous Copies of Phenylacetate-CoA Ligases from the Cystic Fibrosis Pathogen Burkholderia cenocepacia J2315*

PubMed Central

Law, Adrienne; Boulanger, Martin J.

2011-01-01

The phenylacetic acid (PAA) degradation pathway is the sole aerobic route for phenylacetic acid metabolism in bacteria and facilitates degradation of environmental pollutants such as styrene and ethylbenzene. The PAA pathway also is implicated in promoting Burkholderia cenocepacia infections in cystic fibrosis patients. Intriguingly, the first enzyme in the PAA pathway is present in two copies (paaK1 and paaK2), yet each subsequent enzyme is present in only a single copy. Furthermore, sequence divergence indicates that PaaK1 and PaaK2 form a unique subgroup within the adenylate-forming enzyme (AFE) superfamily. To establish a biochemical rationale for the existence of the PaaK paralogs in B. cenocepacia, we present high resolution x-ray crystal structures of a selenomethionine derivative of PaaK1 in complex with ATP and adenylated phenylacetate intermediate complexes of PaaK1 and PaaK2 in distinct conformations. Structural analysis reveals a novel N-terminal microdomain that may serve to recruit subsequent PAA enzymes, whereas a bifunctional role is proposed for the P-loop in stabilizing the C-terminal domain in conformation 2. The potential for different kinetic profiles was suggested by a structurally divergent extension of the aryl substrate pocket in PaaK1 relative to PaaK2. Functional characterization confirmed this prediction, with PaaK1 possessing a lower Km for phenylacetic acid and better able to accommodate 3′ and 4′ substitutions on the phenyl ring. Collectively, these results offer detailed insight into the reaction mechanism of a novel subgroup of the AFE superfamily and provide a clear biochemical rationale for the presence of paralogous copies of PaaK of B. cenocepacia. PMID:21388965

A Complex 6p25 Rearrangement in a Child With Multiple Epiphyseal Dysplasia

PubMed Central

Bedoyan, Jirair K.; Lesperance, Marci M.; Ackley, Todd; Iyer, Ramaswamy K.; Innis, Jeffrey W.; Misra, Vinod K.

2015-01-01

Genomic rearrangements are increasingly recognized as important contributors to human disease. Here we report on an 11½-year-old child with myopia, Duane retraction syndrome, bilateral mixed hearing loss, skeletal anomalies including multiple epiphyseal dysplasia, and global developmental delay, and a complex 6p25 genomic rearrangement. We have employed oligonucleotide-based comparative genomic hybridization arrays (aCGH) of different resolutions (44 and 244K) as well as a 1 M single nucleotide polymorphism (SNP) array to analyze this complex rearrangement. Our analyses reveal a complex rearrangement involving a ~2.21 Mb interstitial deletion, a ~240 kb terminal deletion, and a 70–80 kb region in between these two deletions that shows maintenance of genomic copy number. The interstitial deletion contains eight known genes, including three Forkhead box containing (FOX) transcription factors (FOXQ1, FOXF2, and FOXC1). The region maintaining genomic copy number partly overlaps the dual specificity protein phosphatase 22 (DUSP22) gene. Array analyses suggest a homozygous loss of genomic material at the 5′ end of DUSP22, which was corroborated using TaqMan® copy number analysis. It is possible that this homozygous genomic loss may render both copies of DUSP22 or its products non-functional. Our analysis suggests a rearrangement mechanism distinct from a previously reported replication-based error-prone mechanism without template switching for a specific 6p25 rearrangement with a 1.22 Mb interstitial deletion. Our study demonstrates the utility and limitations of using oligonucleotide-based aCGH and SNP array technologies of increasing resolutions in order to identify complex DNA rearrangements and gene disruptions. PMID:21204225

The complete chloroplast genome sequence of American bird pepper (Capsicum annuum var. glabriusculum).

PubMed

Zeng, Fan-chun; Gao, Cheng-wen; Gao, Li-zhi

2016-01-01

The complete chloroplast genome sequence of American bird pepper (Capsicum annuum var. glabriusculum) is reported and characterized in this study. The genome size is 156,612 bp, containing a pair of inverted repeats (IRs) of 25,776 bp separated by a large single-copy region of 87,213 bp and a small single-copy region of 17,851 bp. The chloroplast genome harbors 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes, and 37 tRNA genes. A total of 18 of these genes are duplicated in the inverted repeat regions, 16 genes contain 1 intron, and 2 genes and one ycf have 2 introns.

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

Recombination-dependent replication and gene conversion homogenize repeat sequences and diversify plastid genome structure.

PubMed

Ruhlman, Tracey A; Zhang, Jin; Blazier, John C; Sabir, Jamal S M; Jansen, Robert K

2017-04-01

There is a misinterpretation in the literature regarding the variable orientation of the small single copy region of plastid genomes (plastomes). The common phenomenon of small and large single copy inversion, hypothesized to occur through intramolecular recombination between inverted repeats (IR) in a circular, single unit-genome, in fact, more likely occurs through recombination-dependent replication (RDR) of linear plastome templates. If RDR can be primed through both intra- and intermolecular recombination, then this mechanism could not only create inversion isomers of so-called single copy regions, but also an array of alternative sequence arrangements. We used Illumina paired-end and PacBio single-molecule real-time (SMRT) sequences to characterize repeat structure in the plastome of Monsonia emarginata (Geraniaceae). We used OrgConv and inspected nucleotide alignments to infer ancestral nucleotides and identify gene conversion among repeats and mapped long (>1 kb) SMRT reads against the unit-genome assembly to identify alternative sequence arrangements. Although M. emarginata lacks the canonical IR, we found that large repeats (>1 kilobase; kb) represent ∼22% of the plastome nucleotide content. Among the largest repeats (>2 kb), we identified GC-biased gene conversion and mapping filtered, long SMRT reads to the M. emarginata unit-genome assembly revealed alternative, substoichiometric sequence arrangements. We offer a model based on RDR and gene conversion between long repeated sequences in the M. emarginata plastome and provide support that both intra-and intermolecular recombination between large repeats, particularly in repeat-rich plastomes, varies unit-genome structure while homogenizing the nucleotide sequence of repeats. © 2017 Botanical Society of America.

Divergent gene copies in the asexual class Bdelloidea (Rotifera) separated before the bdelloid radiation or within bdelloid families.

PubMed

Mark Welch, David B; Cummings, Michael P; Hillis, David M; Meselson, Matthew

2004-02-10

Rotifers of the asexual class Bdelloidea are unusual in possessing two or more divergent copies of every gene that has been examined. Phylogenetic analysis of the heat-shock gene hsp82 and the TATA-box-binding protein gene tbp in multiple bdelloid species suggested that for each gene, each copy belonged to one of two lineages that began to diverge before the bdelloid radiation. Such gene trees are consistent with the two lineages having descended from former alleles that began to diverge after meiotic segregation ceased or from subgenomes of an alloploid ancestor of the bdelloids. However, the original analyses of bdelloid gene-copy divergence used only a single outgroup species and were based on parsimony and neighbor joining. We have now used maximum likelihood and Bayesian inference methods and, for hsp82, multiple outgroups in an attempt to produce more robust gene trees. Here we report that the available data do not unambiguously discriminate between gene trees that root the origin of hsp82 and tbp copy divergence before the bdelloid radiation and those which indicate that the gene copies began to diverge within bdelloid families. The remarkable presence of multiple diverged gene copies in individual genomes is nevertheless consistent with the loss of sex in an ancient ancestor of bdelloids.

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

Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer

PubMed Central

Bailey, Swneke D.; Desai, Kinjal; Kron, Ken J.; Mazrooei, Parisa; Sinnott-Armstrong, Nicholas A.; Treloar, Aislinn E.; Dowar, Mark; Thu, Kelsie L.; Cescon, David W.; Silvester, Jennifer; Yang, S. Y. Cindy; Wu, Xue; Pezo, Rossanna C.; Haibe-Kains, Benjamin; Mak, Tak W.; Bedard, Philippe L.; Pugh, Trevor J.; Sallari, Richard C.; Lupien, Mathieu

2016-01-01

Sustained expression of the oestrogen receptor alpha (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon oestrogen stimulation to establish an oncogenic expression program1. Somatic copy number alterations involving the ESR1 gene occur in approximately 1% of ESR1-positive breast cancers2–5, implying that other mechanisms underlie the persistent expression of ESR1. We report the significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating ESR1 in 7% of ESR1-positive breast cancers. These mutations regulate ESR1 expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by a functional inherited single nucleotide variant (SNV) rs9383590 that accounts for several breast cancer risk-loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer. PMID:27571262

Utilities for master source code distribution: MAX and Friends

NASA Technical Reports Server (NTRS)

Felippa, Carlos A.

1988-01-01

MAX is a program for the manipulation of FORTRAN master source code (MSC). This is a technique by which one maintains one and only one master copy of a FORTRAN program under a program developing system, which for MAX is assumed to be VAX/VMS. The master copy is not intended to be directly compiled. Instead it must be pre-processed by MAX to produce compilable instances. These instances may correspond to different code versions (for example, double precision versus single precision), different machines (for example, IBM, CDC, Cray) or different operating systems (i.e., VAX/VMS versus VAX/UNIX). The advantage os using a master source is more pronounced in complex application programs that are developed and maintained over many years and are to be transported and executed on several computer environments. The version lag problem that plagues many such programs is avoided by this approach. MAX is complemented by several auxiliary programs that perform nonessential functions. The ensemble is collectively known as MAX and Friends. All of these programs, including MAX, are executed as foreign VAX/VMS commands and can easily be hidden in customized VMS command procedures.

Genome-wide screening identifies a KCNIP1 copy number variant as a genetic predictor for atrial fibrillation

PubMed Central

Tsai, Chia-Ti; Hsieh, Chia-Shan; Chang, Sheng-Nan; Chuang, Eric Y.; Ueng, Kwo-Chang; Tsai, Chin-Feng; Lin, Tsung-Hsien; Wu, Cho-Kai; Lee, Jen-Kuang; Lin, Lian-Yu; Wang, Yi-Chih; Yu, Chih-Chieh; Lai, Ling-Ping; Tseng, Chuen-Den; Hwang, Juey-Jen; Chiang, Fu-Tien; Lin, Jiunn-Lee

2016-01-01

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Previous genome-wide association studies had identified single-nucleotide polymorphisms in several genomic regions to be associated with AF. In human genome, copy number variations (CNVs) are known to contribute to disease susceptibility. Using a genome-wide multistage approach to identify AF susceptibility CNVs, we here show a common 4,470-bp diallelic CNV in the first intron of potassium interacting channel 1 gene (KCNIP1) is strongly associated with AF in Taiwanese populations (odds ratio=2.27 for insertion allele; P=6.23 × 10−24). KCNIP1 insertion is associated with higher KCNIP1 mRNA expression. KCNIP1-encoded protein potassium interacting channel 1 (KCHIP1) is physically associated with potassium Kv channels and modulates atrial transient outward current in cardiac myocytes. Overexpression of KCNIP1 results in inducible AF in zebrafish. In conclusions, a common CNV in KCNIP1 gene is a genetic predictor of AF risk possibly pointing to a functional pathway. PMID:26831368

Identification of the first multi-exonic WDR72 deletion in isolated amelogenesis imperfecta, and generation of a WDR72-specific copy number screening tool.

PubMed

Hentschel, Julia; Tatun, Dana; Parkhomchuk, Dmitri; Kurth, Ingo; Schimmel, Bettina; Heinrich-Weltzien, Roswitha; Bertzbach, Sabine; Peters, Hartmut; Beetz, Christian

2016-09-15

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous disorder of tooth development which is due to aberrant deposition or composition of enamel. Both syndromic and isolated forms exist; they may be inherited in an X-linked, autosomal recessive, or autosomal dominant manner. WDR72 is one of ten currently known genes for recessive isolated AI; nine WDR72 mutations affecting single nucleotides have been described to date. Based on whole exome sequencing in a large consanguineous AI pedigree, we obtained evidence for presence of a multi-exonic WDR72 deletion. A home-made multiplex ligation-dependent probe amplification assay was used to confirm the aberration, to narrow its extent, and to identify heterozygous carriers. Our study extends the mutational spectrum for WDR72 to include large deletions, and supports a relevance of the previously proposed loss-of-function mechanism. It also introduces an easy-to-use and highly sensitive tool for detecting WDR72 copy number alterations. Copyright © 2016. Published by Elsevier B.V.

Schizosaccharomyces pombe Retrotransposon Tf2 Mobilizes Primarily through Homologous cDNA Recombination

PubMed Central

Hoff, Eleanor F.; Levin, Henry L.; Boeke, Jef D.

1998-01-01

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5′ untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons. PMID:9774697

Copy Number Variations in Tilapia Genomes.

PubMed

Li, Bi Jun; Li, Hong Lian; Meng, Zining; Zhang, Yong; Lin, Haoran; Yue, Gen Hua; Xia, Jun Hong

2017-02-01

Discovering the nature and pattern of genome variation is fundamental in understanding phenotypic diversity among populations. Although several millions of single nucleotide polymorphisms (SNPs) have been discovered in tilapia, the genome-wide characterization of larger structural variants, such as copy number variation (CNV) regions has not been carried out yet. We conducted a genome-wide scan for CNVs in 47 individuals from three tilapia populations. Based on 254 Gb of high-quality paired-end sequencing reads, we identified 4642 distinct high-confidence CNVs. These CNVs account for 1.9% (12.411 Mb) of the used Nile tilapia reference genome. A total of 1100 predicted CNVs were found overlapping with exon regions of protein genes. Further association analysis based on linear model regression found 85 CNVs ranging between 300 and 27,000 base pairs significantly associated to population types (R 2  > 0.9 and P > 0.001). Our study sheds first insights on genome-wide CNVs in tilapia. These CNVs among and within tilapia populations may have functional effects on phenotypes and specific adaptation to particular environments.

A diffusion approach to approximating preservation probabilities for gene duplicates.

PubMed

O'Hely, Martin

2006-08-01

Consider a haploid population and, within its genome, a gene whose presence is vital for the survival of any individual. Each copy of this gene is subject to mutations which destroy its function. Suppose one member of the population somehow acquires a duplicate copy of the gene, where the duplicate is fully linked to the original gene's locus. Preservation is said to occur if eventually the entire population consists of individuals descended from this one which initially carried the duplicate. The system is modelled by a finite state-space Markov process which in turn is approximated by a diffusion process, whence an explicit expression for the probability of preservation is derived. The event of preservation can be compared to the fixation of a selectively neutral gene variant initially present in a single individual, the probability of which is the reciprocal of the population size. For very weak mutation, this and the probability of preservation are equal, while as mutation becomes stronger, the preservation probability tends to double this reciprocal. This is in excellent agreement with simulation studies.

Strategies for Sharing Seismic Data Among Multiple Computer Platforms

NASA Astrophysics Data System (ADS)

Baker, L. M.; Fletcher, J. B.

2001-12-01

Seismic waveform data is readily available from a variety of sources, but it often comes in a distinct, instrument-specific data format. For example, data may be from portable seismographs, such as those made by Refraction Technology or Kinemetrics, from permanent seismograph arrays, such as the USGS Parkfield Dense Array, from public data centers, such as the IRIS Data Center, or from personal communication with other researchers through e-mail or ftp. A computer must be selected to import the data - usually whichever is the most suitable for reading the originating format. However, the computer best suited for a specific analysis may not be the same. When copies of the data are then made for analysis, a proliferation of copies of the same data results, in possibly incompatible, computer-specific formats. In addition, if an error is detected and corrected in one copy, or some other change is made, all the other copies must be updated to preserve their validity. Keeping track of what data is available, where it is located, and which copy is authoritative requires an effort that is easy to neglect. We solve this problem by importing waveform data to a shared network file server that is accessible to all our computers on our campus LAN. We use a Network Appliance file server running Sun's Network File System (NFS) software. Using an NFS client software package on each analysis computer, waveform data can then be read by our MatLab or Fortran applications without first copying the data. Since there is a single copy of the waveform data in a single location, the NFS file system hierarchy provides an implicit complete waveform data catalog and the single copy is inherently authoritative. Another part of our solution is to convert the original data into a blocked-binary format (known historically as USGS DR100 or VFBB format) that is interpreted by MatLab or Fortran library routines available on each computer so that the idiosyncrasies of each machine are not visible to the user. Commercial software packages, such as MatLab, also have the ability to share data in their own formats across multiple computer platforms. Our Fortran applications can create plot files in Adobe PostScript, Illustrator, and Portable Document Format (PDF) formats. Vendor support for reading these files is readily available on multiple computer platforms. We will illustrate by example our strategies for sharing seismic data among our multiple computer platforms, and we will discuss our positive and negative experiences. We will include our solutions for handling the different byte ordering, floating-point formats, and text file ``end-of-line'' conventions on the various computer platforms we use (6 different operating systems on 5 processor architectures).

Genetic analysis of the ADGF multigene family by homologous recombination and gene conversion in Drosophila.

PubMed

Dolezal, Tomas; Gazi, Michal; Zurovec, Michal; Bryant, Peter J

2003-10-01

Many Drosophila genes exist as members of multigene families and within each family the members can be functionally redundant, making it difficult to identify them by classical mutagenesis techniques based on phenotypic screening. We have addressed this problem in a genetic analysis of a novel family of six adenosine deaminase-related growth factors (ADGFs). We used ends-in targeting to introduce mutations into five of the six ADGF genes, taking advantage of the fact that five of the family members are encoded by a three-gene cluster and a two-gene cluster. We used two targeting constructs to introduce loss-of-function mutations into all five genes, as well as to isolate different combinations of multiple mutations, independent of phenotypic consequences. The results show that (1) it is possible to use ends-in targeting to disrupt gene clusters; (2) gene conversion, which is usually considered a complication in gene targeting, can be used to help recover different mutant combinations in a single screening procedure; (3) the reduction of duplication to a single copy by induction of a double-strand break is better explained by the single-strand annealing mechanism than by simple crossing over between repeats; and (4) loss of function of the most abundantly expressed family member (ADGF-A) leads to disintegration of the fat body and the development of melanotic tumors in mutant larvae.

Multiclonal Invasion in Breast Tumors Identified by Topographic Single Cell Sequencing.

PubMed

Casasent, Anna K; Schalck, Aislyn; Gao, Ruli; Sei, Emi; Long, Annalyssa; Pangburn, William; Casasent, Tod; Meric-Bernstam, Funda; Edgerton, Mary E; Navin, Nicholas E

2018-01-11

Ductal carcinoma in situ (DCIS) is an early-stage breast cancer that infrequently progresses to invasive ductal carcinoma (IDC). Genomic evolution has been difficult to delineate during invasion due to intratumor heterogeneity and the low number of tumor cells in the ducts. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS) to measure genomic copy number profiles of single tumor cells while preserving their spatial context in tissue sections. We applied TSCS to 1,293 single cells from 10 synchronous patients with both DCIS and IDC regions in addition to exome sequencing. Our data reveal a direct genomic lineage between in situ and invasive tumor subpopulations and further show that most mutations and copy number aberrations evolved within the ducts prior to invasion. These results support a multiclonal invasion model, in which one or more clones escape the ducts and migrate into the adjacent tissues to establish the invasive carcinomas. Copyright © 2017 Elsevier Inc. All rights reserved.

DeoR repression at-a-distance only weakly responds to changes in interoperator separation and DNA topology.

PubMed Central

Dandanell, G

1992-01-01

The interoperator distance between a synthetic operator Os and the deoP2O2-galK fusion was varied between 46 and 176 bp. The repression of the deoP2 directed galK expression as a function of the interoperator distance (center-to-center) was measured in vivo in a single-copy system. The results show that the DeoR repressor efficiently can repress transcription at all the interoperator distances tested. The degree of repression depends very little on the spacing between the operators, however, a weak periodic dependency of 8-11 bp may exist. PMID:1437558

Comparison of taxon-specific versus general locus sets for targeted sequence capture in plant phylogenomics.

PubMed

Chau, John H; Rahfeldt, Wolfgang A; Olmstead, Richard G

2018-03-01

Targeted sequence capture can be used to efficiently gather sequence data for large numbers of loci, such as single-copy nuclear loci. Most published studies in plants have used taxon-specific locus sets developed individually for a clade using multiple genomic and transcriptomic resources. General locus sets can also be developed from loci that have been identified as single-copy and have orthologs in large clades of plants. We identify and compare a taxon-specific locus set and three general locus sets (conserved ortholog set [COSII], shared single-copy nuclear [APVO SSC] genes, and pentatricopeptide repeat [PPR] genes) for targeted sequence capture in Buddleja (Scrophulariaceae) and outgroups. We evaluate their performance in terms of assembly success, sequence variability, and resolution and support of inferred phylogenetic trees. The taxon-specific locus set had the most target loci. Assembly success was high for all locus sets in Buddleja samples. For outgroups, general locus sets had greater assembly success. Taxon-specific and PPR loci had the highest average variability. The taxon-specific data set produced the best-supported tree, but all data sets showed improved resolution over previous non-sequence capture data sets. General locus sets can be a useful source of sequence capture targets, especially if multiple genomic resources are not available for a taxon.

The Complete Plastid Genome of Lagerstroemia fauriei and Loss of rpl2 Intron from Lagerstroemia (Lythraceae)

PubMed Central

Gu, Cuihua; Tembrock, Luke R.; Johnson, Nels G.; Simmons, Mark P.; Wu, Zhiqiang

2016-01-01

Lagerstroemia (crape myrtle) is an important plant genus used in ornamental horticulture in temperate regions worldwide. As such, numerous hybrids have been developed. However, DNA sequence resources and genome information for Lagerstroemia are limited, hindering evolutionary inferences regarding interspecific relationships. We report the complete plastid genome of Lagerstroemia fauriei. To our knowledge, this is the first reported whole plastid genome within Lythraceae. This genome is 152,440 bp in length with 38% GC content and consists of two single-copy regions separated by a pair of 25,793 bp inverted repeats. The large single copy and the small single copy regions span 83,921 bp and 16,933 bp, respectively. The genome contains 129 genes, including 17 located in each inverted repeat. Phylogenetic analysis of genera sampled from Geraniaceae, Myrtaceae, and Onagraceae corroborated the sister relationship between Lythraceae and Onagraceae. The plastid genomes of L. fauriei and several other Lythraceae species lack the rpl2 intron, which indicating an early loss of this intron within the Lythraceae lineage. The plastid genome of L. fauriei provides a much needed genetic resource for further phylogenetic research in Lagerstroemia and Lythraceae. Highly variable markers were identified for application in phylogenetic, barcoding and conservation genetic applications. PMID:26950701

Phylogenomic relationship of feijoa (Acca sellowiana (O.Berg) Burret) with other Myrtaceae based on complete chloroplast genome sequences.

PubMed

Machado, Lilian de Oliveira; Vieira, Leila do Nascimento; Stefenon, Valdir Marcos; Oliveira Pedrosa, Fábio de; Souza, Emanuel Maltempi de; Guerra, Miguel Pedro; Nodari, Rubens Onofre

2017-04-01

Given their distribution, importance, and richness, Myrtaceae species comprise a model system for studying the evolution of tropical plant diversity. In addition, chloroplast (cp) genome sequencing is an efficient tool for phylogenetic relationship studies. Feijoa [Acca sellowiana (O. Berg) Burret; CN: pineapple-guava] is a Myrtaceae species that occurs naturally in southern Brazil and northern Uruguay. Feijoa is known for its exquisite perfume and flavorful fruits, pharmacological properties, ornamental value and increasing economic relevance. In the present work, we reported the complete cp genome of feijoa. The feijoa cp genome is a circular molecule of 159,370 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC 88,028 bp) and a Small Single Copy region (SSC 18,598 bp) separated by Inverted Repeat regions (IRs 26,372 bp). The genome structure, gene order, GC content and codon usage are similar to those of typical angiosperm cp genomes. When compared to other cp genome sequences of Myrtaceae, feijoa showed closest relationship with pitanga (Eugenia uniflora L.). Furthermore, a comparison of pitanga synonymous (Ks) and nonsynonymous (Ka) substitution rates revealed extremely low values. Maximum Likelihood and Bayesian Inference analyses produced phylogenomic trees identical in topology. These trees supported monophyly of three Myrtoideae clades.

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

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

Half the entanglement in critical systems is distillable from a single specimen

NASA Astrophysics Data System (ADS)

Orús, R.; Latorre, J. I.; Eisert, J.; Cramer, M.

