Mapping neurofibromatosis 1 homologous loci by fluorescence in situ hybridization

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

Viskochil, D.; Breidenbach, H.H.; Cawthon, R.

Neurofibromatosis 1 maps to chromosome band 17q11.2 and the NF1 gene is comprised of 59 exons that span approximately 335 kb of genomic DNA. In order to further analyze the structure of NF1 from exons 2 through 27b, we isolated a number of cosmid and bacteriophage P-1 genomic clones using NF1-exon probes under high-stringency hybridization conditions. Using tagged, intron-based primers and DNA from various clones as a template, we PCR-amplified and sequenced individual NF1 exons. The exon sequences in PCR products from several genomic clones differed from the exon sequence derived from cloned NF1 cDNAs. Clones with variant sequences weremore » mapped by fluorescence in situ hybridization under high-stringency conditions. Three clones mapped to chromosome band 15q11.2, one mapped to 14q11.2, one mapped to both 2q14.1-14.3 and 14q11.2, one mapped to 2q33-34, and one mapped to both 18q11.2 and 21q21. Even though some PCR-product sequences retained proper splice junctions and open reading frames, we have yet to identify cDNAs that correspond to the variant exon sequences. We are now sequencing clones that map to NF1-homologous loci in order to develop discriminating primer pairs for the exclusive amplification of NF1-specific sequences in our efforts to develop a comprehensive NF1 mutation screen using genomic DNA as template. The role of NF1-homologous sequences may play in neurofibromatosis 1 is not clear.« less

The development and characterisation of a bacterial artificial chromosome library for Fragaria vesca

PubMed Central

Bonet, Julio; Girona, Elena Lopez; Sargent, Daniel J; Muñoz-Torres, Monica C; Monfort, Amparo; Abbott, Albert G; Arús, Pere; Simpson, David W; Davik, Jahn

2009-01-01

Background The cultivated strawberry Fragaria ×ananassa is one of the most economically-important soft-fruit species. Few structural genomic resources have been reported for Fragaria and there exists an urgent need for the development of physical mapping resources for the genus. The first stage in the development of a physical map for Fragaria is the construction and characterisation of a high molecular weight bacterial artificial chromosome (BAC) library. Methods A BAC library, consisting of 18,432 clones was constructed from Fragaria vesca f. semperflorens accession 'Ali Baba'. BAC DNA from individual library clones was pooled to create a PCR-based screening assay for the library, whereby individual clones could be identified with just 34 PCR reactions. These pools were used to screen the BAC library and anchor individual clones to the diploid Fragaria reference map (FV×FN). Findings Clones from the BAC library developed contained an average insert size of 85 kb, representing over seven genome equivalents. The pools and superpools developed were used to identify a set of BAC clones containing 70 molecular markers previously mapped to the diploid Fragaria FV×FN reference map. The number of positive colonies identified for each marker suggests the library represents between 4× and 10× coverage of the diploid Fragaria genome, which is in accordance with the estimate of library coverage based on average insert size. Conclusion This BAC library will be used for the construction of a physical map for F. vesca and the superpools will permit physical anchoring of molecular markers using PCR. PMID:19772672

Rapid in silico cloning of genes using expressed sequence tags (ESTs).

PubMed

Gill, R W; Sanseau, P

2000-01-01

Expressed sequence tags (ESTs) are short single-pass DNA sequences obtained from either end of cDNA clones. These ESTs are derived from a vast number of cDNA libraries obtained from different species. Human ESTs are the bulk of the data and have been widely used to identify new members of gene families, as markers on the human chromosomes, to discover polymorphism sites and to compare expression patterns in different tissues or pathologies states. Information strategies have been devised to query EST databases. Since most of the analysis is performed with a computer, the term "in silico" strategy has been coined. In this chapter we will review the current status of EST databases, the pros and cons of EST-type data and describe possible strategies to retrieve meaningful information.

Analyzing Clonal Variation of Monoclonal Antibody-Producing CHO Cell Lines Using an In Silico Metabolomic Platform

PubMed Central

Ghorbaniaghdam, Atefeh; Chen, Jingkui; Henry, Olivier; Jolicoeur, Mario

2014-01-01

Monoclonal antibody producing Chinese hamster ovary (CHO) cells have been shown to undergo metabolic changes when engineered to produce high titers of recombinant proteins. In this work, we have studied the distinct metabolism of CHO cell clones harboring an efficient inducible expression system, based on the cumate gene switch, and displaying different expression levels, high and low productivities, compared to that of the parental cells from which they were derived. A kinetic model for CHO cell metabolism was further developed to include metabolic regulation. Model calibration was performed using intracellular and extracellular metabolite profiles obtained from shake flask batch cultures. Model simulations of intracellular fluxes and ratios known as biomarkers revealed significant changes correlated with clonal variation but not to the recombinant protein expression level. Metabolic flux distribution mostly differs in the reactions involving pyruvate metabolism, with an increased net flux of pyruvate into the tricarboxylic acid (TCA) cycle in the high-producer clone, either being induced or non-induced with cumate. More specifically, CHO cell metabolism in this clone was characterized by an efficient utilization of glucose and a high pyruvate dehydrogenase flux. Moreover, the high-producer clone shows a high rate of anaplerosis from pyruvate to oxaloacetate, through pyruvate carboxylase and from glutamate to α-ketoglutarate, through glutamate dehydrogenase, and a reduced rate of cataplerosis from malate to pyruvate, through malic enzyme. Indeed, the increase of flux through pyruvate carboxylase was not driven by an increased anabolic demand. It is in fact linked to an increase of the TCA cycle global flux, which allows better regulation of higher redox and more efficient metabolic states. To the best of our knowledge, this is the first time a dynamic in silico platform is proposed to analyze and compare the metabolomic behavior of different CHO clones. PMID:24632968

A triallelic genetic male sterility locus in Brassica napus: an integrative strategy for its physical mapping and possible local chromosome evolution around it

PubMed Central

Lu, Wei; Liu, Jun; Xin, Qiang; Wan, Lili; Hong, Dengfeng; Yang, Guangsheng

2013-01-01

Background and Aims Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5. Methods A large F2 population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5. Key Results BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence. Conclusions This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus. PMID:23243189

i-rDNA: alignment-free algorithm for rapid in silico detection of ribosomal gene fragments from metagenomic sequence data sets.

PubMed

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Chadaram, Sudha; Mande, Sharmila S

2011-11-30

Obtaining accurate estimates of microbial diversity using rDNA profiling is the first step in most metagenomics projects. Consequently, most metagenomic projects spend considerable amounts of time, money and manpower for experimentally cloning, amplifying and sequencing the rDNA content in a metagenomic sample. In the second step, the entire genomic content of the metagenome is extracted, sequenced and analyzed. Since DNA sequences obtained in this second step also contain rDNA fragments, rapid in silico identification of these rDNA fragments would drastically reduce the cost, time and effort of current metagenomic projects by entirely bypassing the experimental steps of primer based rDNA amplification, cloning and sequencing. In this study, we present an algorithm called i-rDNA that can facilitate the rapid detection of 16S rDNA fragments from amongst millions of sequences in metagenomic data sets with high detection sensitivity. Performance evaluation with data sets/database variants simulating typical metagenomic scenarios indicates the significantly high detection sensitivity of i-rDNA. Moreover, i-rDNA can process a million sequences in less than an hour on a simple desktop with modest hardware specifications. In addition to the speed of execution, high sensitivity and low false positive rate, the utility of the algorithmic approach discussed in this paper is immense given that it would help in bypassing the entire experimental step of primer-based rDNA amplification, cloning and sequencing. Application of this algorithmic approach would thus drastically reduce the cost, time and human efforts invested in all metagenomic projects. A web-server for the i-rDNA algorithm is available at http://metagenomics.atc.tcs.com/i-rDNA/

In silico mapping of quantitative trait loci in maize.