2006-06-01

We establish a quantitative relationship between the entanglement content of a single quantum chain at a critical point and the corresponding entropy of entanglement. We find that, surprisingly, the leading critical scaling of the single-copy entanglement with respect to any bipartitioning is exactly one-half of the entropy of entanglement, in a general setting of conformal field theory and quasifree systems. Conformal symmetry imposes that the single-copy entanglement scales as E1(ρL)=(c/6)lnL-(c/6)(π2/lnL)+O(1/L) , where L is the number of constituents in a block of an infinite chain and c denotes the central charge. This shows that from a single specimen of a critical chain, already half the entanglement can be distilled compared to the rate that is asymptotically available. The result is substantiated by a quantitative analysis for all translationally invariant quantum spin chains corresponding to all isotropic quasifree fermionic models. An example of the XY spin chain shows that away from criticality the above relation is maintained only near the quantum phase transition.

Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization▿ †

PubMed Central

Smolina, Irina; Lee, Charles; Frank-Kamenetskii, Maxim

2007-01-01

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes. PMID:17293504

Evidence for a Complex Class of Nonadenylated mRNA in Drosophila

PubMed Central

Zimmerman, J. Lynn; Fouts, David L.; Manning, Jerry E.

1980-01-01

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 x 106 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 x 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 x 104, then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome. PMID:6777246

Transcriptome-Mining for Single-Copy Nuclear Markers in Ferns

PubMed Central

Rothfels, Carl J.; Larsson, Anders; Li, Fay-Wei; Sigel, Erin M.; Huiet, Layne; Burge, Dylan O.; Ruhsam, Markus; Graham, Sean W.; Stevenson, Dennis W.; Wong, Gane Ka-Shu; Korall, Petra; Pryer, Kathleen M.

2013-01-01

Background Molecular phylogenetic investigations have revolutionized our understanding of the evolutionary history of ferns—the second-most species-rich major group of vascular plants, and the sister clade to seed plants. The general absence of genomic resources available for this important group of plants, however, has resulted in the strong dependence of these studies on plastid data; nuclear or mitochondrial data have been rarely used. In this study, we utilize transcriptome data to design primers for nuclear markers for use in studies of fern evolutionary biology, and demonstrate the utility of these markers across the largest order of ferns, the Polypodiales. Principal Findings We present 20 novel single-copy nuclear regions, across 10 distinct protein-coding genes: ApPEFP_C, cryptochrome 2, cryptochrome 4, DET1, gapCpSh, IBR3, pgiC, SQD1, TPLATE, and transducin. These loci, individually and in combination, show strong resolving power across the Polypodiales phylogeny, and are readily amplified and sequenced from our genomic DNA test set (from 15 diploid Polypodiales species). For each region, we also present transcriptome alignments of the focal locus and related paralogs—curated broadly across ferns—that will allow researchers to develop their own primer sets for fern taxa outside of the Polypodiales. Analyses of sequence data generated from our genomic DNA test set reveal strong effects of partitioning schemes on support levels and, to a much lesser extent, on topology. A model partitioned by codon position is strongly favored, and analyses of the combined data yield a Polypodiales phylogeny that is well-supported and consistent with earlier studies of this group. Conclusions The 20 single-copy regions presented here more than triple the single-copy nuclear regions available for use in ferns. They provide a much-needed opportunity to assess plastid-derived hypotheses of relationships within the ferns, and increase our capacity to explore aspects of fern evolution previously unavailable to scientific investigation. PMID:24116189

Single-copy entanglement in critical quantum spin chains

NASA Astrophysics Data System (ADS)

Eisert, J.; Cramer, M.

2005-10-01

We consider the single-copy entanglement as a quantity to assess quantum correlations in the ground state in quantum many-body systems. We show for a large class of models that already on the level of single specimens of spin chains, criticality is accompanied with the possibility of distilling a maximally entangled state of arbitrary dimension from a sufficiently large block deterministically, with local operations and classical communication. These analytical results—which refine previous results on the divergence of block entropy as the rate at which maximally entangled pairs can be distilled from many identically prepared chains—are made quantitative for general isotropic translationally invariant spin chains that can be mapped onto a quasifree fermionic system, and for the anisotropic XY model. For the XX model, we provide the asymptotic scaling of ˜(1/6)log2(L) , and contrast it with the block entropy.

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

DOE PAGES

Cui, Yi; Hu, Dehong; Markillie, Lye Meng; ...

2017-10-04

Here, quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes.more » This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio ( Nkx2- 2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.« less

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

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

Cui, Yi; Hu, Dehong; Markillie, Lye Meng

Here, quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes.more » This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio ( Nkx2- 2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.« less

Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase.

PubMed

Teng, Chang Ying; Dang, Yunkun; Danne, Jillian C; Waller, Ross F; Green, Beverley R

2013-01-01

Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.

In Vivo Characterization of Human APOA5 Haplotypes

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

Ahituv, Nadav; Akiyama, Jennifer; Chapman-Helleboid, Audrey

2006-10-01

Increased plasma triglycerides concentrations are an independent risk factor for cardiovascular disease. Numerous studies support a reproducible genetic association between two minor haplotypes in the human apolipoprotein A5 gene (APOA5) and increased plasma triglyceride concentrations. We thus sought to investigate the effect of these minor haplotypes (APOA5*2 and APOA5*3) on ApoAV plasma levels through the precise insertion of single-copy intact APOA5 haplotypes at a targeted location in the mouse genome. While we found no difference in the amount of human plasma ApoAV in mice containing the common APOA5*1 and minor APOA5*2 haplotype, the introduction of the single APOA5*3 defining allelemore » (19W) resulted in 3-fold lower ApoAV plasma levels consistent with existing genetic association studies. These results indicate that S19W polymorphism is likely to be functional and explain the strong association of this variant with plasma triglycerides supporting the value of sensitive in vivo assays to define the functional nature of human haplotypes.« less

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.

[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.

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

Wang Yinan; Shi Handuo; Xiong Zhaoxi

We present a unified universal quantum cloning machine, which combines several different existing universal cloning machines together, including the asymmetric case. In this unified framework, the identical pure states are projected equally into each copy initially constituted by input and one half of the maximally entangled states. We show explicitly that the output states of those universal cloning machines are the same. One importance of this unified cloning machine is that the cloning procession is always the symmetric projection, which reduces dramatically the difficulties for implementation. Also, it is found that this unified cloning machine can be directly modified tomore » the general asymmetric case. Besides the global fidelity and the single-copy fidelity, we also present all possible arbitrary-copy fidelities.« less

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

Impacts of adding FGDG on the abundance of nitrification and denitrification functional genes during dairy manure and sugarcane pressmud co-composting.

PubMed

Li, Qunliang; Guo, Xiaobo; Lu, Yanyu; Shan, Guangchun; Huang, Junhao

2016-10-01

To investigate the impacts of flue gas desulphurization gypsum (FGDG) amendment on the nitrification and denitrification during composting, dairy manure and sugarcane pressmud co-composting with FGDG (CPG) and without FGDG (CP) were conducted in this work. The physico-chemical parameters and the copies of nitrification and denitrification functional genes with real-time quantitative polymerase chain reaction (qPCR) during composting were analyzed. FGDG amendment displayed an inhibitory effect on the copies of 16S rDNA and delayed the occurrence of the highest gene copies of amoA during composting. The nxrA gene copies was inhibited by FGDG amendment during the mature phase. The addition of FGDG increased the relative content of narG and nirS during composting, contributing to more NO3(-)-N being reduced to NO2(-)-N. The amoA showed significant negative correlation with OM and NH4(+)-N, and positive correlation with NO3(-)-N. The nxrA displayed a negative correlation with temperature. These results demonstrated FGDG amendment significantly affected the copies of nitrification and denitrification functional genes, which changed the nitrogen flux of composting. Taken together, these data shed an insight into FGDG amendment affecting the nitrogen transformation during composting on a molecular level. Copyright © 2016 Elsevier Ltd. All rights reserved.

26. Photographic copy of plant engineer's handdrawn buildings function chart, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

26. Photographic copy of plant engineer's hand-drawn buildings function chart, dated 1967; Ink and pencil on tracing paper; Attributed to GWN, Original in collection of Rath drawings and blueprints owned by Waterloo Community Development Board, Waterloo, Iowa; SHEET THREE; OUTLINES ACTIVITIES TAKING PLANE ON EACH FLOOR OF MAJOR BUILDINGS IN THE RATH COMPLEX - Rath Packing Company, Sycamore Street between Elm & Eighteenth Streets, Waterloo, Black Hawk County, IA

24. Photographic copy of plant engineer's handdrawn buildings function chart, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

24. Photographic copy of plant engineer's hand-drawn buildings function chart, dated 1967; Ink and pencil on tracing paper; Attributed to GWN, Original in collection of Rath drawings and blueprints owned by Waterloo Community Development Board, Waterloo, Iowa; SHEET ONE; OUTLINES ACTIVITIES TAKING PLANE ON EACH FLOOR OF MAJOR BUILDINGS IN THE RATH COMPLEX - Rath Packing Company, Sycamore Street between Elm & Eighteenth Streets, Waterloo, Black Hawk County, IA

25. Photographic copy of plant engineer's handdrawn buildings function chart, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

25. Photographic copy of plant engineer's hand-drawn buildings function chart, dated 1967; Ink and pencil on tracing paper; Attributed to GWN, Original in collection of Rath drawings and blueprints owned by Waterloo Community Development Board, Waterloo, Iowa; SHEET TWO; OUTLINES ACTIVITIES TAKING PLANE ON EACH FLOOR OF MAJOR BUILDINGS IN THE RATH COMPLEX - Rath Packing Company, Sycamore Street between Elm & Eighteenth Streets, Waterloo, Black Hawk County, IA

Exploiting sequence similarity to validate the sensitivity of SNP arrays in detecting fine-scaled copy number variations.

PubMed

Wong, Gerard; Leckie, Christopher; Gorringe, Kylie L; Haviv, Izhak; Campbell, Ian G; Kowalczyk, Adam

2010-04-15

High-density single nucleotide polymorphism (SNP) genotyping arrays are efficient and cost effective platforms for the detection of copy number variation (CNV). To ensure accuracy in probe synthesis and to minimize production costs, short oligonucleotide probe sequences are used. The use of short probe sequences limits the specificity of binding targets in the human genome. The specificity of these short probeset sequences has yet to be fully analysed against a normal reference human genome. Sequence similarity can artificially elevate or suppress copy number measurements, and hence reduce the reliability of affected probe readings. For the purpose of detecting narrow CNVs reliably down to the width of a single probeset, sequence similarity is an important issue that needs to be addressed. We surveyed the Affymetrix Human Mapping SNP arrays for probeset sequence similarity against the reference human genome. Utilizing sequence similarity results, we identified a collection of fine-scaled putative CNVs between gender from autosomal probesets whose sequence matches various loci on the sex chromosomes. To detect these variations, we utilized our statistical approach, Detecting REcurrent Copy number change using rank-order Statistics (DRECS), and showed that its performance was superior and more stable than the t-test in detecting CNVs. Through the application of DRECS on the HapMap population datasets with multi-matching probesets filtered, we identified biologically relevant SNPs in aberrant regions across populations with known association to physical traits, such as height, covered by the span of a single probe. This provided empirical confirmation of the existence of naturally occurring narrow CNVs as well as the sensitivity of the Affymetrix SNP array technology in detecting them. The MATLAB implementation of DRECS is available at http://ww2.cs.mu.oz.au/ approximately gwong/DRECS/index.html.

Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7.

PubMed

Vogler, Amy J; Keys, Christine; Nemoto, Yoshimi; Colman, Rebecca E; Jay, Zack; Keim, Paul

2006-06-01

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.

Organizational strategies mediate nonverbal memory impairment in obsessive-compulsive disorder.

PubMed

Savage, C R; Baer, L; Keuthen, N J; Brown, H D; Rauch, S L; Jenike, M A

1999-04-01

Previous neuropsychological studies of obsessive-compulsive disorder (OCD) have indicated impaired executive functioning and nonverbal memory. The extent to which impaired executive functioning impacts nonverbal memory has not been established. The current study investigated the mediating effects of organizational strategies used when copying a figure on subsequent nonverbal memory for that figure. We examined neuropsychological performance in 20 unmedicated subjects with OCD and 20 matched normal control subjects. Subjects were administered the Rey-Osterrieth Complex Figure Test (RCFT) and neuropsychological tests assessing various aspects of executive function. OCD subjects differed significantly from healthy control subjects in the organizational strategies used to copy the RCFT figure, and they recalled significantly less information on both immediate and delayed testing. Multiple regression analyses indicated that group differences in immediate percent recall were significantly mediated by copy organizational strategies. Further exploratory analyses indicated that organizational problems in OCD may be related to difficulties shifting mental and/or spatial set. Immediate nonverbal memory problems in OCD subjects were mediated by impaired organizational strategies used during the initial copy of the RCFT figure. Thus, the primary deficit was one affecting executive function, which then had a secondary effect on immediate memory. These findings are consistent with current theories proposing frontal-striatal system dysfunction in OCD.

Effect of sustained elevated temperature prior to amplification on template copy number estimation using digital polymerase chain reaction.

PubMed

Bhat, Somanath; McLaughlin, Jacob L H; Emslie, Kerry R

2011-02-21

Digital polymerase chain reaction (dPCR) has the potential to enable accurate quantification of target DNA copy number provided that all target DNA molecules are successfully amplified. Following duplex dPCR analysis from a linear DNA target sequence that contains single copies of two independent template sequences, we have observed that amplification of both templates in a single partition does not always occur. To investigate this finding, we heated the target DNA solution to 95 °C for increasing time intervals and then immediately chilled on ice prior to preparing the dPCR mix. We observed an exponential decline in estimated copy number (R(2)≥ 0.98) of the two template sequences when amplified from either a linearized plasmid or a 388 base pair (bp) amplicon containing the same two template sequences. The distribution of amplifiable templates and the final concentration (copies per µL) were both affected by heat treatment of the samples at 95 °C from 0 s to 30 min. The proportion of target sequences from which only one of the two templates was amplified in a single partition (either 1507 or hmg only) increased over time, while the proportion of target sequences where both templates were amplified (1507 and hmg) in each individual partition declined rapidly from 94% to 52% (plasmid) and 88% to 31% (388 bp amplicon) suggesting an increase in number of targets from which both templates no longer amplify. A 10 min incubation at 95 °C reduced the initial amplifiable template concentration of the plasmid and the 388 bp amplicon by 59% and 91%, respectively. To determine if a similar decrease in amplifiable target occurs during the default pre-activation step of typical PCR amplification protocol, we used mastermixes with a 20 s or 10 min hot-start. The choice of mastermix and consequent pre-activation time did not affect the estimated plasmid concentration. Therefore, we conclude that prolonged exposure of this DNA template to elevated temperatures could lead to significant bias in dPCR measurements. However, care must be taken when designing PCR and non-PCR based experiments by reducing exposure of the DNA template to sustained elevated temperatures in order to improve accuracy in copy number estimation and concentration determination.

The whole chloroplast genome of wild rice (Oryza australiensis).

PubMed

Wu, Zhiqiang; Ge, Song

2016-01-01

The whole chloroplast genome of wild rice (Oryza australiensis) is characterized in this study. The genome size is 135,224  bp, exhibiting a typical circular structure including a pair of 25,776  bp inverted repeats (IRa,b) separated by a large single-copy region (LSC) of 82,212  bp and a small single-copy region (SSC) of 12,470  bp. The overall GC content of the genome is 38.95%. 110 unique genes were annotated, including 76 protein-coding genes, 4 ribosomal RNA genes, and 30t RNA genes. Among these, 18 are duplicated in the inverted repeat regions, 13 genes contain one intron, and 2 genes (rps12 and ycf3) have two introns.

The complete chloroplast genome of a medicinal plant Epimedium koreanum Nakai (Berberidaceae).

PubMed

Lee, Jung-Hoon; Kim, Kyunghee; Kim, Na-Rae; Lee, Sang-Choon; Yang, Tae-Jin; Kim, Young-Dong

2016-11-01

Epimedium koreanum is a perennial medicinal plant distributed in Eastern Asia. The complete chloroplast genome sequences of E. koreanum was obtained by de novo assembly using whole genome next-generation sequences. The chloroplast genome of E. koreanum was 157 218 bp in length and separated into four distinct regions such as large single copy region (89 600 bp), small single copy region (17 222 bp) and a pair of inverted repeat regions (25 198 bp). The genome contained a total of 112 genes including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that E. koreanum is most closely related to Berberis bealei, a traditional medicinal plant in the Berberidaceae family.

The complete chloroplast genome of salt cress (Eutrema salsugineum).

PubMed

Guo, Xinyi; Hao, Guoqian; Ma, Tao

2016-07-01

The complete chloroplast (cp) sequence of the salt cress (Eutrema salsugineum), a plant well-adapted to salt stress, was presented in this study. The circular molecule is 153,407 bp in length and exhibit a typical quadripartite structure containing an 83,894 bp large single copy (LSC) region, a 17,607 bp small single copy (SSC) region, and the two 25,953 bp inverted repeats (IRs). The salt cress cp genome contains 135 known genes, including 87 protein-coding genes, 8 ribosomal RNA genes, and 40 tRNA genes; 21 of these are located in the inverted repeat region. As expected, phylogenetic analysis support the idea that E. salsugineum is sister to Brassiceae species within the Brassicaceae family.

The complete mitochondrial genome sequence of Malus hupehensis var. pinyiensis.

PubMed

Duan, Naibin; Sun, Honghe; Wang, Nan; Fei, Zhangjun; Chen, Xuesen

2016-07-01

The complete mitochondrial genome sequence of Malus hupehensis var. pinyiensis, a widely used apple rootstock, was determined using the Illumina high-throughput sequencing approach. The genome is 422,555 bp in length and has a GC content of 45.21%. It is separated by a pair of inverted repeats of 32,504 bp, to form a large single copy region of 213,055 bp and a small single copy region of 144,492 bp. The genome contains 38 protein-coding genes, four pseudogenes, 25 tRNA genes, and three rRNA genes. The genome is 25,608 bp longer than that of M. domestica, and several structural variations between these two mitogenomes were detected.

24 CFR 291.304 - Bidding process.

Code of Federal Regulations, 2012 CFR

2012-04-01

... DEVELOPMENT HUD-OWNED PROPERTIES DISPOSITION OF HUD-ACQUIRED SINGLE FAMILY PROPERTY Sale of HUD-Held Single... HUD in accordance with instructions in the bid package for a particular sale. (b) Effect of bid. By... bid package. Along with the bid, the bidder must submit an executed copy of the Loan Sale Agreement...

24 CFR 291.304 - Bidding process.

Code of Federal Regulations, 2014 CFR

2014-04-01

... DEVELOPMENT HUD-OWNED PROPERTIES DISPOSITION OF HUD-ACQUIRED SINGLE FAMILY PROPERTY Sale of HUD-Held Single... HUD in accordance with instructions in the bid package for a particular sale. (b) Effect of bid. By... bid package. Along with the bid, the bidder must submit an executed copy of the Loan Sale Agreement...

24 CFR 291.304 - Bidding process.

Code of Federal Regulations, 2010 CFR

2010-04-01

... DEVELOPMENT HUD-OWNED PROPERTIES DISPOSITION OF HUD-ACQUIRED SINGLE FAMILY PROPERTY Sale of HUD-Held Single... HUD in accordance with instructions in the bid package for a particular sale. (b) Effect of bid. By... bid package. Along with the bid, the bidder must submit an executed copy of the Loan Sale Agreement...

SCRIB and PUF60 Are Primary Drivers of the Multisystemic Phenotypes of the 8q24.3 Copy-Number Variant

PubMed Central

Dauber, Andrew; Golzio, Christelle; Guenot, Cécile; Jodelka, Francine M.; Kibaek, Maria; Kjaergaard, Susanne; Leheup, Bruno; Martinet, Danielle; Nowaczyk, Malgorzata J.M.; Rosenfeld, Jill A.; Zeesman, Susan; Zunich, Janice; Beckmann, Jacques S.; Hirschhorn, Joel N.; Hastings, Michelle L.; Jacquemont, Sebastien; Katsanis, Nicholas

2013-01-01

Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions. PMID:24140112

Differential evolution of members of the rhomboid gene family with conservative and divergent patterns.

PubMed

Li, Qi; Zhang, Ning; Zhang, Liangsheng; Ma, Hong

2015-04-01

Rhomboid proteins are intramembrane serine proteases that are involved in a plethora of biological functions, but the evolutionary history of the rhomboid gene family is not clear. We performed a comprehensive molecular evolutionary analysis of the rhomboid gene family and also investigated the organization and sequence features of plant rhomboids in different subfamilies. Our results showed that eukaryotic rhomboids could be divided into five subfamilies (RhoA-RhoD and PARL). Most orthology groups appeared to be conserved only as single or low-copy genes in all lineages in RhoB-RhoD and PARL, whereas RhoA genes underwent several duplication events, resulting in multiple gene copies. These duplication events were due to whole genome duplications in plants and animals and the duplicates might have experienced functional divergence. We also identified a novel group of plant rhomboid (RhoB1) that might have lost their enzymatic activity; their existence suggests that they might have evolved new mechanisms. Plant and animal rhomboids have similar evolutionary patterns. In addition, there are mutations affecting key active sites in RBL8, RBL9 and one of the Brassicaceae PARL duplicates. This study delineates a possible evolutionary scheme for intramembrane proteins and illustrates distinct fates and a mechanism of evolution of gene duplicates. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Functional neural correlates of figure copy and recall task performances in cognitively impaired individuals: an 18F-FDG-PET study.

PubMed

Han, Ji Young; Byun, Min Soo; Seo, Eun Hyun; Yi, Dahyun; Choe, Young Min; Sohn, Bo Kyung; Choi, Hyo Jung; Baek, Hyewon; Lee, Jun Ho; Kim, Hyun Jung; Woo, Jong Inn; Lee, Dong Yong

2015-12-02

Figure copy and recall tasks from the Benton Visual Retention Test (BVRT) and the Consortium to Establish a Registry of Alzheimer's Disease (CERAD) neuropsychological battery are used widely to assess visuospatial function in cognitively impaired (CI) individuals. We aimed to identify functional neural correlates of figure copy and recall task performances as measured by the BVRT and the CERAD constructional praxis (CP) and CP recall (CR) in CI individuals. Both tasks were administered to 64 CI individuals with early or prodromal stage Alzheimer's disease and 36 cognitively normal individuals. Voxel-wise correlations between test scores and regional cerebral glucose metabolism (rCMglc) measured by fluorine-18 fluorodeoxyglucose PET in CI participants were analyzed. BVRT figure copy task performance was associated with rCMglc of the bilateral posterior brain regions including the parieto-temporo-occipital regions, whereas the BVRT figure recall task performance was predominantly correlated with rCMglc of the left parietal and temporo-occipital regions. Meanwhile, CERAD CP performance was associated mainly with rCMglc of the left prefrontal and temporo-occipital areas as well as in the bilateral parietal regions, whereas CERAD CR performance was correlated with rCMglc of the right prefrontal, parietal, and temporal regions. In conclusion, the functional neural correlates of the two tasks were markedly different, suggesting that these tasks might measure different visuospatial functions. Our findings contribute toward understanding the functional neuroanatomical aspects of these tasks, which is useful for both interpreting the task results as well as for more sophisticated utilization of these tasks for probing specific neuroanatomical functions.

Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia1[W][OPEN

PubMed Central

Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S.; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K.; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W.; Gill, Bikram S.

2014-01-01

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations. PMID:25037215

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

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.

Copy number variants and genetic polymorphisms in TBX21, GATA3, Rorc, Foxp3 and susceptibility to Behcet's disease and Vogt-Koyanagi-Harada syndrome.

PubMed

Liao, Dan; Hou, Shengping; Zhang, Jun; Fang, Jing; Liu, Yunjia; Bai, Lin; Cao, Qingfeng; Kijlstra, Aize; Yang, Peizeng

2015-04-15

This study aimed to investigate the role of genetic variants including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) of TBX21, GATA3, Rorc and Foxp3 genes in Behcet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. Genotyping of 25 SNPs was performed by iPLEX system (Sequenom) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). TaqMan real time PCR was used to assess CNVs. The expression of Rorc and Foxp3 were examined by real-time PCR and cytokine production was measured by ELISA. High Rorc CNV was associated with the susceptibility to BD (P = 8.99 × 10(-8), OR = 3.0), and low Foxp3 CNV predisposed to BD in female patients (P = 1.92 × 10(-5), OR = 3.1). CNVs for the investigated genes were not altered in VKH syndrome. Further functional studies demonstrated that the relative mRNA expression levels of Rorc were increased in individuals with high Rorc copy number, but not for Foxp3. Increased production of IL-1β and IL-6 was found in individuals carrying a high CNV of Rorc. Our study showed that high CNVs of Rorc and low CNVs of Foxp3 confer risk for BD but not for VKH syndrome. The tested 25 SNPs in TBX21, GATA3, Rorc and Foxp3 did not associate with BD and VKH syndrome.

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors.

PubMed

Anaka, Matthew; Hudson, Christopher; Lo, Pu-Han; Do, Hongdo; Caballero, Otavia L; Davis, Ian D; Dobrovic, Alexander; Cebon, Jonathan; Behren, Andreas

2013-10-11

Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

Genome-wide comparison of genes involved in the biosynthesis, metabolism, and signaling of juvenile hormone between silkworm and other insects

PubMed Central

Cheng, Daojun; Meng, Meng; Peng, Jian; Qian, Wenliang; Kang, Lixia; Xia, Qingyou

2014-01-01

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects. PMID:25071411

Molecular Evolutionary Characterization of a V1R Subfamily Unique to Strepsirrhine Primates

PubMed Central

Yoder, Anne D.; Chan, Lauren M.; dos Reis, Mario; Larsen, Peter A.; Campbell, C. Ryan; Rasoloarison, Rodin; Barrett, Meredith; Roos, Christian; Kappeler, Peter; Bielawski, Joseph; Yang, Ziheng

2014-01-01

Vomeronasal receptor genes have frequently been invoked as integral to the establishment and maintenance of species boundaries among mammals due to the elaborate one-to-one correspondence between semiochemical signals and neuronal sensory inputs. Here, we report the most extensive sample of vomeronasal receptor class 1 (V1R) sequences ever generated for a diverse yet phylogenetically coherent group of mammals, the tooth-combed primates (suborder Strepsirrhini). Phylogenetic analysis confirms our intensive sampling from a single V1R subfamily, apparently unique to the strepsirrhine primates. We designate this subfamily as V1Rstrep. The subfamily retains extensive repertoires of gene copies that descend from an ancestral gene duplication that appears to have occurred prior to the diversification of all lemuriform primates excluding the basal genus Daubentonia (the aye-aye). We refer to the descendent clades as V1Rstrep-α and V1Rstrep-β. Comparison of the two clades reveals different amino acid compositions corresponding to the predicted ligand-binding site and thus potentially to altered functional profiles between the two. In agreement with previous studies of the mouse lemur (genus, Microcebus), the majority of V1Rstrep gene copies appear to be intact and under strong positive selection, particularly within transmembrane regions. Finally, despite the surprisingly high number of gene copies identified in this study, it is nonetheless probable that V1R diversity remains underestimated in these nonmodel primates and that complete characterization will be limited until high-coverage assembled genomes are available. PMID:24398377

Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers

PubMed Central

Cox, David G.; Simard, Jacques; Sinnett, Daniel; Hamdi, Yosr; Soucy, Penny; Ouimet, Manon; Barjhoux, Laure; Verny-Pierre, Carole; McGuffog, Lesley; Healey, Sue; Szabo, Csilla; Greene, Mark H.; Mai, Phuong L.; Andrulis, Irene L.; Thomassen, Mads; Gerdes, Anne-Marie; Caligo, Maria A.; Friedman, Eitan; Laitman, Yael; Kaufman, Bella; Paluch, Shani S.; Borg, Åke; Karlsson, Per; Stenmark Askmalm, Marie; Barbany Bustinza, Gisela; Nathanson, Katherine L.; Domchek, Susan M.; Rebbeck, Timothy R.; Benítez, Javier; Hamann, Ute; Rookus, Matti A.; van den Ouweland, Ans M.W.; Ausems, Margreet G.E.M.; Aalfs, Cora M.; van Asperen, Christi J.; Devilee, Peter; Gille, Hans J.J.P.; Peock, Susan; Frost, Debra; Evans, D. Gareth; Eeles, Ros; Izatt, Louise; Adlard, Julian; Paterson, Joan; Eason, Jacqueline; Godwin, Andrew K.; Remon, Marie-Alice; Moncoutier, Virginie; Gauthier-Villars, Marion; Lasset, Christine; Giraud, Sophie; Hardouin, Agnès; Berthet, Pascaline; Sobol, Hagay; Eisinger, François; Bressac de Paillerets, Brigitte; Caron, Olivier; Delnatte, Capucine; Goldgar, David; Miron, Alex; Ozcelik, Hilmi; Buys, Saundra; Southey, Melissa C.; Terry, Mary Beth; Singer, Christian F.; Dressler, Anne-Catharina; Tea, Muy-Kheng; Hansen, Thomas V.O.; Johannsson, Oskar; Piedmonte, Marion; Rodriguez, Gustavo C.; Basil, Jack B.; Blank, Stephanie; Toland, Amanda E.; Montagna, Marco; Isaacs, Claudine; Blanco, Ignacio; Gayther, Simon A.; Moysich, Kirsten B.; Schmutzler, Rita K.; Wappenschmidt, Barbara; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Niederacher, Dieter; Sutter, Christian; Gadzicki, Dorothea; Fiebig, Britta; Caldes, Trinidad; Laframboise, Rachel; Nevanlinna, Heli; Chen, Xiaoqing; Beesley, Jonathan; Spurdle, Amanda B.; Neuhausen, Susan L.; Ding, Yuan C.; Couch, Fergus J.; Wang, Xianshu; Peterlongo, Paolo; Manoukian, Siranoush; Bernard, Loris; Radice, Paolo; Easton, Douglas F.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Stoppa-Lyonnet, Dominique; Mazoyer, Sylvie; Sinilnikova, Olga M.

2011-01-01

Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA1 gene would modify the risk of breast cancer in carriers of BRCA1 mutations. A total of 9874 BRCA1 mutation carriers were available in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) for haplotype analyses of BRCA1. Women carrying the rare allele of single nucleotide polymorphism rs16942 on the wild-type copy of BRCA1 were at decreased risk of breast cancer (hazard ratio 0.86, 95% confidence interval 0.77–0.95, P = 0.003). Promoter in vitro assays of the major BRCA1 haplotypes showed that common polymorphisms in the regulatory region alter its activity and that this effect may be attributed to the differential binding affinity of nuclear proteins. In conclusion, variants on the wild-type copy of BRCA1 modify risk of breast cancer among carriers of BRCA1 mutations, possibly by altering the efficiency of BRCA1 transcription. PMID:21890493

ICan: an integrated co-alteration network to identify ovarian cancer-related genes.

PubMed

Zhou, Yuanshuai; Liu, Yongjing; Li, Kening; Zhang, Rui; Qiu, Fujun; Zhao, Ning; Xu, Yan

2015-01-01

Over the last decade, an increasing number of integrative studies on cancer-related genes have been published. Integrative analyses aim to overcome the limitation of a single data type, and provide a more complete view of carcinogenesis. The vast majority of these studies used sample-matched data of gene expression and copy number to investigate the impact of copy number alteration on gene expression, and to predict and prioritize candidate oncogenes and tumor suppressor genes. However, correlations between genes were neglected in these studies. Our work aimed to evaluate the co-alteration of copy number, methylation and expression, allowing us to identify cancer-related genes and essential functional modules in cancer. We built the Integrated Co-alteration network (ICan) based on multi-omics data, and analyzed the network to uncover cancer-related genes. After comparison with random networks, we identified 155 ovarian cancer-related genes, including well-known (TP53, BRCA1, RB1 and PTEN) and also novel cancer-related genes, such as PDPN and EphA2. We compared the results with a conventional method: CNAmet, and obtained a significantly better area under the curve value (ICan: 0.8179, CNAmet: 0.5183). In this paper, we describe a framework to find cancer-related genes based on an Integrated Co-alteration network. Our results proved that ICan could precisely identify candidate cancer genes and provide increased mechanistic understanding of carcinogenesis. This work suggested a new research direction for biological network analyses involving multi-omics data.

ICan: An Integrated Co-Alteration Network to Identify Ovarian Cancer-Related Genes

PubMed Central

Zhou, Yuanshuai; Liu, Yongjing; Li, Kening; Zhang, Rui; Qiu, Fujun; Zhao, Ning; Xu, Yan

2015-01-01

Background Over the last decade, an increasing number of integrative studies on cancer-related genes have been published. Integrative analyses aim to overcome the limitation of a single data type, and provide a more complete view of carcinogenesis. The vast majority of these studies used sample-matched data of gene expression and copy number to investigate the impact of copy number alteration on gene expression, and to predict and prioritize candidate oncogenes and tumor suppressor genes. However, correlations between genes were neglected in these studies. Our work aimed to evaluate the co-alteration of copy number, methylation and expression, allowing us to identify cancer-related genes and essential functional modules in cancer. Results We built the Integrated Co-alteration network (ICan) based on multi-omics data, and analyzed the network to uncover cancer-related genes. After comparison with random networks, we identified 155 ovarian cancer-related genes, including well-known (TP53, BRCA1, RB1 and PTEN) and also novel cancer-related genes, such as PDPN and EphA2. We compared the results with a conventional method: CNAmet, and obtained a significantly better area under the curve value (ICan: 0.8179, CNAmet: 0.5183). Conclusion In this paper, we describe a framework to find cancer-related genes based on an Integrated Co-alteration network. Our results proved that ICan could precisely identify candidate cancer genes and provide increased mechanistic understanding of carcinogenesis. This work suggested a new research direction for biological network analyses involving multi-omics data. PMID:25803614

The Functional Measurement Experiment Builder suite: two Java-based programs to generate and run functional measurement experiments.

PubMed

Mairesse, Olivier; Hofmans, Joeri; Theuns, Peter

2008-05-01

We propose a free, easy-to-use computer program that does not requires prior knowledge of computer programming to generate and run experiments using textual or pictorial stimuli. Although the FM Experiment Builder suite was initially programmed for building and conducting FM experiments, it can also be applied for non-FM experiments that necessitate randomized, single, or multifactorial designs. The program is highly configurable, allowing multilingual use and a wide range of different response formats. The outputs of the experiments are Microsoft Excel compatible .xls files that allow easy copy-paste of the results into Weiss's FM CalSTAT program (2006) or any other statistical package. Its Java-based structure is compatible with both Windows and Macintosh operating systems, and its compactness (< 1 MB) makes it easily distributable over the Internet.

Two potato proteins, including a novel RING finger protein (HIP1), interact with the potyviral multifunctional protein HCpro.

PubMed

Guo, Deyin; Spetz, Carl; Saarma, Mart; Valkonen, Jari P T

2003-05-01

Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins. In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA) (genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system. Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used. Both proteins are encoded by single-copy genes in the potato genome. Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein. The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions.

Biochemical analysis of DNA polymerase η fidelity in the presence of replication protein A.

PubMed

Suarez, Samuel C; Toffton, Shannon M; McCulloch, Scott D

2014-01-01

DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

Genomic Heterogeneity of Osteosarcoma - Shift from Single Candidates to Functional Modules

PubMed Central

Maugg, Doris; Eckstein, Gertrud; Baumhoer, Daniel; Nathrath, Michaela; Korsching, Eberhard

2015-01-01

Osteosarcoma (OS), a bone tumor, exhibit a complex karyotype. On the genomic level a highly variable degree of alterations in nearly all chromosomal regions and between individual tumors is observable. This hampers the identification of common drivers in OS biology. To identify the common molecular mechanisms involved in the maintenance of OS, we follow the hypothesis that all the copy number-associated differences between the patients are intercepted on the level of the functional modules. The implementation is based on a network approach utilizing copy number associated genes in OS, paired expression data and protein interaction data. The resulting functional modules of tightly connected genes were interpreted regarding their biological functions in OS and their potential prognostic significance. We identified an osteosarcoma network assembling well-known and lesser-known candidates. The derived network shows a significant connectivity and modularity suggesting that the genes affected by the heterogeneous genetic alterations share the same biological context. The network modules participate in several critical aspects of cancer biology like DNA damage response, cell growth, and cell motility which is in line with the hypothesis of specifically deregulated but functional modules in cancer. Further, we could deduce genes with possible prognostic significance in OS for further investigation (e.g. EZR, CDKN2A, MAP3K5). Several of those module genes were located on chromosome 6q. The given systems biological approach provides evidence that heterogeneity on the genomic and expression level is ordered by the biological system on the level of the functional modules. Different genomic aberrations are pointing to the same cellular network vicinity to form vital, but already neoplastically altered, functional modules maintaining OS. This observation, exemplarily now shown for OS, has been under discussion already for a longer time, but often in a hypothetical manner, and can here be exemplified for OS. PMID:25848766

The box C/D sRNP dimeric architecture is conserved across domain Archaea

PubMed Central

Bower-Phipps, Kathleen R.; Taylor, David W.; Wang, Hong-Wei; Baserga, Susan J.

2012-01-01

Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2′-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species—Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus—indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain. PMID:22753779

The box C/D sRNP dimeric architecture is conserved across domain Archaea.

PubMed

Bower-Phipps, Kathleen R; Taylor, David W; Wang, Hong-Wei; Baserga, Susan J

2012-08-01

Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2'-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species--Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus--indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain.

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...

22 CFR 1102.2 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... for examination and copying, or furnishing a copy of records. Duplication refers to the process of... organization, functions, policies, decisions, procedures, operations, or other activities of the Government or... make their products available for purchase or subscription by the general public. In the case of...

Mitochondrial Retroprocessing Promoted Functional Transfers of rpl5 to the Nucleus in Grasses.

PubMed

Wu, Zhiqiang; Sloan, Daniel B; Brown, Colin W; Rosenblueth, Mónica; Palmer, Jeffrey D; Ong, Han Chuan

2017-09-01

Functional gene transfers from the mitochondrion to the nucleus are ongoing in angiosperms and have occurred repeatedly for all 15 ribosomal protein genes, but it is not clear why some of these genes are transferred more often than others nor what the balance is between DNA- and RNA-mediated transfers. Although direct insertion of mitochondrial DNA into the nucleus occurs frequently in angiosperms, case studies of functional mitochondrial gene transfer have implicated an RNA-mediated mechanism that eliminates introns and RNA editing sites, which would otherwise impede proper expression of mitochondrial genes in the nucleus. To elucidate the mechanisms that facilitate functional gene transfers and the evolutionary dynamics of the coexisting nuclear and mitochondrial gene copies that are established during these transfers, we have analyzed rpl5 genes from 90 grasses (Poaceae) and related monocots. Multiple lines of evidence indicate that rpl5 has been functionally transferred to the nucleus at least three separate times in the grass family and that at least seven species have intact and transcribed (but not necessarily functional) copies in both the mitochondrion and nucleus. In two grasses, likely functional nuclear copies of rpl5 have been subject to recent gene conversion events via secondarily transferred mitochondrial copies in what we believe are the first described cases of mitochondrial-to-nuclear gene conversion. We show that rpl5 underwent a retroprocessing event within the mitochondrial genome early in the evolution of the grass family, which we argue predisposed the gene towards successful, DNA-mediated functional transfer by generating a "pre-edited" sequence. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

42 CFR 456.612 - Copies of reports.

Code of Federal Regulations, 2011 CFR

2011-10-01

... function, including, if inspection reports concern IMD's, the appropriate State mental health authorities. ... 42 Public Health 4 2011-10-01 2011-10-01 false Copies of reports. 456.612 Section 456.612 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

42 CFR 456.612 - Copies of reports.

Code of Federal Regulations, 2012 CFR

2012-10-01

... function, including, if inspection reports concern IMD's, the appropriate State mental health authorities. ... 42 Public Health 4 2012-10-01 2012-10-01 false Copies of reports. 456.612 Section 456.612 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

42 CFR 456.612 - Copies of reports.

Code of Federal Regulations, 2010 CFR

2010-10-01

... function, including, if inspection reports concern IMD's, the appropriate State mental health authorities. ... 42 Public Health 4 2010-10-01 2010-10-01 false Copies of reports. 456.612 Section 456.612 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

42 CFR 456.612 - Copies of reports.

Code of Federal Regulations, 2014 CFR

2014-10-01

... function, including, if inspection reports concern IMD's, the appropriate State mental health authorities. ... 42 Public Health 4 2014-10-01 2014-10-01 false Copies of reports. 456.612 Section 456.612 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

42 CFR 456.612 - Copies of reports.

Code of Federal Regulations, 2013 CFR

2013-10-01

... function, including, if inspection reports concern IMD's, the appropriate State mental health authorities. ... 42 Public Health 4 2013-10-01 2013-10-01 false Copies of reports. 456.612 Section 456.612 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

The complete chloroplast genome of Cinnamomum camphora and its comparison with related Lauraceae species.

PubMed

Chen, Caihui; Zheng, Yongjie; Liu, Sian; Zhong, Yongda; Wu, Yanfang; Li, Jiang; Xu, Li-An; Xu, Meng

2017-01-01

Cinnamomum camphora , a member of the Lauraceae family, is a valuable aromatic and timber tree that is indigenous to the south of China and Japan. All parts of Cinnamomum camphora have secretory cells containing different volatile chemical compounds that are utilized as herbal medicines and essential oils. Here, we reported the complete sequencing of the chloroplast genome of Cinnamomum camphora using illumina technology. The chloroplast genome of Cinnamomum camphora is 152,570 bp in length and characterized by a relatively conserved quadripartite structure containing a large single copy region of 93,705 bp, a small single copy region of 19,093 bp and two inverted repeat (IR) regions of 19,886 bp. Overall, the genome contained 123 coding regions, of which 15 were repeated in the IR regions. An analysis of chloroplast sequence divergence revealed that the small single copy region was highly variable among the different genera in the Lauraceae family. A total of 40 repeat structures and 83 simple sequence repeats were detected in both the coding and non-coding regions. A phylogenetic analysis indicated that Calycanthus is most closely related to Lauraceae , both being members of Laurales , which forms a sister group to Magnoliids . The complete sequence of the chloroplast of Cinnamomum camphora will aid in in-depth taxonomical studies of the Lauraceae family in the future. The genetic sequence information will also have valuable applications for chloroplast genetic engineering.

Out-of-time-ordered measurements as a probe of quantum dynamics

NASA Astrophysics Data System (ADS)

Bordia, Pranjal; Alet, Fabien; Hosur, Pavan

2018-03-01

Probing the out-of-equilibrium dynamics of quantum matter has gained renewed interest owing to immense experimental progress in artificial quantum systems. Dynamical quantum measures such as the growth of entanglement entropy and out-of-time-ordered correlators (OTOCs) have been shown to provide great insight by exposing subtle quantum features invisible to traditional measures such as mass transport. However, measuring them in experiments requires either identical copies of the system, an ancilla qubit coupled to the whole system, or many measurements on a single copy, thereby making scalability extremely complex and hence, severely limiting their potential. Here, we introduce an alternative quantity, the out-of-time-ordered measurement (OTOM), which involves measuring a single observable on a single copy of the system, while retaining the distinctive features of the OTOCs. We show, theoretically, that OTOMs are closely related to OTOCs in a doubled system with the same quantum statistical properties as the original system. Using exact diagonalization, we numerically simulate classical mass transport, as well as quantum dynamics through computations of the OTOC, the OTOM, and the entanglement entropy in quantum spin chain models in various interesting regimes (including chaotic and many-body localized systems). Our results demonstrate that an OTOM can successfully reveal subtle aspects of quantum dynamics hidden to classical measures and, crucially, provide experimental access to them.

Global population genetic structure and male-mediated gene flow in the green sea turtle (Chelonia mydas): analysis of microsatellite loci.

PubMed Central

Roberts, Mark A; Schwartz, Tonia S; Karl, Stephen A

2004-01-01

We assessed the degree of population subdivision among global populations of green sea turtles, Chelonia mydas, using four microsatellite loci. Previously, a single-copy nuclear DNA study indicated significant male-mediated gene flow among populations alternately fixed for different mitochondrial DNA haplotypes and that genetic divergence between populations in the Atlantic and Pacific Oceans was more common than subdivisions among populations within ocean basins. Even so, overall levels of variation at single-copy loci were low and inferences were limited. Here, the markedly more variable microsatellite loci confirm the presence of male-mediated gene flow among populations within ocean basins. This analysis generally confirms the genetic divergence between the Atlantic and Pacific. As with the previous study, phylogenetic analyses of genetic distances based on the microsatellite loci indicate a close genetic relationship among eastern Atlantic and Indian Ocean populations. Unlike the single-copy study, however, the results here cannot be attributed to an artifact of general low variability and likely represent recent or ongoing migration between ocean basins. Sequence analyses of regions flanking the microsatellite repeat reveal considerable amounts of cryptic variation and homoplasy and significantly aid in our understanding of population connectivity. Assessment of the allele frequency distributions indicates that at least some of the loci may not be evolving by the stepwise mutation model. PMID:15126404

Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system.

PubMed

Sankuntaw, Nipaporn; Sukprasert, Saovaluk; Engchanil, Chulapan; Kaewkes, Wanlop; Chantratita, Wasun; Pairoj, Vantanit; Lulitanond, Viraphong

2011-01-01

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%-3.67% and 0.79%-4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample. Copyright © 2011 Elsevier Ltd. All rights reserved.

Sorting by Cuts, Joins, and Whole Chromosome Duplications.

PubMed

Zeira, Ron; Shamir, Ron

2017-02-01

Genome rearrangement problems have been extensively studied due to their importance in biology. Most studied models assumed a single copy per gene. However, in reality, duplicated genes are common, most notably in cancer. In this study, we make a step toward handling duplicated genes by considering a model that allows the atomic operations of cut, join, and whole chromosome duplication. Given two linear genomes, [Formula: see text] with one copy per gene and [Formula: see text] with two copies per gene, we give a linear time algorithm for computing a shortest sequence of operations transforming [Formula: see text] into [Formula: see text] such that all intermediate genomes are linear. We also show that computing an optimal sequence with fewest duplications is NP-hard.

One- and two-photon states for quantum information

NASA Astrophysics Data System (ADS)

Peters, Nicholas A.

To find expression stability among transgenic lines, the Recombinase Mediated Transgene Integration (RMTI) technology using the Cre/ lox-mediated site-specific gene integration system was used. The objectives were to develop an efficient method of site-specific transgene integration and to test the effectiveness of this method by assaying transgene expression in the RMTI lines. The RMTI technology allows the precise integration of a transgene in a previously placed target genomic location containing a lox site. The efficiency of CRE-mediated site-specific integration in rice by particle bombardment was found to vary from 3 to 28% in nine different experiments. Some hemizygous site-specific integration plants that were derived from homozygous target locus were found to undergo CRE-mediated reversion of the integration locus. No reversion was observed in callus; however, reverting cells may have been excluded due to selection pressure. The expression of the transgene gus was studied in all 40 callus lines, 12 regenerated T0 plants and the T1 and T2 progenies of 5 lines. The isogenic SC lines had an average expression level based on the activity of beta-glucuronidase of 158 +/- 9 units/mg protein (mean +/- SEM; n=3; variance within SC lines are expressed as standard error of the mean SEM) indicating a significantly higher level of expression, as compared to MC lines that had a much lower expression level 44 +/- 8 units/mg protein (mean +/- SEM; n=3) and the imprecise lines that had 22 +/- 8 units/mg protein (mean +/- SEM; n=3). Transgene expression in the callus cells of precise single copy lines varied by ˜3 fold, whereas that in multi-copy lines varied by ˜30 fold. Furthermore, precise single copy lines, on an average, contained ˜3.5 fold higher expression than multi-copy lines. Transgene expression in the plants of precise single-copy lines was highly variable, which was found to be due to the loss of the integration because of CRE-mediated reversion in the locus. (Abstract shortened by UMI.)

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

Dong Yuli; Zou Xubo; Guo Guangcan

We investigate the economical Gaussian cloning of coherent states with the known phase, which produces M copies from N input replica and can be implemented with degenerate parametric amplifiers and beam splitters.The achievable fidelity of single copy is given by 2M{radical}(N)/[{radical}(N)(M-1)+{radical}((1+N)(M{sup 2}+N))], which is bigger than the optimal fidelity of the universal Gaussian cloning. The cloning machine presented here works without ancillary optical modes and can be regarded as the continuous variable generalization of the economical cloning machine for qudits.

Self-sequencing of amino acids and origins of polyfunctional protocells

NASA Technical Reports Server (NTRS)

Fox, S. W.

1984-01-01

The role of proteins in the origin of living things is discussed. It has been experimentally established that amino acids can sequence themselves under simulated geological conditions with highly nonrandom products which accordingly contain diverse information. Multiple copies of each type of macromolecule are formed, resulting in greater power for any protoenzymic molecule than would accrue from a single copy of each type. Thermal proteins are readily incorporated into laboratory protocells. The experimental evidence for original polyfunctional protocells is discussed.

Conference on the Control of the Colorado Beetle, USSR.