PubMed

Parisseaux, B; Bernardo, R

2004-08-01

Quantitative trait loci (QTL) are most often detected through designed mapping experiments. An alternative approach is in silico mapping, whereby genes are detected using existing phenotypic and genomic databases. We explored the usefulness of in silico mapping via a mixed-model approach in maize (Zea mays L.). Specifically, our objective was to determine if the procedure gave results that were repeatable across populations. Multilocation data were obtained from the 1995-2002 hybrid testing program of Limagrain Genetics in Europe. Nine heterotic patterns comprised 22,774 single crosses. These single crosses were made from 1,266 inbreds that had data for 96 simple sequence repeat (SSR) markers. By a mixed-model approach, we estimated the general combining ability effects associated with marker alleles in each heterotic pattern. The numbers of marker loci with significant effects--37 for plant height, 24 for smut [Ustilago maydis (DC.) Cda.] resistance, and 44 for grain moisture--were consistent with previous results from designed mapping experiments. Each trait had many loci with small effects and few loci with large effects. For smut resistance, a marker in bin 8.05 on chromosome 8 had a significant effect in seven (out of a maximum of 18) instances. For this major QTL, the maximum effect of an allele substitution ranged from 5.4% to 41.9%, with an average of 22.0%. We conclude that in silico mapping via a mixed-model approach can detect associations that are repeatable across different populations. We speculate that in silico mapping will be more useful for gene discovery than for selection in plant breeding programs. Copyright 2004 Springer-Verlag

Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome

PubMed Central

Müller, Christina A.; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C. A.; Wellington, Elizabeth M. H.

2015-01-01

Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. PMID:26002894

Map-Based Cloning of Genes Important for Maize Anther Development

NASA Astrophysics Data System (ADS)

Anaya, Y.; Walbot, V.; Nan, G.

2012-12-01

Map-Based cloning for maize mutant MS13 . Scientists still do not understand what decides the fate of a cell in plants. Many maize genes are important for anther development and when they are disrupted, the anthers do not shed pollen, i.e. male sterile. Since the maize genome has been fully sequenced, we conduct map-based cloning using a bulk segregant analysis strategy. Using PCR (polymerase chain reaction), we look for biomarkers that are linked to our gene of interest, Male Sterile 13 (MS13). Recombinations occur more often if the biomarkers are further away from the gene, therefore we can estimate where the gene is and design more PCR primers to get closer to our gene. Genetic and molecular analysis will help distinguish the role of key genes in setting cell fates before meiosis and for being in charge of the switch from mitosis to meiosis.

In silico polypharmacology of natural products.

PubMed

Fang, Jiansong; Liu, Chuang; Wang, Qi; Lin, Ping; Cheng, Feixiong

2017-04-27

Natural products with polypharmacological profiles have demonstrated promise as novel therapeutics for various complex diseases, including cancer. Currently, many gaps exist in our knowledge of which compounds interact with which targets, and experimentally testing all possible interactions is infeasible. Recent advances and developments of systems pharmacology and computational (in silico) approaches provide powerful tools for exploring the polypharmacological profiles of natural products. In this review, we introduce recent progresses and advances of computational tools and systems pharmacology approaches for identifying drug targets of natural products by focusing on the development of targeted cancer therapy. We survey the polypharmacological and systems immunology profiles of five representative natural products that are being considered as cancer therapies. We summarize various chemoinformatics, bioinformatics and systems biology resources for reconstructing drug-target networks of natural products. We then review currently available computational approaches and tools for prediction of drug-target interactions by focusing on five domains: target-based, ligand-based, chemogenomics-based, network-based and omics-based systems biology approaches. In addition, we describe a practical example of the application of systems pharmacology approaches by integrating the polypharmacology of natural products and large-scale cancer genomics data for the development of precision oncology under the systems biology framework. Finally, we highlight the promise of cancer immunotherapies and combination therapies that target tumor ecosystems (e.g. clones or 'selfish' sub-clones) via exploiting the immunological and inflammatory 'side' effects of natural products in the cancer post-genomics era. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Reconstruction of biological pathways and metabolic networks from in silico labeled metabolites.

PubMed

Hadadi, Noushin; Hafner, Jasmin; Soh, Keng Cher; Hatzimanikatis, Vassily

2017-01-01

Reaction atom mappings track the positional changes of all of the atoms between the substrates and the products as they undergo the biochemical transformation. However, information on atom transitions in the context of metabolic pathways is not widely available in the literature. The understanding of metabolic pathways at the atomic level is of great importance as it can deconvolute the overlapping catabolic/anabolic pathways resulting in the observed metabolic phenotype. The automated identification of atom transitions within a metabolic network is a very challenging task since the degree of complexity of metabolic networks dramatically increases when we transit from metabolite-level studies to atom-level studies. Despite being studied extensively in various approaches, the field of atom mapping of metabolic networks is lacking an automated approach, which (i) accounts for the information of reaction mechanism for atom mapping and (ii) is extendable from individual atom-mapped reactions to atom-mapped reaction networks. Hereby, we introduce a computational framework, iAM.NICE (in silico Atom Mapped Network Integrated Computational Explorer), for the systematic atom-level reconstruction of metabolic networks from in silico labelled substrates. iAM.NICE is to our knowledge the first automated atom-mapping algorithm that is based on the underlying enzymatic biotransformation mechanisms, and its application goes beyond individual reactions and it can be used for the reconstruction of atom-mapped metabolic networks. We illustrate the applicability of our method through the reconstruction of atom-mapped reactions of the KEGG database and we provide an example of an atom-level representation of the core metabolic network of E. coli. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Diploid/Polyploid Syntenic Shuttle Mapping and Haplotype-Specific Chromosome Walking Toward a Rust Resistance Gene (Bru1) in Highly Polyploid Sugarcane (2n ∼ 12x ∼ 115)

PubMed Central

Le Cunff, Loïc; Garsmeur, Olivier; Raboin, Louis Marie; Pauquet, Jérome; Telismart, Hugues; Selvi, Athiappan; Grivet, Laurent; Philippe, Romain; Begum, Dilara; Deu, Monique; Costet, Laurent; Wing, Rod; Glaszmann, Jean Christophe; D'Hont, Angélique

2008-01-01

The genome of modern sugarcane cultivars is highly polyploid (∼12x), aneuploid, of interspecific origin, and contains 10 Gb of DNA. Its size and complexity represent a major challenge for the isolation of agronomically important genes. Here we report on the first attempt to isolate a gene from sugarcane by map-based cloning, targeting a durable major rust resistance gene (Bru1). We describe the genomic strategies that we have developed to overcome constraints associated with high polyploidy in the successive steps of map-based cloning approaches, including diploid/polyploid syntenic shuttle mapping with two model diploid species (sorghum and rice) and haplotype-specific chromosome walking. Their applications allowed us (i) to develop a high-resolution map including markers at 0.28 and 0.14 cM on both sides and 13 markers cosegregating with Bru1 and (ii) to develop a physical map of the target haplotype that still includes two gaps at this stage due to the discovery of an insertion specific to this haplotype. These approaches will pave the way for the development of future map-based cloning approaches for sugarcane and other complex polyploid species. PMID:18757946

A high density physical map of chromosome 1BL supports evolutionary studies, map-based cloning and sequencing in wheat

PubMed Central

2013-01-01

Background As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning. Results Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome. Conclusions Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing. PMID:23800011

Self-Organizing Maps for In Silico Screening and Data Visualization.