DTIC Science & Technology

1960-03-25

in 1958, the confarence expressed the desire to establish at the SEV a single joint committee on the control of the Colora- do potato beatle and...i OTS: 60-11,14*1 JHIS: 2U38 25 March I960 • • CONFERENCE ON THE CONTROL OF THE CÖLÖIUDO BEEILE - USSR - ’ by V...NEW T0RK1?, N. If. Reproduced From Best Available Copy k NOTICE THIS DOCUMENT HAS BEEN REPRODUCED FROM THE BEST COPY

Cellulitis

MedlinePlus

... skin. Protect your hands and feet. Wear appropriate footwear and gloves. Promptly treat infections on the skin's ... opportunities Reprint Permissions A single copy of these materials may be reprinted for noncommercial personal use only. " ...

75 FR 19665 - Notice of Action Subject to Intergovernmental Review Under Executive Order 12372

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-15

... the address section. A copy of this notice also is being furnished to the respective State single... area-wide small business assistance plan adopted by a State authorized agency. DATES: A State single..., women, exporters, the disabled, and minorities. SBDC Program Requirements An SBDC must meet programmatic...

Two Functional Copies of the DGCR6 Gene Are Present on Human Chromosome 22q11 Due to a Duplication of an Ancestral Locus

PubMed Central

Edelmann, Lisa; Stankiewicz, Pavel; Spiteri, Elizabeth; Pandita, Raj K.; Shaffer, Lisa; Lupski, James; Morrow, Bernice E.

2001-01-01

The DGCR6 (DiGeorge critical region) gene encodes a putative protein with sequence similarity to gonadal (gdl), a Drosophila melanogaster gene of unknown function. We mapped the DGCR6 gene to chromosome 22q11 within a low copy repeat, termed sc11.1a, and identified a second copy of the gene, DGCR6L, within the duplicate locus, termed sc11.1b. Both sc11.1 repeats are deleted in most persons with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), and they map immediately adjacent and internal to the low copy repeats, termed LCR22, that mediate the deletions associated with VCFS/DGS. We sequenced genomic clones from both loci and determined that the putative initiator methionine is located further upstream than originally described, but in a position similar to the mouse and chicken orthologs. DGCR6L encodes a highly homologous, functional copy of DGCR6, with some base changes rendering amino acid differences. Expression studies of the two genes indicate that both genes are widely expressed in fetal and adult tissues. Evolutionary studies using FISH mapping in several different species of ape combined with sequence analysis of DGCR6 in a number of different primate species indicate that the duplication is at least 12 million years old and may date back to before the divergence of Catarrhines from Platyrrhines, 35 mya. These data suggest that there has been selective evolutionary pressure toward the functional maintenance of both paralogs. Interestingly, a full-length HERV-K provirus integrated into the sc11.1a locus after the divergence of chimpanzees and humans. PMID:11157784

Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases ▿ †

PubMed Central

Zielonka, Jörg; Bravo, Ignacio G.; Marino, Daniela; Conrad, Elea; Perković, Mario; Battenberg, Marion; Cichutek, Klaus; Münk, Carsten

2009-01-01

The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equine and of several of the human and nonprimate A3 proteins against the Equine infectious anemia virus (EIAV), the Simian immunodeficiency virus (SIV), and the Adeno-associated virus type 2 (AAV-2). Hematopoietic cells of horses express at least five different A3s: A3Z1b, A3Z2a-Z2b, A3Z2c-Z2d, A3Z2e, and A3Z3, whereas circulating macrophages, the natural target of EIAV, express only part of the A3 repertoire. The five A3Z2 tandem copies arose after three consecutive, recent duplication events in the horse lineage, after the split between Equidae and Carnivora. The duplicated genes show different antiviral activities against different viruses: equine A3Z3 and A3Z2c-Z2d are potent inhibitors of EIAV while equine A3Z1b, A3Z2a-Z2b, A3Z2e showed only weak anti-EIAV activity. Equine A3Z1b and A3Z3 restricted AAV and all equine A3s, except A3Z1b, inhibited SIV. We hypothesize that the horse A3 genes are undergoing a process of subfunctionalization in their respective viral specificities, which might provide the evolutionary advantage for keeping five copies of the original gene. PMID:19458006

14 CFR 1206.101 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... disclosure (see paragraph (k) of this section). (j) The term duplication means the process of making a copy..., functions, policies, decisions, procedures, operations, or other activities or because of the informational..., a copy of which may be in NASA's possession. (b) The term initial determination means a decision by...

The complete chloroplast genome of Sinopodophyllum hexandrum (Berberidaceae).

PubMed

Li, Huie; Guo, Qiqiang

2016-07-01

The complete chloroplast (cp) genome of the Sinopodophyllum hexandrum (Berberidaceae) was determined in this study. The circular genome is 157,940 bp in size, and comprises a pair of inverted repeat (IR) regions of 26,077 bp each, a large single-copy (LSC) region of 86,460 bp and a small single-copy (SSC) region of 19,326 bp. The GC content of the whole cp genome was 38.5%. A total of 133 genes were identified, including 88 protein-coding genes, 37 tRNA genes and eight rRNA genes. The whole cp genome consists of 114 unique genes, and 19 genes are duplicated in the IR regions. The phylogenetic analysis revealed that S. hexandrum is closely related to Nandina domestica within the family Berberidaceae.

RNA secondary structures of the bacteriophage phi6 packaging regions.

PubMed

Pirttimaa, M J; Bamford, D H

2000-06-01

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

The complete chloroplast genome sequence of Euonymus japonicus (Celastraceae).

PubMed

Choi, Kyoung Su; Park, SeonJoo

2016-09-01

The complete chloroplast (cp) genome sequence of the Euonymus japonicus, the first sequenced of the genus Euonymus, was reported in this study. The total length was 157 637 bp, containing a pair of 26 678 bp inverted repeat region (IR), which were separated by small single copy (SSC) region and large single copy (LSC) region of 18 340 bp and 85 941 bp, respectively. This genome contains 107 unique genes, including 74 coding genes, four rRNA genes, and 29 tRNA genes. Seventeen genes contain intron of E. japonicus, of which three genes (clpP, ycf3, and rps12) include two introns. The maximum likelihood (ML) phylogenetic analysis revealed that E. japonicus was closely related to Manihot and Populus.

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

Zhou Huanqiang; School of Physical Sciences, University of Queensland, Brisbane, Queensland 4072; Barthel, Thomas

We investigate boundary critical phenomena from a quantum-information perspective. Bipartite entanglement in the ground state of one-dimensional quantum systems is quantified using the Renyi entropy S{sub {alpha}}, which includes the von Neumann entropy ({alpha}{yields}1) and the single-copy entanglement ({alpha}{yields}{infinity}) as special cases. We identify the contribution of the boundaries to the Renyi entropy, and show that there is an entanglement loss along boundary renormalization group (RG) flows. This property, which is intimately related to the Affleck-Ludwig g theorem, is a consequence of majorization relations between the spectra of the reduced density matrix along the boundary RG flows. We also pointmore » out that the bulk contribution to the single-copy entanglement is half of that to the von Neumann entropy, whereas the boundary contribution is the same.« less

The complete chloroplast genome of the Dendrobium strongylanthum (Orchidaceae: Epidendroideae).

PubMed

Li, Jing; Chen, Chen; Wang, Zhe-Zhi

2016-07-01

Complete chloroplast genome sequence is very useful for studying the phylogenetic and evolution of species. In this study, the complete chloroplast genome of Dendrobium strongylanthum was constructed from whole-genome Illumina sequencing data. The chloroplast genome is 153 058 bp in length with 37.6% GC content and consists of two inverted repeats (IRs) of 26 316 bp. The IR regions are separated by large single-copy region (LSC, 85 836 bp) and small single-copy (SSC, 14 590 bp) region. A total of 130 chloroplast genes were successfully annotated, including 84 protein coding genes, 38 tRNA genes, and eight rRNA genes. Phylogenetic analyses showed that the chloroplast genome of Dendrobium strongylanthum is related to that of the Dendrobium officinal.

Effects of small particle numbers on long-term behaviour in discrete biochemical systems.

PubMed

Kreyssig, Peter; Wozar, Christian; Peter, Stephan; Veloz, Tomás; Ibrahim, Bashar; Dittrich, Peter

2014-09-01

The functioning of many biological processes depends on the appearance of only a small number of a single molecular species. Additionally, the observation of molecular crowding leads to the insight that even a high number of copies of species do not guarantee their interaction. How single particles contribute to stabilizing biological systems is not well understood yet. Hence, we aim at determining the influence of single molecules on the long-term behaviour of biological systems, i.e. whether they can reach a steady state. We provide theoretical considerations and a tool to analyse Systems Biology Markup Language models for the possibility to stabilize because of the described effects. The theory is an extension of chemical organization theory, which we called discrete chemical organization theory. Furthermore we scanned the BioModels Database for the occurrence of discrete chemical organizations. To exemplify our method, we describe an application to the Template model of the mitotic spindle assembly checkpoint mechanism. http://www.biosys.uni-jena.de/Services.html. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

quenched-smFISH: Counting small RNA in Pathogenic Bacteria

NASA Astrophysics Data System (ADS)

Shepherd, Douglas; Li, Nan; Micheva-Viteva, Sofiya; Munsky, Brian; Hong-Geller, Elizabeth; Werner, James

2014-03-01

Here, we present a modification to single-molecule fluorescence in situ hybridization, quenched smFISH (q-smFISH), that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. Exploiting an automated, multi-color wide-field microscope and GPU-accelerated data analysis package, we analyzed the statistics of sRNA expression in thousands of individual Yersinia pseudotuberculosis and Yersinia pestis bacteria before and during a simulated infection. Before infection, we find only a small fraction of either bacteria express the small RNAs YSR35 or YSP8. The copy numbers of these RNA are increased during simulated infection, suggesting a role in pathogenesis. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in bacterial regulatory networks.

Genetics Home Reference: Léri-Weill dyschondrosteosis

MedlinePlus

... both females (who have two X chromosomes) and males (who have one X and one Y chromosome ) normally have two functional copies of the SHOX gene in each cell. The inheritance pattern of Léri-Weill dyschondrosteosis is described as dominant because one missing or altered copy of the ...

Gene therapy in periodontics

PubMed Central

Chatterjee, Anirban; Singh, Nidhi; Saluja, Mini

2013-01-01

GENES are made of DNA - the code of life. They are made up of two types of base pair from different number of hydrogen bonds AT, GC which can be turned into instruction. Everyone inherits genes from their parents and passes them on in turn to their children. Every person's genes are different, and the changes in sequence determine the inherited differences between each of us. Some changes, usually in a single gene, may cause serious diseases. Gene therapy is ‘the use of genes as medicine’. It involves the transfer of a therapeutic or working gene copy into specific cells of an individual in order to repair a faulty gene copy. Thus it may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favorably modify the clinical course of a condition. It has a promising era in the field of periodontics. Gene therapy has been used as a mode of tissue engineering in periodontics. The tissue engineering approach reconstructs the natural target tissue by combining four elements namely: Scaffold, signaling molecules, cells and blood supply and thus can help in the reconstruction of damaged periodontium including cementum, gingival, periodontal ligament and bone. PMID:23869119

Maternal isodisomy of the telomeric end of chromosome 2 is responsible for a case of primary hyperoxaluria type 1.

PubMed

Chevalier-Porst, Françoise; Rolland, Marie-Odile; Cochat, Pierre; Bozon, Dominique

2005-01-01

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism, in which excessive oxalates are formed by the liver and excreted by the kidneys, causing a wide spectrum of disease, ranging from renal failure in infancy to mere renal stones in late adulthood. This disease is caused by a deficiency of alanine:glyoxylate aminotransferase (AGT), which is encoded by a single copy gene, AGXT, located in 2q37.3. We identified an apparently homozygous, loss-of-function, mutation in a patient; the gene defect was present in the heterozygous mother but not in the patient's father. We performed a microsatellite repeat analysis using 13 specific chromosome 2 markers and non-chromosome 2 minisatellites. Six specific chromosome 2 markers showed an apparently homozygous maternal inheritance while four showed a biparental transmission consistent with paternity (confirmed by minisatellite analysis). Quantitative PCR of AGXT exons 1 and 3 on the patient's and parents genomic DNA revealed the presence of two copies of the gene. This is the first case of PH1 caused by segmental maternal isodisomy of 2q37.3. (c) 2004 Wiley-Liss, Inc.

CNVcaller: highly efficient and widely applicable software for detecting copy number variations in large populations.

PubMed

Wang, Xihong; Zheng, Zhuqing; Cai, Yudong; Chen, Ting; Li, Chao; Fu, Weiwei; Jiang, Yu

2017-12-01

The increasing amount of sequencing data available for a wide variety of species can be theoretically used for detecting copy number variations (CNVs) at the population level. However, the growing sample sizes and the divergent complexity of nonhuman genomes challenge the efficiency and robustness of current human-oriented CNV detection methods. Here, we present CNVcaller, a read-depth method for discovering CNVs in population sequencing data. The computational speed of CNVcaller was 1-2 orders of magnitude faster than CNVnator and Genome STRiP for complex genomes with thousands of unmapped scaffolds. CNV detection of 232 goats required only 1.4 days on a single compute node. Additionally, the Mendelian consistency of sheep trios indicated that CNVcaller mitigated the influence of high proportions of gaps and misassembled duplications in the nonhuman reference genome assembly. Furthermore, multiple evaluations using real sheep and human data indicated that CNVcaller achieved the best accuracy and sensitivity for detecting duplications. The fast generalized detection algorithms included in CNVcaller overcome prior computational barriers for detecting CNVs in large-scale sequencing data with complex genomic structures. Therefore, CNVcaller promotes population genetic analyses of functional CNVs in more species. © The Authors 2017. Published by Oxford University Press.

CNVcaller: highly efficient and widely applicable software for detecting copy number variations in large populations

PubMed Central

Wang, Xihong; Zheng, Zhuqing; Cai, Yudong; Chen, Ting; Li, Chao; Fu, Weiwei

2017-01-01

Abstract Background The increasing amount of sequencing data available for a wide variety of species can be theoretically used for detecting copy number variations (CNVs) at the population level. However, the growing sample sizes and the divergent complexity of nonhuman genomes challenge the efficiency and robustness of current human-oriented CNV detection methods. Results Here, we present CNVcaller, a read-depth method for discovering CNVs in population sequencing data. The computational speed of CNVcaller was 1–2 orders of magnitude faster than CNVnator and Genome STRiP for complex genomes with thousands of unmapped scaffolds. CNV detection of 232 goats required only 1.4 days on a single compute node. Additionally, the Mendelian consistency of sheep trios indicated that CNVcaller mitigated the influence of high proportions of gaps and misassembled duplications in the nonhuman reference genome assembly. Furthermore, multiple evaluations using real sheep and human data indicated that CNVcaller achieved the best accuracy and sensitivity for detecting duplications. Conclusions The fast generalized detection algorithms included in CNVcaller overcome prior computational barriers for detecting CNVs in large-scale sequencing data with complex genomic structures. Therefore, CNVcaller promotes population genetic analyses of functional CNVs in more species. PMID:29220491

The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

PubMed Central

Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire

2014-01-01

Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. PMID:24837284

Gene therapy in periodontics.

PubMed

Chatterjee, Anirban; Singh, Nidhi; Saluja, Mini

2013-03-01

GENES are made of DNA - the code of life. They are made up of two types of base pair from different number of hydrogen bonds AT, GC which can be turned into instruction. Everyone inherits genes from their parents and passes them on in turn to their children. Every person's genes are different, and the changes in sequence determine the inherited differences between each of us. Some changes, usually in a single gene, may cause serious diseases. Gene therapy is 'the use of genes as medicine'. It involves the transfer of a therapeutic or working gene copy into specific cells of an individual in order to repair a faulty gene copy. Thus it may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favorably modify the clinical course of a condition. It has a promising era in the field of periodontics. Gene therapy has been used as a mode of tissue engineering in periodontics. The tissue engineering approach reconstructs the natural target tissue by combining four elements namely: Scaffold, signaling molecules, cells and blood supply and thus can help in the reconstruction of damaged periodontium including cementum, gingival, periodontal ligament and bone.

ACTG A5353: A Pilot Study of Dolutegravir Plus Lamivudine for Initial Treatment of Human Immunodeficiency Virus-1 (HIV-1)-infected Participants With HIV-1 RNA <500000 Copies/mL.

PubMed

Taiwo, Babafemi O; Zheng, Lu; Stefanescu, Andrei; Nyaku, Amesika; Bezins, Baiba; Wallis, Carole L; Godfrey, Catherine; Sax, Paul E; Acosta, Edward; Haas, David; Smith, Kimberly Y; Sha, Beverly; Van Dam, Cornelius; Gulick, Roy M

2018-05-17

Limited data exist on initial human immunodeficiency virus type 1 (HIV-1) treatment with dolutegravir plus lamivudine. A5353 is a phase 2, single-arm, pilot study of once-daily dolutegravir (50 mg) plus lamivudine (300 mg) in treatment-naive participants with HIV-1 RNA ≥1000 and <500000 copies/mL. Exclusion criteria included active hepatitis B or major protease, reverse transcriptase, or integrase resistance. The primary efficacy measure was the proportion with HIV-1 RNA <50 copies/mL (FDA [US Food and Drug Administration] Snapshot) at week 24. Virologic failure (VF) was confirmed HIV-1 RNA >400 copies/mL at week 16/20 or >200 copies/mL at or after week 24. Dolutegravir levels and drug resistance testing were performed at VF. One hundred and twenty participants (87% male, median age 30 years, 37 (31%) HIV-1 RNA >100000 copies/mL) initiated study treatment. Median entry HIV-1 RNA and CD4 count were 4.61 log10 copies/mL and 387 cells/mm3. Virologic efficacy at week 24 was 108/120 (90%, confidence interval [83%, 95%]), with comparable results in the >100000 copies/mL and ≤100000 copies/mL strata, that is, 89% (75%, 97%) and 90% (82%, 96%), respectively. Three participants with VF, had undetected plasma dolutegravir at ≥1 time points; the M184V and R263R/K mutations developed in 1 participant. Two participants experienced grade 3 possible/probable treatment-related adverse events; none discontinued treatment due to adverse events. Dolutegravir plus lamivudine demonstrated efficacy in individuals with pretreatment HIV-1 RNA up to 500000 copies/mL in this pilot trial, but a participant developed resistance mutations. NCT02582684.

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster

PubMed Central

Remnant, Emily J.; Good, Robert T.; Schmidt, Joshua M.; Lumb, Christopher; Robin, Charles; Daborn, Phillip J.; Batterham, Philip

2013-01-01

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala301 to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala301 to Ser resistance mutation and Met360 to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser301 homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser301 change into an Ala301 background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene. PMID:23959864

CNV-RF Is a Random Forest-Based Copy Number Variation Detection Method Using Next-Generation Sequencing.

PubMed

Onsongo, Getiria; Baughn, Linda B; Bower, Matthew; Henzler, Christine; Schomaker, Matthew; Silverstein, Kevin A T; Thyagarajan, Bharat

2016-11-01

Simultaneous detection of small copy number variations (CNVs) (<0.5 kb) and single-nucleotide variants in clinically significant genes is of great interest for clinical laboratories. The analytical variability in next-generation sequencing (NGS) and artifacts in coverage data because of issues with mappability along with lack of robust bioinformatics tools for CNV detection have limited the utility of targeted NGS data to identify CNVs. We describe the development and implementation of a bioinformatics algorithm, copy number variation-random forest (CNV-RF), that incorporates a machine learning component to identify CNVs from targeted NGS data. Using CNV-RF, we identified 12 of 13 deletions in samples with known CNVs, two cases with duplications, and identified novel deletions in 22 additional cases. Furthermore, no CNVs were identified among 60 genes in 14 cases with normal copy number and no CNVs were identified in another 104 patients with clinical suspicion of CNVs. All positive deletions and duplications were confirmed using a quantitative PCR method. CNV-RF also detected heterozygous deletions and duplications with a specificity of 50% across 4813 genes. The ability of CNV-RF to detect clinically relevant CNVs with a high degree of sensitivity along with confirmation using a low-cost quantitative PCR method provides a framework for providing comprehensive NGS-based CNV/single-nucleotide variant detection in a clinical molecular diagnostics laboratory. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

The role of efference copy in striatal learning.

PubMed

Fee, Michale S

2014-04-01

Reinforcement learning requires the convergence of signals representing context, action, and reward. While models of basal ganglia function have well-founded hypotheses about the neural origin of signals representing context and reward, the function and origin of signals representing action are less clear. Recent findings suggest that exploratory or variable behaviors are initiated by a wide array of 'action-generating' circuits in the midbrain, brainstem, and cortex. Thus, in order to learn, the striatum must incorporate an efference copy of action decisions made in these action-generating circuits. Here we review several recent neural models of reinforcement learning that emphasize the role of efference copy signals. Also described are ideas about how these signals might be integrated with inputs signaling context and reward. Copyright © 2014 Elsevier Ltd. All rights reserved.

Length Variation in Mitochondrial DNA of the Minnow Cyprinella Spiloptera

PubMed Central

Broughton, R. E.; Dowling, T. E.

1994-01-01

Length differences in animal mitochondrial DNA (mtDNA) are common, frequently due to variation in copy number of direct tandem duplications. While such duplications appear to form without great difficulty in some taxonomic groups, they appear to be relatively short-lived, as typical duplication products are geographically restricted within species and infrequently shared among species. To better understand such length variation, we have studied a tandem and direct duplication of approximately 260 bp in the control region of the cyprinid fish, Cyprinella spiloptera. Restriction site analysis of 38 individuals was used to characterize population structure and the distribution of variation in repeat copy number. This revealed two length variants, including individuals with two or three copies of the repeat, and little geographic structure among populations. No standard length (single copy) genomes were found and heteroplasmy, a common feature of length variation in other taxa, was absent. Nucleotide sequence of tandem duplications and flanking regions localized duplication junctions in the phenylalanine tRNA and near the origin of replication. The locations of these junctions and the stability of folded repeat copies support the hypothesized importance of secondary structures in models of duplication formation. PMID:8001785

Radiation induces genomic instability and mammary ductal dysplasia in Atm heterozygous mice

NASA Technical Reports Server (NTRS)

Weil, M. M.; Kittrell, F. S.; Yu, Y.; McCarthy, M.; Zabriskie, R. C.; Ullrich, R. L.

2001-01-01

Ataxia-telangiectasia (AT) is a genetic syndrome resulting from the inheritance of two defective copies of the ATM gene that includes among its stigmata radiosensitivity and cancer susceptibility. Epidemiological studies have demonstrated that although women with a single defective copy of ATM (AT heterozygotes) appear clinically normal, they may never the less have an increased relative risk of developing breast cancer. Whether they are at increased risk for radiation-induced breast cancer from medical exposures to ionizing radiation is unknown. We have used a murine model of AT to investigate the effect of a single defective Atm allele, the murine homologue of ATM, on the susceptibility of mammary epithelial cells to radiation-induced transformation. Here we report that mammary epithelial cells from irradiated mice with one copy of Atm truncated in the PI-3 kinase domain were susceptible to radiation-induced genomic instability and generated a 10% incidence of dysplastic mammary ducts when transplanted into syngenic recipients, whereas cells from Atm(+/+) mice were stable and formed only normal ducts. Since radiation-induced ductal dysplasia is a precursor to mammary cancer, the results indicate that AT heterozygosity increases susceptibility to radiogenic breast cancer in this murine model system.

Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate.

PubMed

Ebhardt, H Alexander; Sabidó, Eduard; Hüttenhain, Ruth; Collins, Ben; Aebersold, Ruedi

2012-04-01

Selected or multiple reaction monitoring is a targeted mass spectrometry method (S/MRM-MS), in which many peptides are simultaneously and consistently analyzed during a single liquid chromatography-mass spectrometry (LC-S/MRM-MS) measurement. These capabilities make S/MRM-MS an attractive method to monitor a consistent set of proteins over various experimental conditions. To increase throughput for S/MRM-MS it is advantageous to use scheduled methods and unfractionated protein extracts. Here, we established the practically measurable dynamic range of proteins reliably detectable and quantifiable in an unfractionated protein extract from a human cell line using LC-S/MRM-MS. Initially, we analyzed S/MRM transition peak groups in terms of interfering signals and compared S/MRM transition peak groups to MS1-triggered MS2 spectra using dot-product analysis. Finally, using unfractionated protein extract from human cell lysate, we quantified the upper boundary of copies per cell to be 35 million copies per cell, while 7500 copies per cell represents a lower boundary using a single 35 min linear gradient LC-S/MRM-MS measurement on a current, standard commercial instrument. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

One-loop transition amplitudes in the D1D5 CFT

NASA Astrophysics Data System (ADS)

Carson, Zaq; Hampton, Shaun; Mathur, Samir D.