PubMed

Digles, Daniela; Ecker, Gerhard F

2011-10-01

Self-organizing maps, which are unsupervised artificial neural networks, have become a very useful tool in a wide area of disciplines, including medicinal chemistry. Here, we will focus on two applications of self-organizing maps: the use of self-organizing maps for in silico screening and for clustering and visualisation of large datasets. Additionally, the importance of parameter selection is discussed and some modifications to the original algorithm are summarised. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of predictive pharmacophore model for in silico screening, and 3D QSAR CoMFA and CoMSIA studies for lead optimization, for designing of potent tumor necrosis factor alpha converting enzyme inhibitors

NASA Astrophysics Data System (ADS)

Murumkar, Prashant Revan; Zambre, Vishal Prakash; Yadav, Mange Ram

2010-02-01

A chemical feature-based pharmacophore model was developed for Tumor Necrosis Factor-α converting enzyme (TACE) inhibitors. A five point pharmacophore model having two hydrogen bond acceptors (A), one hydrogen bond donor (D) and two aromatic rings (R) with discrete geometries as pharmacophoric features was developed. The pharmacophore model so generated was then utilized for in silico screening of a database. The pharmacophore model so developed was validated by using four compounds having proven TACE inhibitory activity which were grafted into the database. These compounds mapped well onto the five listed pharmacophoric features. This validated pharmacophore model was also used for alignment of molecules in CoMFA and CoMSIA analysis. The contour maps of the CoMFA/CoMSIA models were utilized to provide structural insight for activity improvement of potential novel TACE inhibitors. The pharmacophore model so developed could be used for in silico screening of any commercial/in house database for identification of TACE inhibiting lead compounds, and the leads so identified could be optimized using the developed CoMSIA model. The present work highlights the tremendous potential of the two mutually complementary ligand-based drug designing techniques (i.e. pharmacophore mapping and 3D-QSAR analysis) using TACE inhibitors as prototype biologically active molecules.

A sequencing-based linkage map of cucumber

USDA-ARS?s Scientific Manuscript database

Genetic maps are important tools for molecular breeding, gene cloning, and study of meiotic recombination. In cucumber (Cucumis sativus L.), the marker density, resolution and genome coverage of previously developed genetic maps using PCR-based molecular markers are relatively low. In this study we ...

The perennial ryegrass GenomeZipper: targeted use of genome resources for comparative grass genomics.

PubMed

Pfeifer, Matthias; Martis, Mihaela; Asp, Torben; Mayer, Klaus F X; Lübberstedt, Thomas; Byrne, Stephen; Frei, Ursula; Studer, Bruno

2013-02-01

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species.

The Perennial Ryegrass GenomeZipper: Targeted Use of Genome Resources for Comparative Grass Genomics1[C][W

PubMed Central

Pfeifer, Matthias; Martis, Mihaela; Asp, Torben; Mayer, Klaus F.X.; Lübberstedt, Thomas; Byrne, Stephen; Frei, Ursula; Studer, Bruno

2013-01-01

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species. PMID:23184232

Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

PubMed

Padaria, Jasdeep Chatrath; Vishwakarma, Harinder; Biswas, Koushik; Jasrotia, Rahul Singh; Singh, Gyanendra Pratap

2014-10-10

Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India. qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42 °C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting. TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome.

PubMed

Müller, Christina A; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C A; Wellington, Elizabeth M H; Berg, Gabriele

2015-08-01

Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Arrhythmic hazard map for a 3D whole-ventricles model under multiple ion channel block.

PubMed

Okada, Jun-Ichi; Yoshinaga, Takashi; Kurokawa, Junko; Washio, Takumi; Furukawa, Tetsushi; Sawada, Kohei; Sugiura, Seiryo; Hisada, Toshiaki

2018-05-10

To date, proposed in silico models for preclinical cardiac safety testing are limited in their predictability and usability. We previously reported a multi-scale heart simulation that accurately predicts arrhythmogenic risk for benchmark drugs. We extend this approach and report the first comprehensive hazard map of drug-induced arrhythmia based on the exhaustive in silico electrocardiogram (ECG) database of drug effects, developed using a petaflop computer. A total of 9075 electrocardiograms constitute the five-dimensional hazard map, with coordinates representing the extent of the block of each of the five ionic currents (rapid delayed rectifier potassium current (IKr), fast (INa) and late (INa,L) components of the sodium current, L-type calcium current (ICa,L) and slow delayed rectifier current (IKs)), involved in arrhythmogenesis. Results of the evaluation of arrhythmogenic risk based on this hazard map agreed well with the risk assessments reported in three references. ECG database also suggested that the interval between the J-point and the T-wave peak is a superior index of arrhythmogenicity compared to other ECG biomarkers including the QT interval. Because concentration-dependent effects on electrocardiograms of any drug can be traced on this map based on in vitro current assay data, its arrhythmogenic risk can be evaluated without performing costly and potentially risky human electrophysiological assays. Hence, the map serves as a novel tool for use in pharmaceutical research and development. This article is protected by copyright. All rights reserved.

A BAC clone fingerprinting approach to the detection of human genome rearrangements

PubMed Central

Krzywinski, Martin; Bosdet, Ian; Mathewson, Carrie; Wye, Natasja; Brebner, Jay; Chiu, Readman; Corbett, Richard; Field, Matthew; Lee, Darlene; Pugh, Trevor; Volik, Stas; Siddiqui, Asim; Jones, Steven; Schein, Jacquie; Collins, Collin; Marra, Marco

2007-01-01

We present a method, called fingerprint profiling (FPP), that uses restriction digest fingerprints of bacterial artificial chromosome clones to detect and classify rearrangements in the human genome. The approach uses alignment of experimental fingerprint patterns to in silico digests of the sequence assembly and is capable of detecting micro-deletions (1-5 kb) and balanced rearrangements. Our method has compelling potential for use as a whole-genome method for the identification and characterization of human genome rearrangements. PMID:17953769

An Efficient Strategy Combining SSR Markers- and Advanced QTL-seq-driven QTL Mapping Unravels Candidate Genes Regulating Grain Weight in Rice

PubMed Central

Daware, Anurag; Das, Sweta; Srivastava, Rishi; Badoni, Saurabh; Singh, Ashok K.; Agarwal, Pinky; Parida, Swarup K.; Tyagi, Akhilesh K.

2016-01-01

Development and use of genome-wide informative simple sequence repeat (SSR) markers and novel integrated genomic strategies are vital to drive genomics-assisted breeding applications and for efficient dissection of quantitative trait loci (QTLs) underlying complex traits in rice. The present study developed 6244 genome-wide informative SSR markers exhibiting in silico fragment length polymorphism based on repeat-unit variations among genomic sequences of 11 indica, japonica, aus, and wild rice accessions. These markers were mapped on diverse coding and non-coding sequence components of known cloned/candidate genes annotated from 12 chromosomes and revealed a much higher amplification (97%) and polymorphic potential (88%) along with wider genetic/functional diversity level (16–74% with a mean 53%) especially among accessions belonging to indica cultivar group, suggesting their utility in large-scale genomics-assisted breeding applications in rice. A high-density 3791 SSR markers-anchored genetic linkage map (IR 64 × Sonasal) spanning 2060 cM total map-length with an average inter-marker distance of 0.54 cM was generated. This reference genetic map identified six major genomic regions harboring robust QTLs (31% combined phenotypic variation explained with a 5.7–8.7 LOD) governing grain weight on six rice chromosomes. One strong grain weight major QTL region (OsqGW5.1) was narrowed-down by integrating traditional QTL mapping with high-resolution QTL region-specific integrated SSR and single nucleotide polymorphism markers-based QTL-seq analysis and differential expression profiling. This led us to delineate two natural allelic variants in two known cis-regulatory elements (RAV1AAT and CARGCW8GAT) of glycosyl hydrolase and serine carboxypeptidase genes exhibiting pronounced seed-specific differential regulation in low (Sonasal) and high (IR 64) grain weight mapping parental accessions. Our genome-wide SSR marker resource (polymorphic within/between diverse cultivar groups) and integrated genomic strategy can efficiently scan functionally relevant potential molecular tags (markers, candidate genes and alleles) regulating complex agronomic traits (grain weight) and expedite marker-assisted genetic enhancement in rice. PMID:27833617

The Release 6 reference sequence of the Drosophila melanogaster genome

DOE PAGES

Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.; ...

2015-01-14

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy andmore » middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.« less

The Release 6 reference sequence of the Drosophila melanogaster genome

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

Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy andmore » middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.« less

A High-throughput AFLP-based Method for Constructing Integrated Genetic and Physical Maps: Progress Toward a Sorghum Genome Map

PubMed Central

Klein, Patricia E.; Klein, Robert R.; Cartinhour, Samuel W.; Ulanch, Paul E.; Dong, Jianmin; Obert, Jacque A.; Morishige, Daryl T.; Schlueter, Shannon D.; Childs, Kevin L.; Ale, Melissa; Mullet, John E.