2017-01-01

We consider the issue of thermalization in the D1D5 CFT. Thermalization is expected to correspond to the formation of a black hole in the dual gravity theory. We start from the orbifold point, where the theory is essentially free, and does not thermalize. In earlier work it was noted that there was no clear thermalization effect when the theory was deformed off the orbifold point to first order in the relevant twist perturbation. In this paper we consider the deformation to second order in the twist, where we do find effects that can cause thermalization of an initial perturbation. We consider a 1-loop process where two untwisted copies of the CFT are twisted to one copy and then again untwisted to two copies. We start with a single oscillator excitation on the initial CFT, and compute the effect of the two twists on this state. We find simple approximate expressions for the Bogoliubov coefficients and the behavior of the single oscillator excitation in the continuum limit, where the mode numbers involved are taken to be much larger than unity. We also prove a number of useful relationships valid for processes with an arbitrary number of twist insertions.

BEAMing LAMP: single-molecule capture and on-bead isothermal amplification for digital detection of hepatitis C virus in plasma.

PubMed

Chen, Jiyun; Xu, Xiaomin; Huang, Zhimei; Luo, Yuan; Tang, Lijuan; Jiang, Jian-Hui

2018-01-02

A novel dNAD platform (BEAMing LAMP) by combining emulsion micro-reactors, single-molecule magnetic capture and on-bead loop-mediated isothermal amplification has been developed for DNA detection, which enables absolute and high-precision quantification of a target with a detection limit of 300 copies.

Skylab S-191 spectrometer single spectral scan analysis program. [user manual

NASA Technical Reports Server (NTRS)

Downes, E. L.

1974-01-01

Documentation and user information for the S-191 single spectral scan analysis program are reported. A breakdown of the computational algorithms is supplied, followed by the program listing and examples of sample output. A copy of the flow chart which describes the driver routine in the body of the main program segment is included.

Diabetic Nephropathy Induced by Increased Ace Gene Dosage Is Associated with High Renal Levels of Angiotensin (1–7) and Bradykinin

PubMed Central

Bertoncello, Nádia; Moreira, Roseli Peres; Arita, Danielle Yuri; Aragão, Danielle S.; Watanabe, Ingrid Kazue Mizuno; Dantas, Patricia S.; Santos, Ralmony; Mattar-Rosa, Rodolfo; Yokota, Rodrigo; Cunha, Tatiana Sousa; Casarini, Dulce Elena

2015-01-01

Population studies have shown an association between diabetic nephropathy (DN) and insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene (ACE in humans, Ace in mice). The aim was to evaluate the modulation of Ace copies number and diabetes mellitus (DM) on renal RAS and correlate it with indicators of kidney function. Increased number of copies of the Ace gene, associated with DM, induces renal dysfunction. The susceptibility to the development of DN in 3 copies of animals is associated with an imbalance in activity of RAS enzymes leading to increased synthesis of Ang II and Ang-(1–7). Increased concentration of renal Ang-(1–7) appears to potentiate the deleterious effects triggered by Ang II on kidney structure and function. Results also show increased bradykinin concentration in 3 copies diabetic group. Taken together, results indicate that the deleterious effects described in 3 copies diabetic group are, at least in part, due to a combination of factors not usually described in the literature. Thus, the data presented here show up innovative and contribute to understanding the complex mechanisms involved in the development of DN, in order to optimize the treatment of patients with this complication. PMID:26442284

SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells.

PubMed

Han, Kyung Yeon; Kim, Kyu-Tae; Joung, Je-Gun; Son, Dae-Soon; Kim, Yeon Jeong; Jo, Areum; Jeon, Hyo-Jeong; Moon, Hui-Sung; Yoo, Chang Eun; Chung, Woosung; Eum, Hye Hyeon; Kim, Sangmin; Kim, Hong Kwan; Lee, Jeong Eon; Ahn, Myung-Ju; Lee, Hae-Ock; Park, Donghyun; Park, Woong-Yang

2018-01-01

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level. © 2018 Han et al.; Published by Cold Spring Harbor Laboratory Press.

SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells

PubMed Central

Han, Kyung Yeon; Kim, Kyu-Tae; Joung, Je-Gun; Son, Dae-Soon; Kim, Yeon Jeong; Jo, Areum; Jeon, Hyo-Jeong; Moon, Hui-Sung; Yoo, Chang Eun; Chung, Woosung; Eum, Hye Hyeon; Kim, Sangmin; Kim, Hong Kwan; Lee, Jeong Eon; Ahn, Myung-Ju; Lee, Hae-Ock; Park, Donghyun; Park, Woong-Yang

2018-01-01

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level. PMID:29208629

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

PubMed

van der Ley, P

1988-11-01

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

Genome fluctuations in cyanobacteria reflect evolutionary, developmental and adaptive traits.

PubMed

Larsson, John; Nylander, Johan Aa; Bergman, Birgitta

2011-06-30

Cyanobacteria belong to an ancient group of photosynthetic prokaryotes with pronounced variations in their cellular differentiation strategies, physiological capacities and choice of habitat. Sequencing efforts have shown that genomes within this phylum are equally diverse in terms of size and protein-coding capacity. To increase our understanding of genomic changes in the lineage, the genomes of 58 contemporary cyanobacteria were analysed for shared and unique orthologs. A total of 404 protein families, present in all cyanobacterial genomes, were identified. Two of these are unique to the phylum, corresponding to an AbrB family transcriptional regulator and a gene that escapes functional annotation although its genomic neighbourhood is conserved among the organisms examined. The evolution of cyanobacterial genome sizes involves a mix of gains and losses in the clade encompassing complex cyanobacteria, while a single event of reduction is evident in a clade dominated by unicellular cyanobacteria. Genome sizes and gene family copy numbers evolve at a higher rate in the former clade, and multi-copy genes were predominant in large genomes. Orthologs unique to cyanobacteria exhibiting specific characteristics, such as filament formation, heterocyst differentiation, diazotrophy and symbiotic competence, were also identified. An ancestral character reconstruction suggests that the most recent common ancestor of cyanobacteria had a genome size of approx. 4.5 Mbp and 1678 to 3291 protein-coding genes, 4%-6% of which are unique to cyanobacteria today. The different rates of genome-size evolution and multi-copy gene abundance suggest two routes of genome development in the history of cyanobacteria. The expansion strategy is driven by gene-family enlargment and generates a broad adaptive potential; while the genome streamlining strategy imposes adaptations to highly specific niches, also reflected in their different functional capacities. A few genomes display extreme proliferation of non-coding nucleotides which is likely to be the result of initial expansion of genomes/gene copy number to gain adaptive potential, followed by a shift to a life-style in a highly specific niche (e.g. symbiosis). This transition results in redundancy of genes and gene families, leading to an increase in junk DNA and eventually to gene loss. A few orthologs can be correlated with specific phenotypes in cyanobacteria, such as filament formation and symbiotic competence; these constitute exciting exploratory targets.

Genome fluctuations in cyanobacteria reflect evolutionary, developmental and adaptive traits

PubMed Central

2011-01-01

Background Cyanobacteria belong to an ancient group of photosynthetic prokaryotes with pronounced variations in their cellular differentiation strategies, physiological capacities and choice of habitat. Sequencing efforts have shown that genomes within this phylum are equally diverse in terms of size and protein-coding capacity. To increase our understanding of genomic changes in the lineage, the genomes of 58 contemporary cyanobacteria were analysed for shared and unique orthologs. Results A total of 404 protein families, present in all cyanobacterial genomes, were identified. Two of these are unique to the phylum, corresponding to an AbrB family transcriptional regulator and a gene that escapes functional annotation although its genomic neighbourhood is conserved among the organisms examined. The evolution of cyanobacterial genome sizes involves a mix of gains and losses in the clade encompassing complex cyanobacteria, while a single event of reduction is evident in a clade dominated by unicellular cyanobacteria. Genome sizes and gene family copy numbers evolve at a higher rate in the former clade, and multi-copy genes were predominant in large genomes. Orthologs unique to cyanobacteria exhibiting specific characteristics, such as filament formation, heterocyst differentiation, diazotrophy and symbiotic competence, were also identified. An ancestral character reconstruction suggests that the most recent common ancestor of cyanobacteria had a genome size of approx. 4.5 Mbp and 1678 to 3291 protein-coding genes, 4%-6% of which are unique to cyanobacteria today. Conclusions The different rates of genome-size evolution and multi-copy gene abundance suggest two routes of genome development in the history of cyanobacteria. The expansion strategy is driven by gene-family enlargment and generates a broad adaptive potential; while the genome streamlining strategy imposes adaptations to highly specific niches, also reflected in their different functional capacities. A few genomes display extreme proliferation of non-coding nucleotides which is likely to be the result of initial expansion of genomes/gene copy number to gain adaptive potential, followed by a shift to a life-style in a highly specific niche (e.g. symbiosis). This transition results in redundancy of genes and gene families, leading to an increase in junk DNA and eventually to gene loss. A few orthologs can be correlated with specific phenotypes in cyanobacteria, such as filament formation and symbiotic competence; these constitute exciting exploratory targets. PMID:21718514

Day-On, Day-Off emtricitabine, tenofovir disoproxil fumarate and efavirenz single tablet regimen (DODO) as maintenance therapy in HIV-infected patients.

PubMed

Costantini, Andrea; Tontini, Chiara; Rocchi, Monia; Martini, Matteo; Butini, Luca

2018-06-01

Reduced dose schedules may be feasible options to simplify antiretroviral therapy (ART) in selected HIV-1-infected individuals. Efficacy and safety of a Day-on, Day-off (DODO) schedule of tenofovir disoproxil fumarate, emtricitabine and efavirenz (FTC/TDF/EFV) single tablet regimen (STR) was assessed. Twenty-seven patients were prescribed the DODO schedule and were monitored for 48 weeks. Switching criteria were: no previous ART failure, no AIDS-defining illnesses, T CD4 cell nadir >200/mmc, and HIV-RNA below detection limit (40 copies/mL) for at least six months. Clinical and laboratory data, including plasma HIV-RNA levels, T CD4 and CD8 counts, liver and kidney function, lipid levels and ultrasensitive C-reactive protein (us-CRP) were assessed at baseline, week 4, 12, 24, and 48. Statistical analysis was performed by paired Student's T-test for comparison between baseline and each time point and Chi square test for CD4/CD8 ratio comparison. In all, 26 out of 27 patients maintained plasma HIV-RNA levels below the detection limit through the entire follow-up. One patient experienced low level plasma HIV-RNA rebound at week 36 (47 copies/ml) and immediately reverted to the conventional dose schedule of FTC/TDF/EFV; plasma HIV-RNA was undetectable after four weeks. No major changes on liver and kidney function tests, lipid levels and us-CRP were observed. Although no profound modifications of T CD4 count were observed during follow-up, the CD4/CD8 ratio increased significantly at week 48 compared to the baseline (p<0.05). In conclusion, 48-week DODO administration of the fixed dose FTC/TDF/EFV STR combination was safe and effective in maintaining HIV viral replication below the detection limit in a selected group of HIV-1-infected individuals.

The Evolution of Pepsinogen C Genes in Vertebrates: Duplication, Loss and Functional Diversification

PubMed Central

Gonçalves, Odete; Wilson, Jonathan Mark

2012-01-01

Background Aspartic proteases comprise a large group of enzymes involved in peptide proteolysis. This collection includes prominent enzymes globally categorized as pepsins, which are derived from pepsinogen precursors. Pepsins are involved in gastric digestion, a hallmark of vertebrate physiology. An important member among the pepsinogens is pepsinogen C (Pgc). A particular aspect of Pgc is its apparent single copy status, which contrasts with the numerous gene copies found for example in pepsinogen A (Pga). Although gene sequences with similarity to Pgc have been described in some vertebrate groups, no exhaustive evolutionary framework has been considered so far. Methodology/Principal Findings By combining phylogenetics and genomic analysis, we find an unexpected Pgc diversity in the vertebrate sub-phylum. We were able to reconstruct gene duplication timings relative to the divergence of major vertebrate clades. Before tetrapod divergence, a single Pgc gene tandemly expanded to produce two gene lineages (Pgbc and Pgc2). These have been differentially retained in various classes. Accordingly, we find Pgc2 in sauropsids, amphibians and marsupials, but not in eutherian mammals. Pgbc was retained in amphibians, but duplicated in the ancestor of amniotes giving rise to Pgb and Pgc1. The latter was retained in mammals and probably in reptiles and marsupials but not in birds. Pgb was kept in all of the amniote clade with independent episodes of loss in some mammalian species. Lineage specific expansions of Pgc2 and Pgbc have also occurred in marsupials and amphibians respectively. We find that teleost and tetrapod Pgc genes reside in distinct genomic regions hinting at a possible translocation. Conclusions We conclude that the repertoire of Pgc genes is larger than previously reported, and that tandem duplications have modelled the history of Pgc genes. We hypothesize that gene expansion lead to functional divergence in tetrapods, coincident with the invasion of terrestrial habitats. PMID:22427897

Omega-3 fatty acid desaturase gene family from two ω-3 sources, Salvia hispanica and Perilla frutescens: Cloning, characterization and expression

PubMed Central

Xue, Yufei; Chen, Baojun; Win, Aung Naing; Fu, Chun; Lian, Jianping; Liu, Xue; Wang, Rui; Zhang, Xingcui

2018-01-01

Omega-3 fatty acid desaturase (ω-3 FAD, D15D) is a key enzyme for α-linolenic acid (ALA) biosynthesis. Both chia (Salvia hispanica) and perilla (Perilla frutescens) contain high levels of ALA in seeds. In this study, the ω-3 FAD gene family was systematically and comparatively cloned from chia and perilla. Perilla FAD3, FAD7, FAD8 and chia FAD7 are encoded by single-copy (but heterozygous) genes, while chia FAD3 is encoded by 2 distinct genes. Only 1 chia FAD8 sequence was isolated. In these genes, there are 1 to 6 transcription start sites, 1 to 8 poly(A) tailing sites, and 7 introns. The 5’UTRs of PfFAD8a/b contain 1 to 2 purine-stretches and 2 pyrimidine-stretches. An alternative splice variant of ShFAD7a/b comprises a 5’UTR intron. Their encoded proteins harbor an FA_desaturase conserved domain together with 4 trans-membrane helices and 3 histidine boxes. Phylogenetic analysis validated their identity of dicot microsomal or plastidial ω-3 FAD proteins, and revealed some important evolutionary features of plant ω-3 FAD genes such as convergent evolution across different phylums, single-copy status in algae, and duplication events in certain taxa. The qRT-PCR assay showed that the ω-3 FAD genes of two species were expressed at different levels in various organs, and they also responded to multiple stress treatments. The functionality of the ShFAD3 and PfFAD3 enzymes was confirmed by yeast expression. The systemic molecular and functional features of the ω-3 FAD gene family from chia and perilla revealed in this study will facilitate their use in future studies on genetic improvement of ALA traits in oilseed crops. PMID:29351555

Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

PubMed Central

Linne, Hannah; Yasaei, Hemad; Marriott, Alison; Harvey, Amanda; Mokbel, Kefah; Newbold, Robert; Roberts, Terry

2017-01-01

Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT-repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells. PMID:28977912

Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression.

PubMed

Linne, Hannah; Yasaei, Hemad; Marriott, Alison; Harvey, Amanda; Mokbel, Kefah; Newbold, Robert; Roberts, Terry

2017-09-22

Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT- repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells.

A pair of new BAC and BIBAC vectors that facilitate BAC/BIBAC library construction and intact large genomic DNA insert exchange.

PubMed

Shi, Xue; Zeng, Haiyang; Xue, Yadong; Luo, Meizhong

2011-10-11

Large-insert BAC and BIBAC libraries are important tools for structural and functional genomics studies of eukaryotic genomes. To facilitate the construction of BAC and BIBAC libraries and the transfer of complete large BAC inserts into BIBAC vectors, which is desired in positional cloning, we developed a pair of new BAC and BIBAC vectors. The new BAC vector pIndigoBAC536-S and the new BIBAC vector BIBAC-S have the following features: 1) both contain two 18-bp non-palindromic I-SceI sites in an inverted orientation at positions that flank an identical DNA fragment containing the lacZ selection marker and the cloning site. Large DNA inserts can be excised from the vectors as single fragments by cutting with I-SceI, allowing the inserts to be easily sized. More importantly, because the two vectors contain different antibiotic resistance genes for transformant selection and produce the same non-complementary 3' protruding ATAA ends by I-SceI that suppress self- and inter-ligations, the exchange of intact large genomic DNA inserts between the BAC and BIBAC vectors is straightforward; 2) both were constructed as high-copy composite vectors. Reliable linearized and dephosphorylated original low-copy pIndigoBAC536-S and BIBAC-S vectors that are ready for library construction can be prepared from the high-copy composite vectors pHZAUBAC1 and pHZAUBIBAC1, respectively, without the need for additional preparation steps or special reagents, thus simplifying the construction of BAC and BIBAC libraries. BIBAC clones constructed with the new BIBAC-S vector are stable in both E. coli and Agrobacterium. The vectors can be accessed through our website http://GResource.hzau.edu.cn. The two new vectors and their respective high-copy composite vectors can largely facilitate the construction and characterization of BAC and BIBAC libraries. The transfer of complete large genomic DNA inserts from one vector to the other is made straightforward.

What Is Hot Yoga (Bikram)?

MedlinePlus

... a vigorous form of yoga performed in a studio that is heated to 105 F (40 C) ... opportunities Reprint Permissions A single copy of these materials may be reprinted for noncommercial personal use only. " ...

Safe Practices for Copy and Paste in the EHR

PubMed Central

Lehmann, Christoph U.; Michel, Jeremy; Solomon, Ronni; Possanza, Lorraine; Gandhi, Tejal

2017-01-01

Summary Background Copy and paste functionality can support efficiency during clinical documentation, but may promote inaccurate documentation with risks for patient safety. The Partnership for Health IT Patient Safety was formed to gather data, conduct analysis, educate, and disseminate safe practices for safer care using health information technology (IT). Objective To characterize copy and paste events in clinical care, identify safety risks, describe existing evidence, and develop implementable practice recommendations for safe reuse of information via copy and paste. Methods The Partnership 1) reviewed 12 reported safety events, 2) solicited expert input, and 3) performed a systematic literature review (2010 to January 2015) to identify publications addressing frequency, perceptions/attitudes, patient safety risks, existing guidance, and potential interventions and mitigation practices. Results The literature review identified 51 publications that were included. Overall, 66% to 90% of clinicians routinely use copy and paste. One study of diagnostic errors found that copy and paste led to 2.6% of errors in which a missed diagnosis required patients to seek additional unplanned care. Copy and paste can promote note bloat, internal inconsistencies, error propagation, and documentation in the wrong patient chart. Existing guidance identified specific responsibilities for authors, organizations, and electronic health record (EHR) developers. Analysis of 12 reported copy and paste safety events was congruent with problems identified from the literature review. Conclusion Despite regular copy and paste use, evidence regarding direct risk to patient safety remains sparse, with significant study limitations. Drawing on existing evidence, the Partnership developed four safe practice recommendations: 1) Provide a mechanism to make copy and paste material easily identifiable; 2) Ensure the provenance of copy and paste material is readily available; 3) Ensure adequate staff training and education; 4) Ensure copy and paste practices are regularly monitored, measured, and assessed. PMID:28074211

Safe Practices for Copy and Paste in the EHR. Systematic Review, Recommendations, and Novel Model for Health IT Collaboration.

PubMed

Tsou, Amy Y; Lehmann, Christoph U; Michel, Jeremy; Solomon, Ronni; Possanza, Lorraine; Gandhi, Tejal

2017-01-11

Copy and paste functionality can support efficiency during clinical documentation, but may promote inaccurate documentation with risks for patient safety. The Partnership for Health IT Patient Safety was formed to gather data, conduct analysis, educate, and disseminate safe practices for safer care using health information technology (IT). To characterize copy and paste events in clinical care, identify safety risks, describe existing evidence, and develop implementable practice recommendations for safe reuse of information via copy and paste. The Partnership 1) reviewed 12 reported safety events, 2) solicited expert input, and 3) performed a systematic literature review (2010 to January 2015) to identify publications addressing frequency, perceptions/attitudes, patient safety risks, existing guidance, and potential interventions and mitigation practices. The literature review identified 51 publications that were included. Overall, 66% to 90% of clinicians routinely use copy and paste. One study of diagnostic errors found that copy and paste led to 2.6% of errors in which a missed diagnosis required patients to seek additional unplanned care. Copy and paste can promote note bloat, internal inconsistencies, error propagation, and documentation in the wrong patient chart. Existing guidance identified specific responsibilities for authors, organizations, and electronic health record (EHR) developers. Analysis of 12 reported copy and paste safety events was congruent with problems identified from the literature review. Despite regular copy and paste use, evidence regarding direct risk to patient safety remains sparse, with significant study limitations. Drawing on existing evidence, the Partnership developed four safe practice recommendations: 1) Provide a mechanism to make copy and paste material easily identifiable; 2) Ensure the provenance of copy and paste material is readily available; 3) Ensure adequate staff training and education; 4) Ensure copy and paste practices are regularly monitored, measured, and assessed.

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.

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.

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors

PubMed Central

2013-01-01

Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma. PMID:24119551

Distinct Trajectories of Massive Recent Gene Gains and Losses in Populations of a Microbial Eukaryotic Pathogen

PubMed Central

Hartmann, Fanny E.; Croll, Daniel

2017-01-01

Abstract Differences in gene content are a significant source of variability within species and have an impact on phenotypic traits. However, little is known about the mechanisms responsible for the most recent gene gains and losses. We screened the genomes of 123 worldwide isolates of the major pathogen of wheat Zymoseptoria tritici for robust evidence of gene copy number variation. Based on orthology relationships in three closely related fungi, we identified 599 gene gains and 1,024 gene losses that have not yet reached fixation within the focal species. Our analyses of gene gains and losses segregating in populations showed that gene copy number variation arose preferentially in subtelomeres and in proximity to transposable elements. Recently lost genes were enriched in virulence factors and secondary metabolite gene clusters. In contrast, recently gained genes encoded mostly secreted protein lacking a conserved domain. We analyzed the frequency spectrum at loci segregating a gene presence–absence polymorphism in four worldwide populations. Recent gene losses showed a significant excess in low-frequency variants compared with genome-wide single nucleotide polymorphism, which is indicative of strong negative selection against gene losses. Recent gene gains were either under weak negative selection or neutral. We found evidence for strong divergent selection among populations at individual loci segregating a gene presence–absence polymorphism. Hence, gene gains and losses likely contributed to local adaptation. Our study shows that microbial eukaryotes harbor extensive copy number variation within populations and that functional differences among recently gained and lost genes led to distinct evolutionary trajectories. PMID:28981698

Copy number variation as a genetic basis for heterotaxy and heterotaxy-spectrum congenital heart defects.

PubMed

Cowan, Jason R; Tariq, Muhammad; Shaw, Chad; Rao, Mitchell; Belmont, John W; Lalani, Seema R; Smolarek, Teresa A; Ware, Stephanie M

2016-12-19

Genomic disorders and rare copy number abnormalities are identified in 15-25% of patients with syndromic conditions, but their prevalence in individuals with isolated birth defects is less clear. A spectrum of congenital heart defects (CHDs) is seen in heterotaxy, a highly heritable and genetically heterogeneous multiple congenital anomaly syndrome resulting from failure to properly establish left-right (L-R) organ asymmetry during early embryonic development. To identify novel genetic causes of heterotaxy, we analysed copy number variants (CNVs) in 225 patients with heterotaxy and heterotaxy-spectrum CHDs using array-based genotyping methods. Clinically relevant CNVs were identified in approximately 20% of patients and encompassed both known and putative heterotaxy genes. Patients were carefully phenotyped, revealing a significant association of abdominal situs inversus with pathogenic or likely pathogenic CNVs, while d-transposition of the great arteries was more frequently associated with common CNVs. Identified cytogenetic abnormalities ranged from large unbalanced translocations to smaller, kilobase-scale CNVs, including a rare, single exon deletion in ZIC3, a gene known to cause X-linked heterotaxy. Morpholino loss-of-function experiments in Xenopus support a role for one of these novel candidates, the platelet isoform of phosphofructokinase-1 (PFKP) in heterotaxy. Collectively, our results confirm a high CNV yield for array-based testing in patients with heterotaxy, and support use of CNV analysis for identification of novel biological processes relevant to human laterality.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

PubMed Central

2013-01-01

Background There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. Results A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. Conclusions We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes. PMID:23758725

The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner

PubMed Central

Vicente, Rebeca L.; Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

2016-01-01

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application. PMID:27977736

Neurophysiological evidence of efference copies to inner speech

PubMed Central

Jack, Bradley N; Pearson, Daniel; Griffiths, Oren; Luque, David; Harris, Anthony WF; Spencer, Kevin M; Le Pelley, Mike E

2017-01-01

Efference copies refer to internal duplicates of movement-producing neural signals. Their primary function is to predict, and often suppress, the sensory consequences of willed movements. Efference copies have been almost exclusively investigated in the context of overt movements. The current electrophysiological study employed a novel design to show that inner speech – the silent production of words in one’s mind – is also associated with an efference copy. Participants produced an inner phoneme at a precisely specified time, at which an audible phoneme was concurrently presented. The production of the inner phoneme resulted in electrophysiological suppression, but only if the content of the inner phoneme matched the content of the audible phoneme. These results demonstrate that inner speech – a purely mental action – is associated with an efference copy with detailed auditory properties. These findings suggest that inner speech may ultimately reflect a special type of overt speech. PMID:29199947

Ovarian Cancer

MedlinePlus

... term use and in large doses. Age when menstruation started and ended. Beginning menstruation at an early age or starting menopause at ... Advertising and sponsorship opportunities Reprint Permissions A single copy of these materials may be reprinted for noncommercial ...