2000-01-01

Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones (∼4× genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified ∼2400 BACs and ordered ∼700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located ∼200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in ∼65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map of the sorghum genome. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF218263.] PMID:10854411

In Silico and Fluorescence In Situ Hybridization Mapping Reveals Collinearity between the Pennisetum squamulatum Apomixis Carrier-Chromosome and Chromosome 2 of Sorghum and Foxtail Millet.

PubMed

Sapkota, Sirjan; Conner, Joann A; Hanna, Wayne W; Simon, Bindu; Fengler, Kevin; Deschamps, Stéphane; Cigan, Mark; Ozias-Akins, Peggy

2016-01-01

Apomixis, or clonal propagation through seed, is a trait identified within multiple species of the grass family (Poaceae). The genetic locus controlling apomixis in Pennisetum squamulatum (syn Cenchrus squamulatus) and Cenchrus ciliaris (syn Pennisetum ciliare, buffelgrass) is the apospory-specific genomic region (ASGR). Previously, the ASGR was shown to be highly conserved but inverted in marker order between P. squamulatum and C. ciliaris based on fluorescence in situ hybridization (FISH) and varied in both karyotype and position of the ASGR on the ASGR-carrier chromosome among other apomictic Cenchrus/Pennisetum species. Using in silico transcript mapping and verification of physical positions of some of the transcripts via FISH, we discovered that the ASGR-carrier chromosome from P. squamulatum is collinear with chromosome 2 of foxtail millet and sorghum outside of the ASGR. The in silico ordering of the ASGR-carrier chromosome markers, previously unmapped in P. squamulatum, allowed for the identification of a backcross line with structural changes to the P. squamulatum ASGR-carrier chromosome derived from gamma irradiated pollen.

In Silico and Fluorescence In Situ Hybridization Mapping Reveals Collinearity between the Pennisetum squamulatum Apomixis Carrier-Chromosome and Chromosome 2 of Sorghum and Foxtail Millet

PubMed Central

Sapkota, Sirjan; Conner, Joann A.; Hanna, Wayne W.; Simon, Bindu; Fengler, Kevin; Deschamps, Stéphane; Cigan, Mark; Ozias-Akins, Peggy

2016-01-01

Apomixis, or clonal propagation through seed, is a trait identified within multiple species of the grass family (Poaceae). The genetic locus controlling apomixis in Pennisetum squamulatum (syn Cenchrus squamulatus) and Cenchrus ciliaris (syn Pennisetum ciliare, buffelgrass) is the apospory-specific genomic region (ASGR). Previously, the ASGR was shown to be highly conserved but inverted in marker order between P. squamulatum and C. ciliaris based on fluorescence in situ hybridization (FISH) and varied in both karyotype and position of the ASGR on the ASGR-carrier chromosome among other apomictic Cenchrus/Pennisetum species. Using in silico transcript mapping and verification of physical positions of some of the transcripts via FISH, we discovered that the ASGR-carrier chromosome from P. squamulatum is collinear with chromosome 2 of foxtail millet and sorghum outside of the ASGR. The in silico ordering of the ASGR-carrier chromosome markers, previously unmapped in P. squamulatum, allowed for the identification of a backcross line with structural changes to the P. squamulatum ASGR-carrier chromosome derived from gamma irradiated pollen. PMID:27031857

Physical mapping of complex genomes

DOEpatents

Evans, G.A.

1993-06-15

A method for the simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts in the pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert in the common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed.

Comparison of peanut gentics and physical maps provided insights on collinearity, reversions and translocations

USDA-ARS?s Scientific Manuscript database

Genetic and physical maps are the valuable resources for peanut research community in understanding genome organization and serving as the basis for map-based cloning and marker-assisted selection. Physical maps of two diploid wild peanut progenitor species, Arachis duranensis (A genome) and A. ipae...

SNPs in genes functional in starch-sugar interconversion associate with natural variation of tuber starch and sugar content of potato (Solanum tuberosum L.).

PubMed

Schreiber, Lena; Nader-Nieto, Anna Camila; Schönhals, Elske Maria; Walkemeier, Birgit; Gebhardt, Christiane

2014-07-31

Starch accumulation and breakdown are vital processes in plant storage organs such as seeds, roots, and tubers. In tubers of potato (Solanum tuberosum L.) a small fraction of starch is converted into the reducing sugars glucose and fructose. Reducing sugars accumulate in response to cold temperatures. Even small quantities of reducing sugars affect negatively the quality of processed products such as chips and French fries. Tuber starch and sugar content are inversely correlated complex traits that are controlled by multiple genetic and environmental factors. Based on in silico annotation of the potato genome sequence, 123 loci are involved in starch-sugar interconversion, approximately half of which have been previously cloned and characterized. By means of candidate gene association mapping, we identified single-nucleotide polymorphisms (SNPs) in eight genes known to have key functions in starch-sugar interconversion, which were diagnostic for increased tuber starch and/or decreased sugar content and vice versa. Most positive or negative effects of SNPs on tuber-reducing sugar content were reproducible in two different collections of potato cultivars. The diagnostic SNP markers are useful for breeding applications. An allele of the plastidic starch phosphorylase PHO1a associated with increased tuber starch content was cloned as full-length cDNA and characterized. The PHO1a-HA allele has several amino acid changes, one of which is unique among all known starch/glycogen phosphorylases. This mutation might cause reduced enzyme activity due to impaired formation of the active dimers, thereby limiting starch breakdown. Copyright © 2014 Schreiber et al.

SNPs in Genes Functional in Starch-Sugar Interconversion Associate with Natural Variation of Tuber Starch and Sugar Content of Potato (Solanum tuberosum L.)

PubMed Central

Schreiber, Lena; Nader-Nieto, Anna Camila; Schönhals, Elske Maria; Walkemeier, Birgit; Gebhardt, Christiane

2014-01-01

Starch accumulation and breakdown are vital processes in plant storage organs such as seeds, roots, and tubers. In tubers of potato (Solanum tuberosum L.) a small fraction of starch is converted into the reducing sugars glucose and fructose. Reducing sugars accumulate in response to cold temperatures. Even small quantities of reducing sugars affect negatively the quality of processed products such as chips and French fries. Tuber starch and sugar content are inversely correlated complex traits that are controlled by multiple genetic and environmental factors. Based on in silico annotation of the potato genome sequence, 123 loci are involved in starch-sugar interconversion, approximately half of which have been previously cloned and characterized. By means of candidate gene association mapping, we identified single-nucleotide polymorphisms (SNPs) in eight genes known to have key functions in starch-sugar interconversion, which were diagnostic for increased tuber starch and/or decreased sugar content and vice versa. Most positive or negative effects of SNPs on tuber-reducing sugar content were reproducible in two different collections of potato cultivars. The diagnostic SNP markers are useful for breeding applications. An allele of the plastidic starch phosphorylase PHO1a associated with increased tuber starch content was cloned as full-length cDNA and characterized. The PHO1a-HA allele has several amino acid changes, one of which is unique among all known starch/glycogen phosphorylases. This mutation might cause reduced enzyme activity due to impaired formation of the active dimers, thereby limiting starch breakdown. PMID:25081979

Novel SSR Markers from BAC-End Sequences, DArT Arrays and a Comprehensive Genetic Map with 1,291 Marker Loci for Chickpea (Cicer arietinum L.)

PubMed Central

Nayak, Spurthi N.; Varghese, Nicy; Shah, Trushar M.; Penmetsa, R. Varma; Thirunavukkarasu, Nepolean; Gudipati, Srivani; Gaur, Pooran M.; Kulwal, Pawan L.; Upadhyaya, Hari D.; KaviKishor, Polavarapu B.; Winter, Peter; Kahl, Günter; Town, Christopher D.; Kilian, Andrzej; Cook, Douglas R.; Varshney, Rajeev K.