A PLATFORM FOR CONTEXTUAL MOBILE PRIVACY

DTIC Science & Technology

2017-12-01

cen- tralizes all permission management functionality. “Streamline the interface to require less touches to find the information about permissions...APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED STINFO COPY AIR FORCE RESEARCH LABORATORY INFORMATION DIRECTORATE...public, including foreign nationals. Copies may be obtained from the Defense Technical Information Center (DTIC) (http://www.dtic.mil). AFRL

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...

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…

The positioning logic and copy number control of genes in bacteria under stress

NASA Astrophysics Data System (ADS)

Zhang, Qiucen; Austin, Robert; Vyawahare, Saurabh; Lau, Alexandra

2013-03-01

Escherichia coli (E. coli) cells when challenged with sublethal concentrations of the genotoxic antibiotic ciprofloxacin cease to divide and form long filaments which contain multiple bacterial chromosomes. These filaments are individual mesoscopic environmental niches which provide protection for a community of chromosomes (as opposed to cells) under mutagenic stress and can provide an evolutionary fitness advantage within the niche. We use comparative genomic hybridization to show that the mesoscopic niche evolves within 20 minutes of ciprofloxacin exposure via replication of multiple copies of genes expressing ATP dependent transporters. We show that this rapid genomic amplification is done in a time efficient manner via placement of the genes encoding the pumps near the origin of replication on the bacterial chromosome. The de-amplification of multiple copies back to the wild type number is a function of the duration is a function of the ciprofloxacin exposure duration: the longer the exposure, the slower the removal of the multiple copies. The project described was supported by the National Science Foundation and the National Cancer Institute

The complete chloroplast genome of North American ginseng, Panax quinquefolius.

PubMed

Han, Zeng-Jie; Li, Wei; Liu, Yuan; Gao, Li-Zhi

2016-09-01

We report complete nucleotide sequence of the Panax quinquefolius chloroplast genome using next-generation sequencing technology. The genome size is 156 359 bp, including two inverted repeats (IRs) of 52 153 bp, separated by the large single-copy (LSC 86 184 bp) and small single-copy (SSC 18 081 bp) regions. This cp genome encodes 114 unigenes (80 protein-coding genes, four rRNA genes, and 30 tRNA genes), in which 18 are duplicated in the IR regions. Overall GC content of the genome is 38.08%. A phylogenomic analysis of the 10 complete chloroplast genomes from Araliaceae using Daucus carota from Apiaceae as outgroup showed that P. quinquefolius is closely related to the other two members of the genus Panax, P. ginseng and P. notoginseng.

EvolMarkers: a database for mining exon and intron markers for evolution, ecology and conservation studies.

PubMed

Li, Chenhong; Riethoven, Jean-Jack M; Naylor, Gavin J P

2012-09-01

Recent innovations in next-generation sequencing have lowered the cost of genome projects. Nevertheless, sequencing entire genomes for all representatives in a study remains expensive and unnecessary for most studies in ecology, evolution and conservation. It is still more cost-effective and efficient to target and sequence single-copy nuclear gene markers for such studies. Many tools have been developed for identifying nuclear markers, but most of these have focused on particular taxonomic groups. We have built a searchable database, EvolMarkers, for developing single-copy coding sequence (CDS) and exon-primed-intron-crossing (EPIC) markers that is designed to work across a broad range of phylogenetic divergences. The database is made up of single-copy CDS derived from BLAST searches of a variety of metazoan genomes. Users can search the database for different types of markers (CDS or EPIC) that are common to different sets of input species with different divergence characteristics. EvolMarkers can be applied to any taxonomic group for which genome data are available for two or more species. We included 82 genomes in the first version of EvolMarkers and have found the methods to be effective across Placozoa, Cnidaria, Arthropod, Nematoda, Annelida, Mollusca, Echinodermata, Hemichordata, Chordata and plants. We demonstrate the effectiveness of searching for CDS markers within annelids and show how to find potentially useful intronic markers within the lizard Anolis. © 2012 Blackwell Publishing Ltd.

IL-TIF/IL-22: genomic organization and mapping of the human and mouse genes.

PubMed

Dumoutier, L; Van Roost, E; Ameye, G; Michaux, L; Renauld, J C

2000-12-01

IL-TIF is a new cytokine originally identified as a gene induced by IL-9 in murine T lymphocytes, and showing 22% amino acid identity with IL-10. Here, we report the sequence and organization of the mouse and human IL-TIF genes, which both consist of 6 exons spreading over approximately 6 Kb. The IL-TIF gene is a single copy gene in humans, and is located on chromosome 12q15, at 90 Kb from the IFN gamma gene, and at 27 Kb from the AK155 gene, which codes for another IL-10-related cytokine. In the mouse, the IL-TIF gene is located on chromosome 10, also in the same region as the IFN gamma gene. Although it is a single copy gene in BALB/c and DBA/2 mice, the IL-TIF gene is duplicated in other strains such as C57Bl/6, FVB and 129. The two copies, which show 98% nucleotide identity in the coding region, were named IL-TIF alpha and IL-TIF beta. Beside single nucleotide variations, they differ by a 658 nucleotide deletion in IL-TIF beta, including the first non-coding exon and 603 nucleotides from the promoter. A DNA fragment corresponding to this deletion was sufficient to confer IL-9-regulated expression of a luciferase reporter plasmid, suggesting that the IL-TIF beta gene is either differentially regulated, or not expressed at all.

Detection of clinically relevant copy-number variants by exome sequencing in a large cohort of genetic disorders

PubMed Central

Pfundt, Rolph; del Rosario, Marisol; Vissers, Lisenka E.L.M.; Kwint, Michael P.; Janssen, Irene M.; de Leeuw, Nicole; Yntema, Helger G.; Nelen, Marcel R.; Lugtenberg, Dorien; Kamsteeg, Erik-Jan; Wieskamp, Nienke; Stegmann, Alexander P.A.; Stevens, Servi J.C.; Rodenburg, Richard J.T.; Simons, Annet; Mensenkamp, Arjen R.; Rinne, Tuula; Gilissen, Christian; Scheffer, Hans; Veltman, Joris A.; Hehir-Kwa, Jayne Y.

2017-01-01

Purpose: Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10–20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test. Methods: We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting. Results: In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to –5.8% per disorder). Conclusions: This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics. Genet Med advance online publication 27 October 2016 PMID:28574513

The complete chloroplast genome sequence of Dodonaea viscosa: comparative and phylogenetic analyses.

PubMed

Saina, Josphat K; Gichira, Andrew W; Li, Zhi-Zhong; Hu, Guang-Wan; Wang, Qing-Feng; Liao, Kuo

2018-02-01

The plant chloroplast (cp) genome is a highly conserved structure which is beneficial for evolution and systematic research. Currently, numerous complete cp genome sequences have been reported due to high throughput sequencing technology. However, there is no complete chloroplast genome of genus Dodonaea that has been reported before. To better understand the molecular basis of Dodonaea viscosa chloroplast, we used Illumina sequencing technology to sequence its complete genome. The whole length of the cp genome is 159,375 base pairs (bp), with a pair of inverted repeats (IRs) of 27,099 bp separated by a large single copy (LSC) 87,204 bp, and small single copy (SSC) 17,972 bp. The annotation analysis revealed a total of 115 unique genes of which 81 were protein coding, 30 tRNA, and four ribosomal RNA genes. Comparative genome analysis with other closely related Sapindaceae members showed conserved gene order in the inverted and single copy regions. Phylogenetic analysis clustered D. viscosa with other species of Sapindaceae with strong bootstrap support. Finally, a total of 249 SSRs were detected. Moreover, a comparison of the synonymous (Ks) and nonsynonymous (Ka) substitution rates in D. viscosa showed very low values. The availability of cp genome reported here provides a valuable genetic resource for comprehensive further studies in genetic variation, taxonomy and phylogenetic evolution of Sapindaceae family. In addition, SSR markers detected will be used in further phylogeographic and population structure studies of the species in this genus.

CNVinspector: a web-based tool for the interactive evaluation of copy number variations in single patients and in cohorts.

PubMed

Knierim, Ellen; Schwarz, Jana Marie; Schuelke, Markus; Seelow, Dominik

2013-08-01

Many genetic disorders are caused by copy number variations (CNVs) in the human genome. However, the large number of benign CNV polymorphisms makes it difficult to delineate causative variants for a certain disease phenotype. Hence, we set out to create software that accumulates and visualises locus-specific knowledge and enables clinicians to study their own CNVs in the context of known polymorphisms and disease variants. CNV data from healthy cohorts (Database of Genomic Variants) and from disease-related databases (DECIPHER) were integrated into a joint resource. Data are presented in an interactive web-based application that allows inspection, evaluation and filtering of CNVs in single individuals or in entire cohorts. CNVinspector provides simple interfaces to upload CNV data, compare them with own or published control data and visualise the results in graphical interfaces. Beyond choosing control data from different public studies, platforms and methods, dedicated filter options allow the detection of CNVs that are either enriched in patients or depleted in controls. Alternatively, a search can be restricted to those CNVs that appear in individuals of similar clinical phenotype. For each gene of interest within a CNV, we provide a link to NCBI, ENSEMBL and the GeneDistiller search engine to browse for potential disease-associated genes. With its user-friendly handling, the integration of control data and the filtering options, CNVinspector will facilitate the daily work of clinical geneticists and accelerate the delineation of new syndromes and gene functions. CNVinspector is freely accessible under http://www.cnvinspector.org.

The complete chloroplast genome sequence of Epipremnum aureum and its comparative analysis among eight Araceae species

PubMed Central

Han, Limin; Chen, Chen; Wang, Zhezhi

2018-01-01

Epipremnum aureum is an important foliage plant in the Araceae family. In this study, we have sequenced the complete chloroplast genome of E. aureum by using Illumina Hiseq sequencing platforms. This genome is a double-stranded circular DNA sequence of 164,831 bp that contains 35.8% GC. The two inverted repeats (IRa and IRb; 26,606 bp) are spaced by a small single-copy region (22,868 bp) and a large single-copy region (88,751 bp). The chloroplast genome has 131 (113 unique) functional genes, including 86 (79 unique) protein-coding genes, 37 (30 unique) tRNA genes, and eight (four unique) rRNA genes. Tandem repeats comprise the majority of the 43 long repetitive sequences. In addition, 111 simple sequence repeats are present, with mononucleotides being the most common type and di- and tetranucleotides being infrequent events. Positive selection pressure on rps12 in the E. aureum chloroplast has been demonstrated via synonymous and nonsynonymous substitution rates and selection pressure sites analyses. Ycf15 and infA are pseudogenes in this species. We constructed a Maximum Likelihood phylogenetic tree based on the complete chloroplast genomes of 38 species from 13 families. Those results strongly indicated that E. aureum is positioned as the sister of Colocasia esculenta within the Araceae family. This work may provide information for further study of the molecular phylogenetic relationships within Araceae, as well as molecular markers and breeding novel varieties by chloroplast genetic-transformation of E. aureum in particular. PMID:29529038

Identification of Rare, Single-Nucleotide Mutations in NDE1 and Their Contributions to Schizophrenia Susceptibility

PubMed Central

Kimura, Hiroki; Tsuboi, Daisuke; Wang, Chenyao; Kushima, Itaru; Koide, Takayoshi; Ikeda, Masashi; Iwayama, Yoshimi; Toyota, Tomoko; Yamamoto, Noriko; Kunimoto, Shohko; Nakamura, Yukako; Yoshimi, Akira; Banno, Masahiro; Xing, Jingrui; Takasaki, Yuto; Yoshida, Mami; Aleksic, Branko; Uno, Yota; Okada, Takashi; Iidaka, Tetsuya; Inada, Toshiya; Suzuki, Michio; Ujike, Hiroshi; Kunugi, Hiroshi; Kato, Tadafumi; Yoshikawa, Takeo; Iwata, Nakao; Kaibuchi, Kozo; Ozaki, Norio

2015-01-01

Background: Nuclear distribution E homolog 1 (NDE1), located within chromosome 16p13.11, plays an essential role in microtubule organization, mitosis, and neuronal migration and has been suggested by several studies of rare copy number variants to be a promising schizophrenia (SCZ) candidate gene. Recently, increasing attention has been paid to rare single-nucleotide variants (SNVs) discovered by deep sequencing of candidate genes, because such SNVs may have large effect sizes and their functional analysis may clarify etiopathology. Methods and Results: We conducted mutation screening of NDE1 coding exons using 433 SCZ and 145 pervasive developmental disorders samples in order to identify rare single nucleotide variants with a minor allele frequency ≤5%. We then performed genetic association analysis using a large number of unrelated individuals (3554 SCZ, 1041 bipolar disorder [BD], and 4746 controls). Among the discovered novel rare variants, we detected significant associations between SCZ and S214F (P = .039), and between BD and R234C (P = .032). Furthermore, functional assays showed that S214F affected axonal outgrowth and the interaction between NDE1 and YWHAE (14-3-3 epsilon; a neurodevelopmental regulator). Conclusions: This study strengthens the evidence for association between rare variants within NDE1 and SCZ, and may shed light into the molecular mechanisms underlying this severe psychiatric disorder. PMID:25332407

Genetic Regulation of Charged Particle Mutagenesis in Human Cells

NASA Technical Reports Server (NTRS)

Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

1999-01-01

Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

Effect of Repeat Copy Number on Variable-Number Tandem Repeat Mutations in Escherichia coli O157:H7

PubMed Central

Vogler, Amy J.; Keys, Christine; Nemoto, Yoshimi; Colman, Rebecca E.; Jay, Zack; Keim, Paul

2006-01-01

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 × 10−4 mutations/generation and a combined 28-locus rate of 6.4 × 10−4 mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2 = 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2 = 0.833, P < 0.0001) or excluded (r2 = 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data. PMID:16740932

Cognitive predictors of copying and drawing from memory of the Rey-Osterrieth complex figure in 7- to 10-year-old children.

PubMed

Senese, Vincenzo Paolo; De Lucia, Natascia; Conson, Massimiliano

2015-01-01

Cognitive models of drawing are mainly based on assessment of copying performance of adults, whereas only a few studies have verified these models in young children. Moreover, developmental investigations have only rarely performed a systematic examination of the contribution of perceptual and representational visuo-spatial processes to copying and drawing from memory. In this study we investigated the role of visual perception and mental representation in both copying and drawing from memory skills in a sample of 227 typically developing children (53% females) aged 7-10 years. Participants underwent a neuropsychological assessment and the Rey-Osterrieth Complex Figure (ROCF). The fit and invariance of the predictive model considering visuo-spatial abilities, working memory, and executive functions were tested by means of hierarchical regressions and path analysis. Results showed that, in a gender invariant way, visual perception abilities and spatial mental representation had a direct effect on copying performance, whereas copying performance was the only specific predictor for drawing from memory. These effects were independent from age and socioeconomic status, and showed that cognitive models of drawing built up for adults could be considered for predicting copying and drawing from memory in children.

Mineral Resource Team 2010 Activities Summary

DTIC Science & Technology

2011-01-29

survey Afghanistan. Many of the resulting reports existed only at the Afghan Geological Survey in single paper copies - and the majority of the documents...Deposit: Copper ores associated with intrusive igneous rocks, and the fluids that accompany them, during magmatic emplacement and crystallization ...important commodities for agriculture and commercial industrial production. Thus, from a single massif, the Khanneshin carbonatite could be a source

37 CFR 201.20 - Methods of affixation and positions of the copyright notice on various types of works.

Code of Federal Regulations, 2014 CFR

2014-07-01

... music. (e) Single-leaf works. In the case of single-leaf works, a notice reproduced on the copies... the first page of music of the contribution; (4) As an alternative to placing the notice on one of the... optical enlargement (such as film, filmstrips, slide films, and works published in any variety of...

The singular behavior of a β-type semi-synthetic two branched polypeptide: three-dimensional structure and mode of action.

PubMed

Manzo, Giorgia; Serra, Ilaria; Pira, Alessandro; Pintus, Manuela; Ceccarelli, Matteo; Casu, Mariano; Rinaldi, Andrea C; Scorciapino, Mariano Andrea

2016-11-16

Dendrimeric peptides make a versatile group of bioactive peptidomimetics and a potential new class of antimicrobial agents to tackle the pressing threat of multi-drug resistant pathogens. These are branched supramolecular assemblies where multiple copies of the bioactive unit are linked to a central core. Beyond their antimicrobial activity, dendrimeric peptides could also be designed to functionalize the surface of nanoparticles or materials for other medical uses. Despite these properties, however, little is known about the structure-function relationship of such compounds, which is key to unveil the fundamental physico-chemical parameters and design analogues with desired attributes. To close this gap, we focused on a semi-synthetic, two-branched peptide, SB056, endowed with remarkable activity against both Gram-positive and Gram-negative bacteria and limited cytotoxicity. SB056 can be considered the smallest prototypical dendrimeric peptide, with the core restricted to a single lysine residue and only two copies of the same highly cationic 10-mer polypeptide; an octanamide tail is present at the C-terminus. Combining NMR and Molecular Dynamics simulations, we have determined the 3D structure of two analogues. Fluorescence spectroscopy was applied to investigate the water-bilayer partition in the presence of vesicles of variable charge. Vesicle leakage assays were also performed and the experimental data were analyzed by applying an iterative Monte Carlo scheme to estimate the minimum number of bound peptides needed to achieve the release. We unveiled a singular beta hairpin-type structure determined by the peptide chains only, with the octanamide tail available for further functionalization to add new potential properties without affecting the structure.

Non-biased and efficient global amplification of a single-cell cDNA library

PubMed Central

Huang, Huan; Goto, Mari; Tsunoda, Hiroyuki; Sun, Lizhou; Taniguchi, Kiyomi; Matsunaga, Hiroko; Kambara, Hideki

2014-01-01

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively. PMID:24141095

Genome stability of programmed stem cell products.

PubMed

Martin, Ulrich

2017-10-01

Inherited and acquired genomic abnormalities are known to cause genetic diseases and contribute to cancer formation. Recent studies demonstrated a substantial mutational load in mouse and human embryonic and induced pluripotent stem cells (ESCs and iPSCs). Single nucleotide variants, copy number variations, and larger chromosomal abnormalities may influence the differentiation capacity of pluripotent stem cells and the functionality of their derivatives in disease modeling and drug screening, and are considered a serious risk for cellular therapies based on ESC or iPSC derivatives. This review discusses the types and origins of different genetic abnormalities in pluripotent stem cells, methods for their detection, and the mechanisms of development and enrichment during reprogramming and culture expansion. Copyright © 2017 Elsevier B.V. All rights reserved.

Hormone Replacement Therapy: Can It Cause Vaginal Bleeding?

MedlinePlus

... hormone therapy for menopause symptoms, and my monthly menstrual periods have returned. Is this normal? Answers from ... Advertising and sponsorship opportunities Reprint Permissions A single copy of these materials may be reprinted for noncommercial ...

Older Americans 2016: Key Indicators of Well-Being

MedlinePlus

... Contacts FAQ Search X Home | Data Data Current Report Older Americans 2016: Key Indicators of Well-Being ... Figures PPTX How to obtain a hardcopy of Report Single copies of this report are available at ...

Quantitative imaging of single mRNA splice variants in living cells

NASA Astrophysics Data System (ADS)

Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

2014-06-01

Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

Three kinds of particles on a single rationally parameterized world line

NASA Astrophysics Data System (ADS)

Kassandrov, V. V.; Markova, N. V.

2016-10-01

We consider the light cone (`retardation') equation (LCE) of an inertially moving observer and a single worldline parameterized by arbitrary rational functions. Then a set of apparent copies, R- or C-particles, defined by the (real or complex conjugate) roots of the LCE will be detected by the observer. For any rational worldline the collective R-C dynamics is manifestly Lorentz-invariant and conservative; the latter property follows directly from the structure of Vieta formulas for the LCE roots. In particular, two Lorentz invariants, the square of total 4-momentum and total rest mass, are distinct and both integer-valued. Asymptotically, at large values of the observer's proper time, one distinguishes three types of the LCE roots and associated R-C particles, with specific locations and evolutions; each of three kinds of particles can assemble into compact large groups - clusters. Throughout the paper, we make no use of differential equations of motion, field equations, etc.: the collective R-C dynamics is purely algebraic

Counting numbers of synaptic proteins: absolute quantification and single molecule imaging techniques

PubMed Central

Patrizio, Angela; Specht, Christian G.

2016-01-01

Abstract. The ability to count molecules is essential to elucidating cellular mechanisms, as these often depend on the absolute numbers and concentrations of molecules within specific compartments. Such is the case at chemical synapses, where the transmission of information from presynaptic to postsynaptic terminals requires complex interactions between small sets of molecules. Be it the subunit stoichiometry specifying neurotransmitter receptor properties, the copy numbers of scaffold proteins setting the limit of receptor accumulation at synapses, or protein packing densities shaping the molecular organization and plasticity of the postsynaptic density, all of these depend on exact quantities of components. A variety of proteomic, electrophysiological, and quantitative imaging techniques have yielded insights into the molecular composition of synaptic complexes. In this review, we compare the different quantitative approaches and consider the potential of single molecule imaging techniques for the quantification of synaptic components. We also discuss specific neurobiological data to contextualize the obtained numbers and to explain how they aid our understanding of synaptic structure and function. PMID:27335891

Counting numbers of synaptic proteins: absolute quantification and single molecule imaging techniques.

PubMed

Patrizio, Angela; Specht, Christian G

2016-10-01

The ability to count molecules is essential to elucidating cellular mechanisms, as these often depend on the absolute numbers and concentrations of molecules within specific compartments. Such is the case at chemical synapses, where the transmission of information from presynaptic to postsynaptic terminals requires complex interactions between small sets of molecules. Be it the subunit stoichiometry specifying neurotransmitter receptor properties, the copy numbers of scaffold proteins setting the limit of receptor accumulation at synapses, or protein packing densities shaping the molecular organization and plasticity of the postsynaptic density, all of these depend on exact quantities of components. A variety of proteomic, electrophysiological, and quantitative imaging techniques have yielded insights into the molecular composition of synaptic complexes. In this review, we compare the different quantitative approaches and consider the potential of single molecule imaging techniques for the quantification of synaptic components. We also discuss specific neurobiological data to contextualize the obtained numbers and to explain how they aid our understanding of synaptic structure and function.

Drosophila germ granules are structured and contain homotypic mRNA clusters

PubMed Central

Trcek, Tatjana; Grosch, Markus; York, Andrew; Shroff, Hari; Lionnet, Timothée; Lehmann, Ruth

2015-01-01

Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. Here we show, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into ‘homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules. PMID:26242323

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.

Expression of Duplicate msa Genes in the Salmonid Pathogen Renibacterium salmoninarum

PubMed Central

Rhodes, Linda D.; Coady, Alison M.; Strom, Mark S.

2002-01-01

Renibacterium salmoninarum is a gram-positive bacterium responsible for bacterial kidney disease of salmon and trout. R. salmoninarum has two identical copies of the gene encoding major soluble antigen (MSA), an immunodominant, extracellular protein. To determine whether one or both copies of msa are expressed, reporter plasmids encoding a fusion of MSA and green fluorescent protein controlled by 0.6 kb of promoter region from msa1 or msa2 were constructed and introduced into R. salmoninarum. Single copies of the reporter plasmids integrated into the chromosome by homologous recombination. Expression of mRNA and protein from the integrated plasmids was detected, and transformed cells were fluorescent, demonstrating that both msa1 and msa2 are expressed under in vitro conditions. This is the first report of successful transformation and homologous recombination in R. salmoninarum. PMID:12406741

Verifiable Measurement-Only Blind Quantum Computing with Stabilizer Testing.