2011-01-01

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes. PMID:22102885

DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

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

Lu, Chun-Mei; Kwan, Johnson; Baumgartner, Adolf

2009-01-30

Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpointmore » mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome (BAC) clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multi-clone and multi-color mapping experiments do not generate additional information. Our pooling protocol described here with examples from thyroid cancer research and PGD accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as three to four days.« less

Chromosomal Mapping of Canine-Derived BAC Clones to the Red Fox and American Mink Genomes

PubMed Central

Vorobieva, Nadegda V.; Beklemisheva, Violetta R.; Johnson, Jennifer L.; Temnykh, Svetlana V.; Yudkin, Dmitry V.; Trut, Lyudmila N.; Andre, Catherine; Galibert, Francis; Aguirre, Gustavo D.; Acland, Gregory M.; Graphodatsky, Alexander S.

2009-01-01

High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene–containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations. PMID:19546120

Chromosomal mapping of canine-derived BAC clones to the red fox and American mink genomes.

PubMed

Kukekova, Anna V; Vorobieva, Nadegda V; Beklemisheva, Violetta R; Johnson, Jennifer L; Temnykh, Svetlana V; Yudkin, Dmitry V; Trut, Lyudmila N; Andre, Catherine; Galibert, Francis; Aguirre, Gustavo D; Acland, Gregory M; Graphodatsky, Alexander S

2009-01-01

High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene-containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations.

An "in Silico" DNA Cloning Experiment for the Biochemistry Laboratory

ERIC Educational Resources Information Center

Elkins, Kelly M.

2011-01-01

This laboratory exercise introduces students to concepts in recombinant DNA technology while accommodating a major semester project in protein purification, structure, and function in a biochemistry laboratory for junior- and senior-level undergraduate students. It is also suitable for forensic science courses focused in DNA biology and advanced…

Reconstruction of an SSR-based Magnaporthe oryzae physical map to locate avirulence gene AvrPi12.

PubMed

Li, Tonghui; Wen, Jianqiang; Zhang, Yaling; Correll, James; Wang, Ling; Pan, Qinghua

2018-05-31

Pathogen avirulence (Avr) genes can evolve rapidly when challenged by the widespread deployment of host genes for resistance. They can be effectively isolated by positional cloning provided a robust and well-populated genetic map is available. An updated, SSR-based physical map of the rice blast pathogen Magnaporthe oryzae (Mo) has been constructed based on 116 of the 120 SSRs used to assemble the last map, along with 18 newly developed ones. A comparison between the two versions of the map has revealed an altered marker content and order within most of the Mo chromosomes. The avirulence gene AvrPi12 was mapped in a population of 219 progeny derived from a cross between the two Mo isolates CHL42 and CHL357. A bulked segregant analysis indicated that the gene was located on chromosome 6, a conclusion borne out by an analysis of the pattern of segregation shown by individual isolates. Six additional PCR-based markers were developed to improve the map resolution in the key region. AvrPi12 was finally located within the sub-telomeric region of chromosome 6, distal to the SSR locus LSM6-5. The improved SSR-based linkage map should be useful as a platform for gene mapping and isolation in Mo. It was used to establish the location of AvrPi12, thereby providing a starting point for its positional cloning.

Creation of BAC genomic resources for cocoa ( Theobroma cacao L.) for physical mapping of RGA containing BAC clones.

PubMed

Clément, D; Lanaud, C; Sabau, X; Fouet, O; Le Cunff, L; Ruiz, E; Risterucci, A M; Glaszmann, J C; Piffanelli, P

2004-05-01

We have constructed and validated the first cocoa ( Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp ( palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.

Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

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

Arondel, V.; Lemieux, B.; Hwang, I.

1992-11-20

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1more » tabs.« less

High-throughput gene mapping in Caenorhabditis elegans.

PubMed

Swan, Kathryn A; Curtis, Damian E; McKusick, Kathleen B; Voinov, Alexander V; Mapa, Felipa A; Cancilla, Michael R

2002-07-01

Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 +/- 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18.

Physical mapping of complex genomes

DOEpatents

Evans, Glen A.

1993-01-01

Method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts int he pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert int he common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed. In other preferred embodiments, the cosmid clones are arranged in a three dimensional matrix, pooled and compared in threes according to intersecting planes of the three dimensional matrix. Arrangements corresponding to geometries of higher dimensions may also be prepared and used to simultaneously identify overlapping clones in highly complex libraries with relatively few hybridization reactions.

Saturation of an intra-gene pool linkage map: toward unified consensus linkage map in common bean

USDA-ARS?s Scientific Manuscript database

Map-based cloning to find genes of interest and marker assisted selection (MAS) requires good genetic maps with high reproducible markers. In this study, we saturated the linkage map of the intra-gene pool population of common bean DOR364×BAT477 (DB) by evaluating 2,706 molecular markers in includin...

In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate.

PubMed

Cravo, Pedro; Machado, Renato B; Leite, Juliana A; Leda, Taizy; Suwanarusk, Rossarin; Bittencourt, Najara; Albrecht, Letusa; Judice, Carla; Lopes, Stefanie C P; Lacerda, Marcus V G; Ferreira, Marcelo U; Soares, Irene S; Goh, Yun Shan; Bargieri, Daniel Y; Nosten, François; Russell, Bruce; Rénia, Laurent; Costa, Fabio T M

2018-01-10

Technical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated. Detailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity. The MAEBL antigen is promising candidate towards the development of a malaria vaccine.

Development of a D genome specific marker resource for diploid and hexaploid wheat

USDA-ARS?s Scientific Manuscript database

Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those of the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of po...

Imperfect duplicate insertions type of mutations in plasmepsin V modulates binding properties of PEXEL motifs of export proteins in Indian Plasmodium vivax.

PubMed

Rawat, Manmeet; Vijay, Sonam; Gupta, Yash; Tiwari, Pramod Kumar; Sharma, Arun

2013-01-01

Plasmepsin V (PM-V) have functionally conserved orthologues across the Plasmodium genus who's binding and antigenic processing at the PEXEL motifs for export about 200-300 essential proteins is important for the virulence and viability of the causative Plasmodium species. This study was undertaken to determine P. vivax plasmepsin V Ind (PvPM-V-Ind) PEXEL motif export pathway for pathogenicity-related proteins/antigens export thereby altering plasmodium exportome during erythrocytic stages. We identify and characterize Plasmodium vivax plasmepsin-V-Ind (mutant) gene by cloning, sequence analysis, in silico bioinformatic protocols and structural modeling predictions based on docking studies on binding capacity with PEXEL motifs processing in terms of binding and accessibility of export proteins. Cloning and sequence analysis for genetic diversity demonstrates PvPM-V-Ind (mutant) gene is highly conserved among all isolates from different geographical regions of India. Imperfect duplicate insertion types of mutations (SVSE from 246-249 AA and SLSE from 266-269 AA) were identified among all Indian isolates in comparison to P.vivax Sal-1 (PvPM-V-Sal 1) isolate. In silico bioinformatics interaction studies of PEXEL peptide and active enzyme reveal that PvPM-V-Ind (mutant) is only active in endoplasmic reticulum lumen and membrane embedding is essential for activation of plasmepsin V. Structural modeling predictions based on docking studies with PEXEL motif show significant variation in substrate protein binding of these imperfect mutations with data mined PEXEL sequences. The predicted variation in the docking score and interacting amino acids of PvPM-V-Ind (mutant) proteins with PEXEL and lopinavir suggests a modulation in the activity of PvPM-V in terms of binding and accessibility at these sites. Our functional modeled validation of PvPM-V-Ind (mutant) imperfect duplicate insertions with data mined PEXEL sequences leading to altered binding and substrate accessibility of the enzyme makes it a plausible target to investigate export mechanisms for in silico virtual screening and novel pharmacophore designing.

Imperfect Duplicate Insertions Type of Mutations in Plasmepsin V Modulates Binding Properties of PEXEL Motifs of Export Proteins in Indian Plasmodium vivax

PubMed Central

Rawat, Manmeet; Vijay, Sonam; Gupta, Yash; Tiwari, Pramod Kumar; Sharma, Arun

2013-01-01

Introduction Plasmepsin V (PM-V) have functionally conserved orthologues across the Plasmodium genus who's binding and antigenic processing at the PEXEL motifs for export about 200–300 essential proteins is important for the virulence and viability of the causative Plasmodium species. This study was undertaken to determine P. vivax plasmepsin V Ind (PvPM-V-Ind) PEXEL motif export pathway for pathogenicity-related proteins/antigens export thereby altering plasmodium exportome during erythrocytic stages. Method We identify and characterize Plasmodium vivax plasmepsin-V-Ind (mutant) gene by cloning, sequence analysis, in silico bioinformatic protocols and structural modeling predictions based on docking studies on binding capacity with PEXEL motifs processing in terms of binding and accessibility of export proteins. Results Cloning and sequence analysis for genetic diversity demonstrates PvPM-V-Ind (mutant) gene is highly conserved among all isolates from different geographical regions of India. Imperfect duplicate insertion types of mutations (SVSE from 246–249 AA and SLSE from 266–269 AA) were identified among all Indian isolates in comparison to P.vivax Sal-1 (PvPM-V-Sal 1) isolate. In silico bioinformatics interaction studies of PEXEL peptide and active enzyme reveal that PvPM-V-Ind (mutant) is only active in endoplasmic reticulum lumen and membrane embedding is essential for activation of plasmepsin V. Structural modeling predictions based on docking studies with PEXEL motif show significant variation in substrate protein binding of these imperfect mutations with data mined PEXEL sequences. The predicted variation in the docking score and interacting amino acids of PvPM-V-Ind (mutant) proteins with PEXEL and lopinavir suggests a modulation in the activity of PvPM-V in terms of binding and accessibility at these sites. Conclusion/Significance Our functional modeled validation of PvPM-V-Ind (mutant) imperfect duplicate insertions with data mined PEXEL sequences leading to altered binding and substrate accessibility of the enzyme makes it a plausible target to investigate export mechanisms for in silico virtual screening and novel pharmacophore designing. PMID:23555891

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA

PubMed Central

Zozaya-Valdés, Enrique; Porter, Jessica L.; Coventry, John; Fyfe, Janet A. M.; Carter, Glen P.; Gonçalves da Silva, Anders; Schultz, Mark B.; Seemann, Torsten; Johnson, Paul D. R.; Stewardson, Andrew J.; Bastian, Ivan; Roberts, Sally A.; Howden, Benjamin P.; Williamson, Deborah A.

2017-01-01

ABSTRACT Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera. Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera. PMID:28381604

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

PubMed

Zozaya-Valdés, Enrique; Porter, Jessica L; Coventry, John; Fyfe, Janet A M; Carter, Glen P; Gonçalves da Silva, Anders; Schultz, Mark B; Seemann, Torsten; Johnson, Paul D R; Stewardson, Andrew J; Bastian, Ivan; Roberts, Sally A; Howden, Benjamin P; Williamson, Deborah A; Stinear, Timothy P

2017-06-01

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera . Copyright © 2017 American Society for Microbiology.

In silico cloning, expression of Rieske-like apoprotein gene and protein subcellular localization in the Pacific oyster, Crassostrea gigas.

PubMed

He, Xiaocui; Zhang, Yang; Yu, Ziniu

2010-10-01

Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined, respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5'- and 3'-untranslated regions of 35 and 161 bp, respectively, with an open reading frame of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe-2S] cluster binding sites and highly matched-pair tertiary structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences, box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological function.

A first generation BAC-based physical map of the rainbow trout genome

PubMed Central

Palti, Yniv; Luo, Ming-Cheng; Hu, Yuqin; Genet, Carine; You, Frank M; Vallejo, Roger L; Thorgaard, Gary H; Wheeler, Paul A; Rexroad, Caird E

2009-01-01

Background Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. A bacterial artificial chromosome (BAC) physical map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) for improving rainbow trout aquaculture production. This resource will also facilitate efforts to obtain and assemble a whole-genome reference sequence for this species. Results The physical map was constructed from DNA fingerprinting of 192,096 BAC clones using the 4-color high-information content fingerprinting (HICF) method. The clones were assembled into physical map contigs using the finger-printing contig (FPC) program. The map is composed of 4,173 contigs and 9,379 singletons. The total number of unique fingerprinting fragments (consensus bands) in contigs is 1,185,157, which corresponds to an estimated physical length of 2.0 Gb. The map assembly was validated by 1) comparison with probe hybridization results and agarose gel fingerprinting contigs; and 2) anchoring large contigs to the microsatellite-based genetic linkage map. Conclusion The production and validation of the first BAC physical map of the rainbow trout genome is described in this paper. We are currently integrating this map with the NCCCWA genetic map using more than 200 microsatellites isolated from BAC end sequences and by identifying BACs that harbor more than 300 previously mapped markers. The availability of an integrated physical and genetic map will enable detailed comparative genome analyses, fine mapping of QTL, positional cloning, selection of positional candidate genes for economically important traits and the incorporation of MAS into rainbow trout breeding programs. PMID:19814815

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

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

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with atmore » least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.« less

Mapping Flagellar Genes in Chlamydomonas Using Restriction Fragment Length Polymorphisms

PubMed Central

Ranum, LPW.; Thompson, M. D.; Schloss, J. A.; Lefebvre, P. A.; Silflow, C. D.

1988-01-01

To correlate cloned nuclear DNA sequences with previously characterized mutations in Chlamydomonas and, to gain insight into the organization of its nuclear genome, we have begun to map molecular markers using restriction fragment length polymorphisms (RFLPs). A Chlamydomonas reinhardtii strain (CC-29) containing phenotypic markers on nine of the 19 linkage groups was crossed to the interfertile species Chlamydomonas smithii. DNA from each member of 22 randomly selected tetrads was analyzed for the segregation of RFLPs associated with cloned genes detected by hybridization with radioactive DNA probes. The current set of markers allows the detection of linkage to new molecular markers over approximately 54% of the existing genetic map. This study focused on mapping cloned flagellar genes and genes whose transcripts accumulate after deflagellation. Twelve different molecular clones have been assigned to seven linkage groups. The α-1 tubulin gene maps to linkage group III and is linked to the genomic sequence homologous to pcf6-100, a cDNA clone whose corresponding transcript accumulates after deflagellation. The α-2 tubulin gene maps to linkage group IV. The two β-tubulin genes are linked, with the β-1 gene being approximately 12 cM more distal from the centromere than the β-2 gene. A clone corresponding to a 73-kD dynein protein maps to the opposite arm of the same linkage group. The gene corresponding to the cDNA clone pcf6-187, whose mRNA accumulates after deflagellation, maps very close to the tightly linked pf-26 and pf-1 mutations on linkage group V. PMID:2906025

Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.)

PubMed Central

Asha, Srinivasan; Soniya, Eppurath V.

2016-01-01

Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5′tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5′tRFs in the infected leaf and root. The abundance of 5′tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5′AlaCGC tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5′Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper. PMID:27313593

Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

PubMed

Asha, Srinivasan; Soniya, Eppurath V

2016-01-01

Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper.

Isolation and characterization of a cDNA clone specific for avian vitellogenin II.

PubMed Central

Protter, A A; Wang, S Y; Shelness, G S; Ostapchuk, P; Williams, D L

1982-01-01

A clone for vitellogenin, a major avian, estrogen responsive egg yolk protein, was isolated from the cDNA library of estrogen-induced rooster liver. Two forms of plasma vitellogenin, vitellogenin I (VTG I) and vitellogenin II (VTG II), distinguishable on the basis of their unique partial proteolysis maps, have been characterized and their corresponding hepatic precursor forms identified. We have used this criterion to specifically characterize which vitellogenin protein had been cloned. Partial proteolysis maps of BTG I and VTG II standards, synthesized in vivo, were compared to maps of protein synthesized in vitro using RNA hybrid-selected by the vitellogenin plasmid. Eight major digest fragments were found common to the in vitro synthesized vitellogenin and the VTG II standard while no fragments were observed to correspond to the VTG I map. A restriction map of the VTG II cDNA clone permits comparison to previously described cDNA and genomic vitellogenin clones. Images PMID:6182527

Optically Mapping Multiple Bacterial Genomes Simultaneously in a Single Run

DTIC Science & Technology

2011-11-21

sequence orientation. We have demonstrated mapping of Shigella dysenteriae and Escherichia coli simultaneously, despite their very close phylogenetic...relationship ( Shigella and Escherichia coli are generally considered to be within a single species, but are segregated at the genus level for historical...reasons [4]); two clones of Shigella would likely not map together successfully using the mixed DNA method. Similarly, based on reference maps being

Designing B- and T-cell multi-epitope based subunit vaccine using immunoinformatics approach to control Zika virus infection.