PubMed

Hayashi, Masahito; Morimae, Tomoyuki

2015-11-27

We introduce a simple protocol for verifiable measurement-only blind quantum computing. Alice, a client, can perform only single-qubit measurements, whereas Bob, a server, can generate and store entangled many-qubit states. Bob generates copies of a graph state, which is a universal resource state for measurement-based quantum computing, and sends Alice each qubit of them one by one. Alice adaptively measures each qubit according to her program. If Bob is honest, he generates the correct graph state, and, therefore, Alice can obtain the correct computation result. Regarding the security, whatever Bob does, Bob cannot get any information about Alice's computation because of the no-signaling principle. Furthermore, malicious Bob does not necessarily send the copies of the correct graph state, but Alice can check the correctness of Bob's state by directly verifying the stabilizers of some copies.

Experimental quantum forgery of quantum optical money

NASA Astrophysics Data System (ADS)

Bartkiewicz, Karol; Černoch, Antonín; Chimczak, Grzegorz; Lemr, Karel; Miranowicz, Adam; Nori, Franco

2017-03-01

Unknown quantum information cannot be perfectly copied (cloned). This statement is the bedrock of quantum technologies and quantum cryptography, including the seminal scheme of Wiesner's quantum money, which was the first quantum-cryptographic proposal. Surprisingly, to our knowledge, quantum money has not been tested experimentally yet. Here, we experimentally revisit the Wiesner idea, assuming a banknote to be an image encoded in the polarization states of single photons. We demonstrate that it is possible to use quantum states to prepare a banknote that cannot be ideally copied without making the owner aware of only unauthorized actions. We provide the security conditions for quantum money by investigating the physically-achievable limits on the fidelity of 1-to-2 copying of arbitrary sequences of qubits. These results can be applied as a security measure in quantum digital right management.

Verifiable Measurement-Only Blind Quantum Computing with Stabilizer Testing

NASA Astrophysics Data System (ADS)

Hayashi, Masahito; Morimae, Tomoyuki

2015-11-01

We introduce a simple protocol for verifiable measurement-only blind quantum computing. Alice, a client, can perform only single-qubit measurements, whereas Bob, a server, can generate and store entangled many-qubit states. Bob generates copies of a graph state, which is a universal resource state for measurement-based quantum computing, and sends Alice each qubit of them one by one. Alice adaptively measures each qubit according to her program. If Bob is honest, he generates the correct graph state, and, therefore, Alice can obtain the correct computation result. Regarding the security, whatever Bob does, Bob cannot get any information about Alice's computation because of the no-signaling principle. Furthermore, malicious Bob does not necessarily send the copies of the correct graph state, but Alice can check the correctness of Bob's state by directly verifying the stabilizers of some copies.

Dead Element Replicating: Degenerate R2 Element Replication and rDNA Genomic Turnover in the Bacillus rossius Stick Insect (Insecta: Phasmida)

PubMed Central

Martoni, Francesco; Eickbush, Danna G.; Scavariello, Claudia; Luchetti, Andrea; Mantovani, Barbara

2015-01-01

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5’ end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution. PMID:25799008

Copy number variants calling for single cell sequencing data by multi-constrained optimization.

PubMed

Xu, Bo; Cai, Hongmin; Zhang, Changsheng; Yang, Xi; Han, Guoqiang

2016-08-01

Variations in DNA copy number carry important information on genome evolution and regulation of DNA replication in cancer cells. The rapid development of single-cell sequencing technology allows one to explore gene expression heterogeneity among single-cells, thus providing important cancer cell evolution information. Single-cell DNA/RNA sequencing data usually have low genome coverage, which requires an extra step of amplification to accumulate enough samples. However, such amplification will introduce large bias and makes bioinformatics analysis challenging. Accurately modeling the distribution of sequencing data and effectively suppressing the bias influence is the key to success variations analysis. Recent advances demonstrate the technical noises by amplification are more likely to follow negative binomial distribution, a special case of Poisson distribution. Thus, we tackle the problem CNV detection by formulating it into a quadratic optimization problem involving two constraints, in which the underling signals are corrupted by Poisson distributed noises. By imposing the constraints of sparsity and smoothness, the reconstructed read depth signals from single-cell sequencing data are anticipated to fit the CNVs patterns more accurately. An efficient numerical solution based on the classical alternating direction minimization method (ADMM) is tailored to solve the proposed model. We demonstrate the advantages of the proposed method using both synthetic and empirical single-cell sequencing data. Our experimental results demonstrate that the proposed method achieves excellent performance and high promise of success with single-cell sequencing data. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Single-cell analysis of transcription kinetics across the cell cycle

PubMed Central

Skinner, Samuel O; Xu, Heng; Nagarkar-Jaiswal, Sonal; Freire, Pablo R; Zwaka, Thomas P; Golding, Ido

2016-01-01

Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation. DOI: http://dx.doi.org/10.7554/eLife.12175.001 PMID:26824388

Children and Television Violence--a Survey of the Literature

ERIC Educational Resources Information Center

Greagg, L.

1977-01-01

Describes the findings and some of the difficulties in researching the effects of television on children. Available from: Australian College of Education, 916 Swanston Street, Carlton, Victoria 3053, Australia, $2.50 single copy. (Author/MLF)

Changing Patterns in Illinois' School Tort Immunity.

ERIC Educational Resources Information Center

Gardner, Shelley B.

1979-01-01

It appears that Illinois law no longer grants full parental immunity to school districts and their employees. Available from IIT/Chicago-Kent College of Law, 77 South Wacker Drive, Chicago, IL 60606; single copies $5.00. (Author)

Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus.

PubMed

Yang, G S; Banks, K G; Bonaguro, R J; Wilson, G; Dreolini, L; de Leeuw, C N; Liu, L; Swanson, D J; Goldowitz, D; Holt, R A; Simpson, E M

2009-03-01

We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1(b-m3) locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.

VCS: Tool for Visualizing Copy Number Variation and Single Nucleotide Polymorphism.

PubMed

Kim, HyoYoung; Sung, Samsun; Cho, Seoae; Kim, Tae-Hun; Seo, Kangseok; Kim, Heebal

2014-12-01

Copy number variation (CNV) or single nucleotide phlyorphism (SNP) is useful genetic resource to aid in understanding complex phenotypes or deseases susceptibility. Although thousands of CNVs and SNPs are currently avaliable in the public databases, they are somewhat difficult to use for analyses without visualization tools. We developed a web-based tool called the VCS (visualization of CNV or SNP) to visualize the CNV or SNP detected. The VCS tool can assist to easily interpret a biological meaning from the numerical value of CNV and SNP. The VCS provides six visualization tools: i) the enrichment of genome contents in CNV; ii) the physical distribution of CNV or SNP on chromosomes; iii) the distribution of log2 ratio of CNVs with criteria of interested; iv) the number of CNV or SNP per binning unit; v) the distribution of homozygosity of SNP genotype; and vi) cytomap of genes within CNV or SNP region.

Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

PubMed

Beiter, Thomas; Zimmermann, Martina; Fragasso, Annunziata; Armeanu, Sorin; Lauer, Ulrich M; Bitzer, Michael; Su, Hua; Young, William L; Niess, Andreas M; Simon, Perikles

2008-01-01

So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

The complete chloroplast genome of an irreplaceable dietary and model crop, foxtail millet (Setaria italica).

PubMed

Wang, Shuo; Gao, Li-Zhi

2016-11-01

The complete chloroplast genome sequence of foxtail millet (Setaria italica), an important food and fodder crop in the family Poaceae, is first reported in this study. The genome consists of 1 35 516 bp containing a pair of inverted repeats (IRs) of 21 804 bp separated by a large single-copy (LSC) region and a small single-copy (SSC) region of 79 896 bp and 12 012 bp, respectively. Coding sequences constitute 58.8% of the genome harboring 111 unique genes, 71 of which are protein-coding genes, 4 are rRNA genes, and 36 are tRNA genes. Phylogenetic analysis indicated foxtail millet clustered with Panicum virgatum and Echinochloa crus-galli belonging to the tribe Paniceae of the subfamily Panicoideae. This newly determined chloroplast genome will provide valuable information for the future breeding programs of valuable cereal crops in the family Poaceae.

FlaME: Flash Molecular Editor - a 2D structure input tool for the web.

PubMed

Dallakian, Pavel; Haider, Norbert

2011-02-01

So far, there have been no Flash-based web tools available for chemical structure input. The authors herein present a feasibility study, aiming at the development of a compact and easy-to-use 2D structure editor, using Adobe's Flash technology and its programming language, ActionScript. As a reference model application from the Java world, we selected the Java Molecular Editor (JME). In this feasibility study, we made an attempt to realize a subset of JME's functionality in the Flash Molecular Editor (FlaME) utility. These basic capabilities are: structure input, editing and depiction of single molecules, data import and export in molfile format. The result of molecular diagram sketching in FlaME is accessible in V2000 molfile format. By integrating the molecular editor into a web page, its communication with the HTML elements on this page is established using the two JavaScript functions, getMol() and setMol(). In addition, structures can be copied to the system clipboard. A first attempt was made to create a compact single-file application for 2D molecular structure input/editing on the web, based on Flash technology. With the application examples presented in this article, it could be demonstrated that the Flash methods are principally well-suited to provide the requisite communication between the Flash object (application) and the HTML elements on a web page, using JavaScript functions.

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.

Complete mitochondrial genome of endangered Yellow-shouldered Amazon (Amazona barbadensis): two control region copies in parrot species of the Amazona genus.

PubMed

Urantowka, Adam Dawid; Hajduk, Kacper; Kosowska, Barbara

2013-08-01

Amazona barbadensis is an endangered species of parrot living in northern coastal Venezuela and in several Caribbean islands. In this study, we sequenced full mitochondrial genome of the considered species. The total length of the mitogenome was 18,983 bp and contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, duplicated control region, and degenerate copies of ND6 and tRNA (Glu) genes. High degree of identity between two copies of control region suggests their coincident evolution and functionality. Comparative analysis of both the control region sequences from four Amazona species revealed their 89.1% identity over a region of 1300 bp and indicates the presence of distinctive parts of two control region copies.

Complete chloroplast genome sequences of Drimys, Liriodendron, andPiper: Implications for the phylogeny of magnoliids and the evolution ofGC content

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

Zhengqiu, C.; Penaflor, C.; Kuehl, J.V.

2006-06-01

The magnoliids represent the largest basal angiosperm clade with four orders, 19 families and 8,500 species. Although several recent angiosperm molecular phylogenies have supported the monophyly of magnoliids and suggested relationships among the orders, the limited number of genes examined resulted in only weak support, and these issues remain controversial. Furthermore, considerable incongruence has resulted in phylogenies supporting three different sets of relationships among magnoliids and the two large angiosperm clades, monocots and eudicots. This is one of the most important remaining issues concerning relationships among basal angiosperms. We sequenced the chloroplast genomes of three magnoliids, Drimys (Canellales), Liriodendron (Magnoliales),more » and Piper (Piperales), and used these data in combination with 32 other completed angiosperm chloroplast genomes to assess phylogenetic relationships among magnoliids. The Drimys and Piper chloroplast genomes are nearly identical in size at 160,606 and 160,624 bp, respectively. The genomes include a pair of inverted repeats of 26,649 bp (Drimys) and 27,039 (Piper), separated by a small single copy region of 18,621 (Drimys) and 18,878 (Piper) and a large single copy region of 88,685 bp (Drimys) and 87,666 bp (Piper). The gene order of both taxa is nearly identical to many other unrearranged angiosperm chloroplast genomes, including Calycanthus, the other published magnoliid genome. Comparisons of angiosperm chloroplast genomes indicate that GC content is not uniformly distributed across the genome. Overall GC content ranges from 34-39%, and coding regions have a substantially higher GC content than non-coding regions (both intergenic spacers and introns). Among protein-coding genes, GC content varies by codon position with 1st codon > 2nd codon > 3rd codon, and it varies by functional group with photosynthetic genes having the highest percentage and NADH genes the lowest. Across the genome, GC content is highest in the inverted repeat due to the presence of rRNA genes and lowest in the small single copy region where most NADH genes are located. Phylogenetic analyses using maximum parsimony and maximum likelihood methods were performed on DNA sequences of 61 protein-coding genes. Trees from both analyses provided strong support for the monophyly of magnoliids and two strongly supported groups were identified, the Canellales/Piperales and the Laurales/Magnoliales. The phylogenies also provided moderate to strong support for the basal position of Amborella, and a sister relationship of magnoliids to a clade that includes monocots and eudicots. The complete sequences of three magnoliid chloroplast genomes provide new data from the largest basal angiosperm clade. Evolutionary comparisons of these new genome sequences, combined with other published angiosperm genome, confirm that GC content is unevenly distributed across the genome by location, codon position, and functional group. Furthermore, phylogenetic analyses provide the strongest support so far for the hypothesis that the magnoliids are sister to a large clade that includes both monocots and eudicots.« less

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

Explicit formulae for Yang-Mills-Einstein amplitudes from the double copy

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

Chiodaroli, Marco; Günaydin, Murat; Johansson, Henrik

Using the double-copy construction of Yang-Mills-Einstein theories formulated in our earlier work, we obtain compact presentations for single-trace Yang-Mills-Einstein tree amplitudes with up to five external gravitons and an arbitrary number of gluons. These are written as linear combinations of color-ordered Yang-Mills trees, where the coefficients are given by color/kinematics-satisfying numerators in a Yang-Mills + φ 3 theory. The construction outlined in this paper holds in general dimension and extends straightforwardly to supergravity theories. For one, two, and three external gravitons, our expressions give identical or simpler presentations of amplitudes already constructed through string-theory considerations or the scattering equations formalism.more » Our results are based on color/kinematics duality and gauge invariance, and strongly hint at a recursive structure underlying the single-trace amplitudes with an arbitrary number of gravitons. We also present explicit expressions for all-loop single-graviton Einstein-Yang-Mills amplitudes in terms of Yang-Mills amplitudes and, through gauge invariance, derive new all-loop amplitude relations for Yang-Mills theory.« less

Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.

PubMed

Liu, Hui; Dong, Peng; Ioannou, Maria S; Li, Li; Shea, Jamien; Pasolli, H Amalia; Grimm, Jonathan B; Rivlin, Patricia K; Lavis, Luke D; Koyama, Minoru; Liu, Zhe

2018-01-09

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of ∼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially restricted subnuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

Explicit formulae for Yang-Mills-Einstein amplitudes from the double copy

DOE PAGES

Chiodaroli, Marco; Günaydin, Murat; Johansson, Henrik; ...

2017-07-03

Using the double-copy construction of Yang-Mills-Einstein theories formulated in our earlier work, we obtain compact presentations for single-trace Yang-Mills-Einstein tree amplitudes with up to five external gravitons and an arbitrary number of gluons. These are written as linear combinations of color-ordered Yang-Mills trees, where the coefficients are given by color/kinematics-satisfying numerators in a Yang-Mills + φ 3 theory. The construction outlined in this paper holds in general dimension and extends straightforwardly to supergravity theories. For one, two, and three external gravitons, our expressions give identical or simpler presentations of amplitudes already constructed through string-theory considerations or the scattering equations formalism.more » Our results are based on color/kinematics duality and gauge invariance, and strongly hint at a recursive structure underlying the single-trace amplitudes with an arbitrary number of gravitons. We also present explicit expressions for all-loop single-graviton Einstein-Yang-Mills amplitudes in terms of Yang-Mills amplitudes and, through gauge invariance, derive new all-loop amplitude relations for Yang-Mills theory.« less

One-loop transition amplitudes in the D1D5 CFT

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

Carson, Zaq; Hampton, Shaun; Mathur, Samir D.

We consider the issue of thermalization in the D1D5 CFT. Thermalization is expected to correspond to the formation of a black hole in the dual gravity theory. We start from the orbifold point, where the theory is essentially free, and does not thermalize. In earlier work it was noted that there was no clear thermalization effect when the theory was deformed of the orbifold point to first order in the relevant twist perturbation. In this paper we consider the deformation to second order in the twist, where we do find effects that can cause thermalization of an initial perturbation. Wemore » consider a 1-loop process where two untwisted copies of the CFT are twisted to one copy and then again untwisted to two copies. We start with a single oscillator excitation on the initial CFT, and compute the effect of the two twists on this state. We find simple approximate expressions for the Bogoliubov coeffcients and the behavior of the single oscillator excitation in the continuum limit, where the mode numbers involved are taken to be much larger than unity. We also prove a number of useful relationships valid for processes with an arbitrary number of twist insertions.« less

One-loop transition amplitudes in the D1D5 CFT

DOE PAGES

Carson, Zaq; Hampton, Shaun; Mathur, Samir D.

2017-01-02

We consider the issue of thermalization in the D1D5 CFT. Thermalization is expected to correspond to the formation of a black hole in the dual gravity theory. We start from the orbifold point, where the theory is essentially free, and does not thermalize. In earlier work it was noted that there was no clear thermalization effect when the theory was deformed of the orbifold point to first order in the relevant twist perturbation. In this paper we consider the deformation to second order in the twist, where we do find effects that can cause thermalization of an initial perturbation. Wemore » consider a 1-loop process where two untwisted copies of the CFT are twisted to one copy and then again untwisted to two copies. We start with a single oscillator excitation on the initial CFT, and compute the effect of the two twists on this state. We find simple approximate expressions for the Bogoliubov coeffcients and the behavior of the single oscillator excitation in the continuum limit, where the mode numbers involved are taken to be much larger than unity. We also prove a number of useful relationships valid for processes with an arbitrary number of twist insertions.« less

HPV circulating tumor DNA to monitor the efficacy of anti-PD-1 therapy in metastatic squamous cell carcinoma of the anal canal: A case report.

PubMed

Cabel, Luc; Bidard, François-Clément; Servois, Vincent; Cacheux, Wulfran; Mariani, Pascale; Romano, Emanuela; Minsat, Mathieu; Bieche, Ivan; Farkhondeh, Fereshteh; Jeannot, Emmanuelle; Buecher, Bruno

2017-10-15

Squamous cell carcinoma of the anal canal (SCCA) is a rare HPV-associated cancer with limited sensitivity to standard chemotherapy. In a phase 2 study, nivolumab, an anti PD-1 immune checkpoint inhibitor, demonstrated significant efficacy as single-agent therapy in metastatic SCCA patients. Nevertheless, imaging assessment by standard RECIST criteria of the efficacy of immune therapy can be difficult in some patients due to tumor immune cell infiltration, and biomarkers of treatment efficacy are needed. We have previously developed a quantitative droplet digital PCR (ddPCR) technique to detect HPV circulating tumor DNA (HPV ctDNA), with excellent sensitivity and specificity. Here, we report, for the first time, the kinetics of HPV ctDNA during therapy in a patient with metastatic SCCA, who obtained sustained partial response to single-agent nivolumab. We observed an early and very significant decrease of HPV ctDNA during therapy from the baseline level of 3713 copies/ml plasma to 564 copies/ml plasma at 4 weeks, and 156 copies/ml at 6 weeks, followed by a plateau. This observation provides proof-of-concept that HPV ctDNA can be used as a noninvasive early dynamic biomarker to monitor the efficacy of new immunotherapy agents. © 2017 UICC.

Molecular counting of membrane receptor subunits with single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Krüger, Carmen; Fricke, Franziska; Karathanasis, Christos; Dietz, Marina S.; Malkusch, Sebastian; Hummer, Gerhard; Heilemann, Mike

2017-02-01

We report on quantitative single-molecule localization microscopy, a method that next to super-resolved images of cellular structures provides information on protein copy numbers in protein clusters. This approach is based on the analysis of blinking cycles of single fluorophores, and on a model-free description of the distribution of the number of blinking events. We describe the experimental and analytical procedures, present cellular data of plasma membrane proteins and discuss the applicability of this method.

Anti-CDR3 Therapy for B-Cell Malignancies

DTIC Science & Technology

2014-10-01

are happy to summarize substantial completion of Task 3. Fig 3 shows a schematic of an M13 phage displaying a single chain Fv. The Tomlinson I phage...the M13 bacteriophage displaying a single chain Fv fused with one copy of the pIII protein. HC LC T A G CDR3 CDR2 CDR1 CDR2 CDR3 CDR1 p

Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

PubMed Central

Carver-Brown, Rachel K.; Reis, Arthur H.; Rice, Lisa M.; Czajka, John W.; Wangh, Lawrence J.

2012-01-01

Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis. PMID:23326668

A mathematical model of single target site location by Brownian movement in subcellular compartments.

PubMed

Kuthan, Hartmut

2003-03-07

The location of distinct sites is mandatory for many cellular processes. In the subcompartments of the cell nucleus, only very small numbers of diffusing macromolecules and specific target sites of some types may be present. In this case, we are faced with the Brownian movement of individual macromolecules and their "random search" for single/few specific target sites, rather than bulk-averaged diffusion and multiple sites. In this article, I consider the location of a distant central target site, e.g. a globular protein, by individual macromolecules executing unbiased (i.e. drift-free) random walks in a spherical compartment. For this walk-and-capture model, the closed-form analytic solution of the first passage time probability density function (p.d.f.) has been obtained as well as the first and second moment. In the limit of a large ratio of the radii of the spherical diffusion space and central target, well-known relations for the variance and the first two moments for the exponential p.d.f. were found to hold with high accuracy. These calculations reinforce earlier numerical results and Monte Carlo simulations. A major implication derivable from the model is that non-directed random movement is an effective means for locating single sites in submicron-sized compartments, even when the diffusion coefficients are comparatively small and the diffusing species are present in one copy only. These theoretical conclusions are underscored numerically for effective diffusion constants ranging from 0.5 to 10.0 microm(2) s(-1), which have been reported for a couple of nuclear proteins in their physiological environment. Spherical compartments of submicron size are, for example, the Cajal bodies (size: 0.1-1.0 microm), which are present in 1-5 copies in the cell nucleus. Within a small Cajal body of radius 0.1 microm a single diffusing protein molecule (with D=0.5 microm(2) s(-1)) would encounter a medium-sized protein of radius 2.5 nm within 1 s with a probability near certainty (p=0.98).

Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response.

PubMed

Xue, Qiong; Bettini, Emily; Paczkowski, Patrick; Ng, Colin; Kaiser, Alaina; McConnell, Timothy; Kodrasi, Olja; Quigley, Máire F; Heath, James; Fan, Rong; Mackay, Sean; Dudley, Mark E; Kassim, Sadik H; Zhou, Jing

2017-11-21

It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods. We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors. Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.

Fiber-optics implementation of an asymmetric phase-covariant quantum cloner.

PubMed

Bartůsková, Lucie; Dusek, Miloslav; Cernoch, Antonín; Soubusta, Jan; Fiurásek, Jaromír

2007-09-21

We present the experimental realization of optimal symmetric and asymmetric phase-covariant 1-->2 cloning of qubit states using fiber optics. The state of each qubit is encoded into a single photon which can propagate through two optical fibers. The operation of our device is based on one- and two-photon interference. We have demonstrated the creation of two copies for a wide range of qubit states from the equator of the Bloch sphere. The measured fidelities of both copies are close to the theoretical values and they surpass the theoretical maximum obtainable with the universal cloner.

Photographic copy of circa 1935, black and white, 10” x ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Photographic copy of circa 1935, black and white, 10” x 14” photograph. Located in box 14 of Huey P. Long Bridge folder (Greater New Orleans), at the National Museum of American History, Smithsonian Institution, Archives Center, Work and Industry Division, Washington, D.C. Original Photographer unknown. PHOTOGRAPH OF COMPLETED BRIDGE TAKEN IN CENTER LOOKING SOUTHEAST AT EAST BANK SINGLE THROUGH TRUSS SPAN SHOWING PERIOD AUTOMOBILE AND WOMAN ON BICYCLE WEST BOUND. - Huey P. Long Bridge, Spanning Mississippi River approximately midway between nine & twelve mile points upstream from & west of New Orleans, Jefferson, Jefferson Parish, LA

[Evaluation of Consistency in detection of epidermal growth factor receptor gene T790M mutation in plasma and tumor specimens of patients with lung adenocarcinoma].