PubMed

Kumar Pandey, Rajan; Ojha, Rupal; Mishra, Amit; Kumar Prajapati, Vijay

2018-06-14

The Zika virus is a rapidly spreading Aedes mosquito-borne sickness, which creates an unanticipated linkage birth deformity and neurological turmoil. This study represents the use of the combinatorial immunoinformatics approach to develop a multiepitope subunit vaccine using the structural and nonstructural proteins of the Zika virus. The designed subunit vaccine consists of cytotoxic T-lymphocyte and helper T-lymphocyte epitopes accompanied by suitable adjuvant and linkers. The presence of humoral immune response specific B-cell epitopes was also confirmed by B-cell epitope mapping among vaccine protein. Further, the vaccine protein was characterized for its allergenicity, antigenicity, and physiochemical parameters and found to be safe and immunogenic. Molecular docking and molecular dynamics studies of the vaccine protein with the toll-like receptor-3 were performed to ensure the binding affinity and stability of their complex. Finally, in silico cloning was performed for the effective expression of vaccine construct in the microbial system (Escherichia coli K12 strain). Aforementioned approaches result in the multiepitope subunit vaccine which may have the ability to induce cellular as well as humoral immune response. Moreover, this study needs the experimental validation to prove the immunogenic and protective behavior of the developed subunit vaccine. © 2018 Wiley Periodicals, Inc., A Wiley Company.

A framework linkage map of perennial ryegrass based on SSR markers

Treesearch

G.P. Gill; P.L. Wilcox; D.J. Whittaker; R.A. Winz; P. Bickerstaff; Craig E. Echt; J. Kent; M.O. Humphreys; K.M. Elborough; R.C. Gardner

2006-01-01

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third ( 124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci...

Quantitative DNA fiber mapping

DOEpatents

Gray, Joe W.; Weier, Heinz-Ulrich G.

1998-01-01

The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

Genome-Wide Discovery of Microsatellite Markers from Diploid Progenitor Species, Arachis duranensis and A. ipaensis, and Their Application in Cultivated Peanut (A. hypogaea)

PubMed Central

Zhao, Chuanzhi; Qiu, Jingjing; Agarwal, Gaurav; Wang, Jiangshan; Ren, Xuezhen; Xia, Han; Guo, Baozhu; Ma, Changle; Wan, Shubo; Bertioli, David J.; Varshney, Rajeev K.; Pandey, Manish K.; Wang, Xingjun

2017-01-01

Despite several efforts in the last decade toward development of simple sequence repeat (SSR) markers in peanut, there is still a need for more markers for conducting different genetic and breeding studies. With the effort of the International Peanut Genome Initiative, the availability of reference genome for both the diploid progenitors of cultivated peanut allowed us to identify 135,529 and 199,957 SSRs from the A (Arachis duranensis) and B genomes (Arachis ipaensis), respectively. Genome sequence analysis showed uneven distribution of the SSR motifs across genomes with variation in parameters such as SSR type, repeat number, and SSR length. Using the flanking sequences of identified SSRs, primers were designed for 51,354 and 60,893 SSRs with densities of 49 and 45 SSRs per Mb in A. duranensis and A. ipaensis, respectively. In silico PCR analysis of these SSR markers showed high transferability between wild and cultivated Arachis species. Two physical maps were developed for the A genome and the B genome using these SSR markers, and two reported disease resistance quantitative trait loci (QTLs), qF2TSWV5 for tomato spotted wilt virus (TSWV) and qF2LS6 for leaf spot (LS), were mapped in the 8.135 Mb region of chromosome A04 of A. duranensis. From this genomic region, 719 novel SSR markers were developed, which provide the possibility for fine mapping of these QTLs. In addition, this region also harbors 652 genes and 49 of these are defense related genes, including two NB-ARC genes, three LRR receptor-like genes and three WRKY transcription factors. These disease resistance related genes could contribute to resistance to viral (such as TSWV) and fungal (such as LS) diseases in peanut. In summary, this study not only provides a large number of molecular markers for potential use in peanut genetic map development and QTL mapping but also for map-based gene cloning and molecular breeding. PMID:28769940

Randomly picked cosmid clones overlap the pyrB and oriC gap in the physical map of the E. coli chromosome.

PubMed Central

Knott, V; Rees, D J; Cheng, Z; Brownlee, G G

1988-01-01

Sets of overlapping cosmid clones generated by random sampling and fingerprinting methods complement data at pyrB (96.5') and oriC (84') in the published physical map of E. coli. A new cloning strategy using sheared DNA, and a low copy, inducible cosmid vector were used in order to reduce bias in libraries, in conjunction with micro-methods for preparing cosmid DNA from a large number of clones. Our results are relevant to the design of the best approach to the physical mapping of large genomes. PMID:2834694

Identification of Biomarkers for Resistance to Fusarium oxysporum f. sp. cubense Infection and in Silico Studies in Musa paradisiaca Cultivar Puttabale through Proteomic Approach.

PubMed

Ramu, Venkatesh; Venkatarangaiah, Krishna; Krishnappa, Pradeepa; Shimoga Rajanna, Santosh Kumar; Deeplanaik, Nagaraja; Chandra Pal, Anup; Kini, Kukkundoor Ramachandra

2016-02-24

Panama wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the major disease constraints of banana production. Previously, we reported the disease resistance Musa paradisiaca cv. puttabale clones developed from Ethylmethanesulfonate and Foc culture filtrate against Foc inoculation. Here, the same resistant clones and susceptible clones were used for the study of protein accumulation against Foc inoculation by two-dimensional gel electrophoresis (2-DE), their expression pattern and an in silico approach. The present investigation revealed mass-spectrometry identified 16 proteins that were over accumulated and 5 proteins that were under accumulated as compared to the control. The polyphosphoinositide binding protein ssh2p (PBPssh2p) and Indoleacetic acid-induced-like (IAA) protein showed significant up-regulation and down-regulation. The docking of the pathogenesis-related protein (PR) with the fungal protein endopolygalacturonase (PG) exemplify the three ionic interactions and seven hydrophobic residues that tends to good interaction at the active site of PG with free energy of assembly dissociation (1.5 kcal/mol). The protein-ligand docking of the Peptide methionine sulfoxide reductase chloroplastic-like protein (PMSRc) with the ligand β-1,3 glucan showed minimum binding energy (-6.48 kcal/mol) and docking energy (-8.2 kcal/mol) with an interaction of nine amino-acid residues. These explorations accelerate the research in designing the host pathogen interaction studies for the better management of diseases.