PubMed

Du, J; Wang, Z; Yang, L; Di, J; Zhang, J G; Wang, T Y; Liu, D G

2018-01-23

Objective: To evaluate the consistency in detection of T790M mutation of epidermal growth factor receptor gene (EGFR) in plasma and tumor samples of patients with lung adenocarcinoma. Methods: The tumor tissues or cytological specimens of 12 patients with operable lung adenocarcinoma(stage Ⅰ-ⅢA) and 100 patients with advanced stage ⅢB-Ⅳ lung adenocarcinoma were collected, among which 11 patients showed acquired resistance for gefitinib (11/100). In the same period, peripheral blood samples were collected from all patients and 50 healthy volunteers. Amplification refractory mutation system (ARMS) was used to detect EGFR mutations in tumor specimens. Next Generation Sequencing(NGS) based circulating single-molecule amplification and resequencing technology (cSMART)was performed to quantitatively detect the EGFR mutations in circulating tumor DNA (ctDNA) from plasma specimens. Results: The sensitivity, specificity and concordance rate of EGFR T790M mutation between plasma and tissue specimens from 100 advanced stage patients were 50.0%, 72.9% and 72.0%, respectively. For L858R mutation and exon 19 deletion mutations, the above mentioned sensitivity, specificity and concordance rate were 91.7%, 100.0%, and 98.0%, as well as 79.2%, 100.0% and 95.0%, respectively. The L858R mutation and exon 19 deletion mutations were not detected in plasma of 50 healthy volunteers, whereasT790M mutation(1.0±0.0 copies) was found in 7 individuals(7/50, 14.0%). Similarly, in 12 resectable patients, 4 (4/12, 33.3%) T790M mutations were found in plasma (1.2±0.2 copies), but no L858R mutation and 19 exon deletion mutations. In comparison, 28.0% of patients with advanced lung adenocarcinoma (28/100)had detectable T790M mutation in plasma with copy numbers (34.0±22.7 copies). Furthermore, the copy numbers of T790M were 268.2±119.9 in plasma of 5 cases with acquired gefitinib-resistance. Conclusions: In patients with advanced stages of lung adenocarcinoma, the detection of T790M mutation in plasma and tumor specimens is low. The T790M mutation also exists in the plasma of some healthy controls, suggesting that T790M mutation participates in EGFR signaling pathway and it might function in healthy population.

Technological Developments in Journalism: The Impact of the Computer in the Newsroom.

ERIC Educational Resources Information Center

Garrison, Bruce

A review of the literature for the past 7 years reveals that the computer serves several key functions in the newsroom. Its more dominant role is in word processing, or internal copy processing regardless of the source of the copy. Computers are also useful in reviewing documents for content analysis, for survey research in public opinion polls…

A Role of DLPFC in the Learning Process of Human Mate Copying

PubMed Central

Zhuang, Jin-Ying; Xie, Jiajia; Hu, Die; Fan, Mingxia; Zheng, Li

2016-01-01

In the current study, we conducted a behavioral experiment to test the mate coping effect and a functional magnetic resonance imaging (fMRI) experiment to test the neural basis involved in the social learning process of mate copying. In the behavioral experiment, participants were asked to rate the attractiveness of isolated opposite-sex (potential mates) facial photographs, then shown the targets associating with a neutral-faced model with textual cues indicating the models’ attitude (interested vs. not-interested) toward the potential mates, and then asked to re-evaluate the potential mates’ attractiveness. Using a similar procedure as the behavioral experiment, participants were scanned while observing the compound images in the fMRI experiment. The mate copying effect was confirmed in the behavioral experiment –greater increase in attractiveness ratings was observed for opposite-sex photographs in the interested than in the not-interested condition. The fMRI results showed that the dorsolateral prefrontal gyrus (DLPFC) was significantly active in the comparison of interested > not-interested condition, suggesting that a cognitive integration and selection function may be involved when participants process information from conditions related to mate copying. PMID:27148151

SNPitty: An Intuitive Web Application for Interactive B-Allele Frequency and Copy Number Visualization of Next-Generation Sequencing Data.

PubMed

van Riet, Job; Krol, Niels M G; Atmodimedjo, Peggy N; Brosens, Erwin; van IJcken, Wilfred F J; Jansen, Maurice P H M; Martens, John W M; Looijenga, Leendert H; Jenster, Guido; Dubbink, Hendrikus J; Dinjens, Winand N M; van de Werken, Harmen J G

2018-03-01

Exploration and visualization of next-generation sequencing data are crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions of interest coupled with dynamic heuristic filtering of genetic aberrations is, however, lacking. Therefore, the authors developed the web application SNPitty that allows interactive visualization and interrogation of variant call format files by using B-allele frequencies of single-nucleotide polymorphisms and single-nucleotide variants, coverage metrics, and copy numbers analysis results. SNPitty displays variant alleles and allelic imbalances with a focus on loss of heterozygosity and copy number variation using genome-wide heterozygous markers and somatic mutations. In addition, SNPitty is capable of generating predefined reports that summarize and highlight disease-specific targets of interest. SNPitty was validated for diagnostic interpretation of somatic events by showcasing a serial dilution series of glioma tissue. Additionally, SNPitty is demonstrated in four cancer-related scenarios encountered in daily clinical practice and on whole-exome sequencing data of peripheral blood from a Down syndrome patient. SNPitty allows detection of loss of heterozygosity, chromosomal and gene amplifications, homozygous or heterozygous deletions, somatic mutations, or any combination thereof in regions or genes of interest. Furthermore, SNPitty can be used to distinguish molecular relationships between multiple tumors from a single patient. On the basis of these data, the authors demonstrate that SNPitty is robust and user friendly in a wide range of diagnostic scenarios. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

PubMed Central

Wiszniewska, Joanna; Bi, Weimin; Shaw, Chad; Stankiewicz, Pawel; Kang, Sung-Hae L; Pursley, Amber N; Lalani, Seema; Hixson, Patricia; Gambin, Tomasz; Tsai, Chun-hui; Bock, Hans-Georg; Descartes, Maria; Probst, Frank J; Scaglia, Fernando; Beaudet, Arthur L; Lupski, James R; Eng, Christine; Wai Cheung, Sau; Bacino, Carlos; Patel, Ankita

2014-01-01

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner. PMID:23695279

Detection of genome-wide copy number variants in myeloid malignancies using next-generation sequencing.

PubMed

Shen, Wei; Paxton, Christian N; Szankasi, Philippe; Longhurst, Maria; Schumacher, Jonathan A; Frizzell, Kimberly A; Sorrells, Shelly M; Clayton, Adam L; Jattani, Rakhi P; Patel, Jay L; Toydemir, Reha; Kelley, Todd W; Xu, Xinjie

2018-04-01

Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Chromosome-specific physical localisation of expressed sequence tag loci in Corchorus olitorius L.

PubMed

Joshi, A; Das, S K; Samanta, P; Paria, P; Sen, S K; Basu, A

2014-11-01

Jute (Corchorus spp.), as a natural fibre-producing species, ranks next only to cotton. Inadequate understanding of its genetic architecture is a major lacuna for genetic improvement of this crop in terms of yield and quality. Establishment of a physical map provides a genomic tool that helps in positional cloning of valuable genes. In this report, an attempt was initiated to study association and localisation of single copy expressed sequence tag (EST) loci in the genome of Corchorus olitorius. The chromosome-specific association of EST was determined based on the appearance of an extra signal for a single copy cDNA probe in mitotic interphase nuclei of specific trisomic(s) for fluorescence in situ hybridisation, and validated using a cDNA fragment of the 26S rRNA gene (600 bp) as molecular probe. The probe exhibited three signals in meiotic interphase nuclei of trisomic 5, instead of two as observed in diploids and other trisomics, indicating its association with chromosome 5. Subsequent hybridisation of the same probe on the pachytene chromosomes of diploids confirmed that 26S rRNA occupies the terminal end of the short arm of chromosome 5 in C. olitorius. Subsequently, chromosome-specific association of 63 single copy EST and their physical localisation were determined on chromosomes 2, 4, 5 and 7. The study describes chromosome-specific physical localisation of genes in jute. The approach used here could be a step towards construction of genome-wide physical maps for any recalcitrant plant species like jute. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Two sequence-ready contigs spanning the two copies of a 200-kb duplication on human 21q: partial sequence and polymorphisms.

PubMed

Potier, M; Dutriaux, A; Orti, R; Groet, J; Gibelin, N; Karadima, G; Lutfalla, G; Lynn, A; Van Broeckhoven, C; Chakravarti, A; Petersen, M; Nizetic, D; Delabar, J; Rossier, J

1998-08-01

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications. Copyright 1998 Academic Press.

International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons

PubMed Central

Olson, Nathan D.; Lund, Steven P.; Zook, Justin M.; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S.; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B.

2015-01-01

This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

Esthetic Rehabilitation of Anterior Teeth with Copy-Milled Restorations: A Report of Two Cases.

PubMed

Rani, Sapna; Devi, Jyoti; Jain, Chandan; Mutneja, Parul; Verma, Mahesh

2017-01-01

Digitalization has become part and parcel of contemporary prosthodontics with the probability of most of the procedures being based on the digital techniques in the near future. This digital revolution started in the latter half of the 20th century by converting analog objects/signals into digital bits and bytes. Recent developments in all-ceramic materials and systems of computer-aided designing and computer-aided manufacturing (CAD/CAM), copy milling, and so forth offer excellent esthetics and superb biocompatibility. Copy milling system for ceramics enables milling of the zirconia cores of all-ceramic restorations precisely and also if this system is properly used the procedure for fabricating all-ceramic restorations can be substantially simplified. This case report presents fabrication of all-ceramic Maryland Bridge and post-core with a copy milling system for esthetics and preservation of integrity of tooth. For both of the patients, the use of biologic, all-ceramic, copy-milled restorations resulted in clinical success and recovered function and esthetics.

Esthetic Rehabilitation of Anterior Teeth with Copy-Milled Restorations: A Report of Two Cases

PubMed Central

Jain, Chandan; Mutneja, Parul; Verma, Mahesh

2017-01-01

Digitalization has become part and parcel of contemporary prosthodontics with the probability of most of the procedures being based on the digital techniques in the near future. This digital revolution started in the latter half of the 20th century by converting analog objects/signals into digital bits and bytes. Recent developments in all-ceramic materials and systems of computer-aided designing and computer-aided manufacturing (CAD/CAM), copy milling, and so forth offer excellent esthetics and superb biocompatibility. Copy milling system for ceramics enables milling of the zirconia cores of all-ceramic restorations precisely and also if this system is properly used the procedure for fabricating all-ceramic restorations can be substantially simplified. This case report presents fabrication of all-ceramic Maryland Bridge and post-core with a copy milling system for esthetics and preservation of integrity of tooth. For both of the patients, the use of biologic, all-ceramic, copy-milled restorations resulted in clinical success and recovered function and esthetics. PMID:28326203

Effects of Transgenic Bt+CpTI cotton on the abundance and diversity of rhizosphere ammonia oxidizing bacteria and archaea.

PubMed

Dong, Lianhua; Meng, Ying; Wang, Jing; Sun, Guoqing

2016-09-01

Genetically modified crops (GMCs) hold great promise for improving agricultural output, but at the same time present challenges in terms of environmental safety assessment. Ammonia oxidizers, including ammonia oxidizing bacteria (AOB) and archaea (AOA), are very important functional microbial groups in nitrogen cycle. The abundance and diversity of AOA and AOB in the rhizosphere of genetically modified cotton (SGK321) and non-GM cotton (SY321) across growth stages were investigated using real time quantitative PCR (qPCR) and terminal restriction fragment length polymorphism (T-RFLP). Results showed that cotton genotype had a significant effect on the change in abundance of AOA and AOB, as indicated by amoA copy number. Variations in AOB abundance in rhizosphere of SY321 differed from those in SGK321. The number of AOB in the rhizosphere of SY321 fluctuated considerably: It dramatically decreased from 1.2?106 copies g-1 dry soil to 3?105 copies g-1 dry soil during the flowering stage and then increased to 1.1?106 copies g-1 and 1.5?106 copies g-1 at the belling and boll opening stages, respectively. However, abundance of AOB in the rhizosphere of SGK321 was relatively stable during all the stages of growth. The effect of SGK321 and SY321 on AOA number was quite similar to that of AOB: AOA abundance in SGK321 increased smoothly from 1.0 ?105 copies g-1 dry soil to 1.4?106 copies g-1 dry soil during growth, but that in SY321 fluctuated. Correspondence analysis (CA), canonical CA (CCA), and partial CCA (pCCA) of T-RFLP profiles of AOA and AOB showed that AOB community changed across growth stages in both cotton genotypes, and cotton genotype was the most important factor affecting the AOA community. In conclusion, the current findings indicated no adverse effect of GM cotton on functional microorganisms.

Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG).

PubMed

Pentheroudakis, George; Kotoula, Vassiliki; Eleftheraki, Anastasia G; Tsolaki, Eleftheria; Wirtz, Ralph M; Kalogeras, Konstantine T; Batistatou, Anna; Bobos, Mattheos; Dimopoulos, Meletios A; Timotheadou, Eleni; Gogas, Helen; Christodoulou, Christos; Papadopoulou, Kyriaki; Efstratiou, Ioannis; Scopa, Chrisoula D; Papaspyrou, Irene; Vlachodimitropoulos, Dimitrios; Linardou, Helena; Samantas, Epaminontas; Pectasides, Dimitrios; Pavlidis, Nicholas; Fountzilas, George

2013-01-01

Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes. In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01). ESR1 clusters were observed in 9.5% (57 gain, 38 amplification) of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression) in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification) fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003). Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066) and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029). ESR1 gene deletion and amplification do not constitute per se prognostic markers, instead they can be classified to distinct prognostic groups according to their protein-mediated functional status.

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.

Organization and transient expression of the gene for human U11 snRNA

PubMed Central

Clemens, Suter-Crazzolara; Walter, Keller

1991-01-01

The nucleotide sequence of U11 small nuclear RNA, a minor U RNA from HeLa cells, was determined. Computer analysis of the sequence (135 residues) predicts two strong hairpin loops which are separated by seventeen nucleotides containing an Sm binding site (AAUUUUUUGG). A synthetic gene was constructed in which the coding region of U11 RNA is under the control of a T7 promoter. This vector can be used to produce U11 RNA in vitro. Southern hybridization and PCR analysis of HeLa genomic DNA suggest that U11 RNA is encoded by a single copy gene, and that at least three genomic regions could be U11 RNA pseudogenes. A HeLa genomic copy of a U11 gene was isolated by inverted PCR. This gene contains the U11 RNA coding sequence and several sequence elements unique for the U RNA genes. These include a Distal Sequence Element (DSE, ATTTGCATA) present between positions −215 and −223 relative to the start of transcription; a Proximal Sequence Element (PSE, TTCACCTTTACCAAAAATG) located between positions −43 and −63 ; and a 3′box (GTTAGGCGAAATATTA) between positions +150 and +166. Transfection of HeLa cells with this gene revealed that it is functioning in vivo and can produce U11 RNA. PMID:1820214

A social activity and physical contact-based routing algorithm in mobile opportunistic networks for emergency response to sudden disasters

NASA Astrophysics Data System (ADS)

Wang, Xiaoming; Lin, Yaguang; Zhang, Shanshan; Cai, Zhipeng

2017-05-01

Sudden disasters such as earthquake, flood and hurricane necessitate the employment of communication networks to carry out emergency response activities. Routing has a significant impact on the functionality, performance and flexibility of communication networks. In this article, the routing problem is studied considering the delivery ratio of messages, the overhead ratio of messages and the average delay of messages in mobile opportunistic networks (MONs) for enterprise-level emergency response communications in sudden disaster scenarios. Unlike the traditional routing methods for MONS, this article presents a new two-stage spreading and forwarding dynamic routing algorithm based on the proposed social activity degree and physical contact factor for mobile customers. A new modelling method for describing a dynamic evolving process of the topology structure of a MON is first proposed. Then a multi-copy spreading strategy based on the social activity degree of nodes and a single-copy forwarding strategy based on the physical contact factor between nodes are designed. Compared with the most relevant routing algorithms such as Epidemic, Prophet, Labelled-sim, Dlife-comm and Distribute-sim, the proposed routing algorithm can significantly increase the delivery ratio of messages, and decrease the overhead ratio and average delay of messages.

Cores Of Recurrent Events (CORE) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

CORE is a statistically supported computational method for finding recurrently targeted regions in massive collections of genomic intervals, such as those arising from DNA copy number analysis of single tumor cells or bulk tumor tissues.

Fast Food Can Be Profitable

ERIC Educational Resources Information Center

Frederick, Len

1978-01-01

The Clark County School District of Las Vegas, Nevada, has increased student participation in the school lunch program, and makes a profit. Available from Educational Economics, Capla Associates, 18 Overlook Avenue, Rochelle Park, N.J. 07662; Single copy $2.50. (Author/MLF)

The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes

PubMed Central

Pombert, Jean-François; Lemieux, Claude; Turmel, Monique

2006-01-01

Background The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA) sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae), in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR) featuring an inverted rRNA operon and a small single-copy (SSC) region containing 14 genes normally found in the large single-copy (LSC) region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage. Results The 151,933 bp IR-containing genome of Oltmannsiellopsis differs considerably from Pseudendoclonium and other chlorophyte cpDNAs in intron content and gene order, but shares close similarities with its ulvophyte homologue at the levels of quadripartite architecture, gene content and gene density. Oltmannsiellopsis cpDNA encodes 105 genes, contains five group I introns, and features many short dispersed repeats. As in Pseudendoclonium cpDNA, the rRNA genes in the IR are transcribed toward the single copy region featuring the genes typically found in the ancestral LSC region, and the opposite single copy region harbours genes characteristic of both the ancestral SSC and LSC regions. The 52 genes that were transferred from the ancestral LSC to SSC region include 12 of those observed in Pseudendoclonium cpDNA. Surprisingly, the overall gene organization of Oltmannsiellopsis cpDNA more closely resembles that of Chlorella (Trebouxiophyceae) cpDNA. Conclusion The chloroplast genome of the last common ancestor of Oltmannsiellopsis and Pseudendoclonium contained a minimum of 108 genes, carried only a few group I introns, and featured a distinctive quadripartite architecture. Numerous changes were experienced by the chloroplast genome in the lineages leading to Oltmannsiellopsis and Pseudendoclonium. Our comparative analyses of chlorophyte cpDNAs support the notion that the Ulvophyceae is sister to the Chlorophyceae. PMID:16472375

The complete chloroplast genome sequence of the chlorophycean green alga Scenedesmus obliquus reveals a compact gene organization and a biased distribution of genes on the two DNA strands

PubMed Central

de Cambiaire, Jean-Charles; Otis, Christian; Lemieux, Claude; Turmel, Monique

2006-01-01

Background The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. While the basal position of the Prasinophyceae is well established, the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae (UTC) remains uncertain. The five complete chloroplast DNA (cpDNA) sequences currently available for representatives of these classes display considerable variability in overall structure, gene content, gene density, intron content and gene order. Among these genomes, that of the chlorophycean green alga Chlamydomonas reinhardtii has retained the least ancestral features. The two single-copy regions, which are separated from one another by the large inverted repeat (IR), have similar sizes, rather than unequal sizes, and differ radically in both gene contents and gene organizations relative to the single-copy regions of prasinophyte and ulvophyte cpDNAs. To gain insights into the various changes that underwent the chloroplast genome during the evolution of chlorophycean green algae, we have sequenced the cpDNA of Scenedesmus obliquus, a member of a distinct chlorophycean lineage. Results The 161,452 bp IR-containing genome of Scenedesmus features single-copy regions of similar sizes, encodes 96 genes, i.e. only two additional genes (infA and rpl12) relative to its Chlamydomonas homologue and contains seven group I and two group II introns. It is clearly more compact than the four UTC algal cpDNAs that have been examined so far, displays the lowest proportion of short repeats among these algae and shows a stronger bias in clustering of genes on the same DNA strand compared to Chlamydomonas cpDNA. Like the latter genome, Scenedesmus cpDNA displays only a few ancestral gene clusters. The two chlorophycean genomes share 11 gene clusters that are not found in previously sequenced trebouxiophyte and ulvophyte cpDNAs as well as a few genes that have an unusual structure; however, their single-copy regions differ considerably in gene content. Conclusion Our results underscore the remarkable plasticity of the chlorophycean chloroplast genome. Owing to this plasticity, only a sketchy portrait could be drawn for the chloroplast genome of the last common ancestor of Scenedesmus and Chlamydomonas. PMID:16638149

Effects of small particle numbers on long-term behaviour in discrete biochemical systems

PubMed Central

Ibrahim, Bashar; Dittrich, Peter

2014-01-01

Motivation: The functioning of many biological processes depends on the appearance of only a small number of a single molecular species. Additionally, the observation of molecular crowding leads to the insight that even a high number of copies of species do not guarantee their interaction. How single particles contribute to stabilizing biological systems is not well understood yet. Hence, we aim at determining the influence of single molecules on the long-term behaviour of biological systems, i.e. whether they can reach a steady state. Results: We provide theoretical considerations and a tool to analyse Systems Biology Markup Language models for the possibility to stabilize because of the described effects. The theory is an extension of chemical organization theory, which we called discrete chemical organization theory. Furthermore we scanned the BioModels Database for the occurrence of discrete chemical organizations. To exemplify our method, we describe an application to the Template model of the mitotic spindle assembly checkpoint mechanism. Availability and implementation: http://www.biosys.uni-jena.de/Services.html. Contact: bashar.ibrahim@uni-jena.de or dittrich@minet.uni-jena.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25161236

Mismatch and G-Stack Modulated Probe Signals on SNP Microarrays

PubMed Central

Binder, Hans; Fasold, Mario; Glomb, Torsten

2009-01-01

Background Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. We analyze the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates. Methodology/Principal Findings The probe design of GeneChip SNP arrays enables us to disentangle different sources of intensity modulations such as the number of mismatches per duplex, matched and mismatched base pairings including nearest and next-nearest neighbors and their position along the probe sequence. The effect of probe sequence was estimated in terms of triple-motifs with central matches and mismatches which include all 256 combinations of possible base pairings. The probe/target interactions on the chip can be decomposed into nearest neighbor contributions which correlate well with free energy terms of DNA/DNA-interactions in solution. The effect of mismatches is about twice as large as that of canonical pairings. Runs of guanines (G) and the particular type of mismatched pairings formed in cross-allelic probe/target duplexes constitute sources of systematic biases of the probe signals with consequences for genotyping and copy number estimates. The poly-G effect seems to be related to the crowded arrangement of probes which facilitates complex formation of neighboring probes with at minimum three adjacent G's in their sequence. Conclusions The applied method of “triple-averaging” represents a model-free approach to estimate the mean intensity contributions of different sequence motifs which can be applied in calibration algorithms to correct signal values for sequence effects. Rules for appropriate sequence corrections are suggested. PMID:19924253

Distinct Trajectories of Massive Recent Gene Gains and Losses in Populations of a Microbial Eukaryotic Pathogen.

PubMed

Hartmann, Fanny E; Croll, Daniel

2017-11-01

Differences in gene content are a significant source of variability within species and have an impact on phenotypic traits. However, little is known about the mechanisms responsible for the most recent gene gains and losses. We screened the genomes of 123 worldwide isolates of the major pathogen of wheat Zymoseptoria tritici for robust evidence of gene copy number variation. Based on orthology relationships in three closely related fungi, we identified 599 gene gains and 1,024 gene losses that have not yet reached fixation within the focal species. Our analyses of gene gains and losses segregating in populations showed that gene copy number variation arose preferentially in subtelomeres and in proximity to transposable elements. Recently lost genes were enriched in virulence factors and secondary metabolite gene clusters. In contrast, recently gained genes encoded mostly secreted protein lacking a conserved domain. We analyzed the frequency spectrum at loci segregating a gene presence-absence polymorphism in four worldwide populations. Recent gene losses showed a significant excess in low-frequency variants compared with genome-wide single nucleotide polymorphism, which is indicative of strong negative selection against gene losses. Recent gene gains were either under weak negative selection or neutral. We found evidence for strong divergent selection among populations at individual loci segregating a gene presence-absence polymorphism. Hence, gene gains and losses likely contributed to local adaptation. Our study shows that microbial eukaryotes harbor extensive copy number variation within populations and that functional differences among recently gained and lost genes led to distinct evolutionary trajectories. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Saccharomyces cerevisiae Bat1 and Bat2 Aminotransferases Have Functionally Diverged from the Ancestral-Like Kluyveromyces lactis Orthologous Enzyme

PubMed Central

Colón, Maritrini; Hernández, Fabiola; López, Karla; Quezada, Héctor; González, James; López, Geovani; Aranda, Cristina; González, Alicia

2011-01-01

Background Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism. Principal Findings Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs). This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1), while catabolic substrates are accumulated in the cytosol (Bat2). Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition. Conclusions Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the biosynthetic and catabolic roles of the ancestral BCAT in two isozymes improving BCAAs metabolism and constituting an adaptation to facultative metabolism. PMID:21267457