Identification of Biomarkers for Resistance to Fusarium oxysporum f. sp. cubense Infection and in Silico Studies in Musa paradisiaca Cultivar Puttabale through Proteomic Approach

PubMed Central

Ramu, Venkatesh; Venkatarangaiah, Krishna; Krishnappa, Pradeepa; Shimoga Rajanna, Santosh Kumar; Deeplanaik, Nagaraja; Chandra Pal, Anup; Kini, Kukkundoor Ramachandra

2016-01-01

Panama wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the major disease constraints of banana production. Previously, we reported the disease resistance Musa paradisiaca cv. puttabale clones developed from Ethylmethanesulfonate and Foc culture filtrate against Foc inoculation. Here, the same resistant clones and susceptible clones were used for the study of protein accumulation against Foc inoculation by two-dimensional gel electrophoresis (2-DE), their expression pattern and an in silico approach. The present investigation revealed mass-spectrometry identified 16 proteins that were over accumulated and 5 proteins that were under accumulated as compared to the control. The polyphosphoinositide binding protein ssh2p (PBPssh2p) and Indoleacetic acid-induced-like (IAA) protein showed significant up-regulation and down-regulation. The docking of the pathogenesis-related protein (PR) with the fungal protein endopolygalacturonase (PG) exemplify the three ionic interactions and seven hydrophobic residues that tends to good interaction at the active site of PG with free energy of assembly dissociation (1.5 kcal/mol). The protein-ligand docking of the Peptide methionine sulfoxide reductase chloroplastic-like protein (PMSRc) with the ligand β-1,3 glucan showed minimum binding energy (−6.48 kcal/mol) and docking energy (−8.2 kcal/mol) with an interaction of nine amino-acid residues. These explorations accelerate the research in designing the host pathogen interaction studies for the better management of diseases. PMID:28248219

Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

PubMed Central

Marraccini, Pierre; Vinecky, Felipe; Alves, Gabriel S.C.; Ramos, Humberto J.O.; Elbelt, Sonia; Vieira, Natalia G.; Carneiro, Fernanda A.; Sujii, Patricia S.; Alekcevetch, Jean C.; Silva, Vânia A.; DaMatta, Fábio M.; Ferrão, Maria A.G.; Leroy, Thierry; Pot, David; Vieira, Luiz G.E.; da Silva, Felipe R.; Andrade, Alan C.

2012-01-01

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora. PMID:22511801

Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members

PubMed Central

Dolinšek, Jan; Dorninger, Christiane; Lagkouvardos, Ilias; Wagner, Michael

2013-01-01

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer “Candidatus Nitrospira defluvii.” In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition. PMID:23263968

In Silico Prediction of Neuropeptides/Peptide Hormone Transcripts in the Cheilostome Bryozoan Bugula neritina

PubMed Central

Zhang, Gen; He, Li-Sheng; Qian, Pei-Yuan

2016-01-01

The bryozoan Bugula neritina has a biphasic life cycle that consists of a planktonic larval stage and a sessile juvenile/adult stage. The transition between these two stages is crucial for the development and recruitment of B. neritina. Metamorphosis in B. neritina is mediated by both the nervous system and the release of developmental signals. However, no research has been conducted to investigate the expression of neuropeptides (NP)/peptide hormones in B. neritina larvae. Here, we report a comprehensive study of the NP/peptide hormones in the marine bryozoan B. neritina based on in silico identification methods. We recovered 22 transcripts encompassing 11 NP/peptide hormone precursor transcript sequences. The transcript sequences of the 11 isolated NP precursors were validated by cDNA cloning using gene-specific primers. We also examined the expression of three peptide hormone precursor transcripts (BnFDSIG, BnILP1, BnGPB) in the coronate larvae of B. neritina, demonstrating their distinct expression patterns in the larvae. Overall, our findings serve as an important foundation for subsequent investigations of the peptidergic control of bryozoan larval behavior and settlement. PMID:27537380

In silico modeling techniques for predicting the tertiary structure of human H4 receptor.

PubMed

Zaid, Hilal; Raiyn, Jamal; Osman, Midhat; Falah, Mizied; Srouji, Samer; Rayan, Anwar

2016-01-01

First cloned in 2000, the human Histamine H4 Receptor (hH4R) is the last member of the histamine receptors family discovered so far, it belongs to the GPCR super-family and is involved in a wide variety of immunological and inflammatory responses. Potential hH4R antagonists are proposed to have therapeutic potential for the treatment of allergies, inflammation, asthma and colitis. So far, no hH4R ligands have been successfully introduced to the pharmaceutical market, which creates a strong demand for new selective ligands to be developed. in silico techniques and structural based modeling are likely to facilitate the achievement of this goal. In this review paper we attempt to cover the fundamental concepts of hH4R structure modeling and its implementations in drug discovery and development, especially those that have been experimentally tested and to highlight some ideas that are currently being discussed on the dynamic nature of hH4R and GPCRs, in regards to computerized techniques for 3-D structure modeling.

Chromosomal localization of actin genes in the malaria mosquito Anopheles darlingi

PubMed Central

BRIDI, L. C.; SHARAKHOVA, M. V.; SHARAKHOV, I. V.; CORDEIRO, J.; AZEVEDO, G. M.; TADEI, W. P.; RAFAEL, M. S.

2012-01-01

Physical and genetic maps have been used for chromosomal localization of genes in vectors of infectious diseases. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to precisely map genes of interest. We report physical mapping of two actin genes on polytene chromosomes of the major malaria vector in Amazon Anopheles darlingi. The clones with the actin genes sequences were obtained from a cDNA library constructed from RNA isolated from adult females and males of An. darlingi. Each of the two clones was mapped to a unique site on the chromosomal arm 2L in subdivisions 21A (clone pl05-A04) and 23B (clone pl17-G06). The obtained results together with previous mapping data provide a suitable basis for comparative genomics and for establishing chromosomal homologies among major malaria vectors. PMID:22804344

Transcriptome Sequencing of Hevea brasiliensis for Development of Microsatellite Markers and Construction of a Genetic Linkage Map

PubMed Central

Triwitayakorn, Kanokporn; Chatkulkawin, Pornsupa; Kanjanawattanawong, Supanath; Sraphet, Supajit; Yoocha, Thippawan; Sangsrakru, Duangjai; Chanprasert, Juntima; Ngamphiw, Chumpol; Jomchai, Nukoon; Therawattanasuk, Kanikar; Tangphatsornruang, Sithichoke

2011-01-01

To obtain more information on the Hevea brasiliensis genome, we sequenced the transcriptome from the vegetative shoot apex yielding 2 311 497 reads. Clustering and assembly of the reads produced a total of 113 313 unique sequences, comprising 28 387 isotigs and 84 926 singletons. Also, 17 819 expressed sequence tag (EST)-simple sequence repeats (SSRs) were identified from the data set. To demonstrate the use of this EST resource for marker development, primers were designed for 430 of the EST-SSRs. Three hundred and twenty-three primer pairs were amplifiable in H. brasiliensis clones. Polymorphic information content values of selected 47 SSRs among 20 H. brasiliensis clones ranged from 0.13 to 0.71, with an average of 0.51. A dendrogram of genetic similarities between the 20 H. brasiliensis clones using these 47 EST-SSRs suggested two distinct groups that correlated well with clone pedigree. These novel EST-SSRs together with the published SSRs were used for the construction of an integrated parental linkage map of H. brasiliensis based on 81 lines of an F1 mapping population. The map consisted of 97 loci, consisting of 37 novel EST-SSRs and 60 published SSRs, distributed on 23 linkage groups and covered 842.9 cM with a mean interval of 11.9 cM and ∼4 loci per linkage group. Although the numbers of linkage groups exceed the haploid number (18), but with several common markers between homologous linkage groups with the previous map indicated that the F1 map in this study is appropriate for further study in marker-assisted selection. PMID:22086998

A physical map of a BAC clone contig covering the entire autosome insertion between ovine MHC Class IIa and IIb

PubMed Central

2012-01-01

Background The ovine Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of the host to infectious diseases. Compared to human and mouse, the ovine MHC is interrupted by a large piece of autosome insertion via a hypothetical chromosome inversion that constitutes ~25% of ovine chromosome 20. The evolutionary consequence of such an inversion and an insertion (inversion/insertion) in relation to MHC function remains unknown. We previously constructed a BAC clone physical map for the ovine MHC exclusive of the insertion region. Here we report the construction of a high-density physical map covering the autosome insertion in order to address the question of what the inversion/insertion had to do with ruminants during the MHC evolution. Results A total of 119 pairs of comparative bovine oligo primers were utilized to screen an ovine BAC library for positive clones and the orders and overlapping relationships of the identified clones were determined by DNA fingerprinting, BAC-end sequencing, and sequence-specific PCR. A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb. Therefore, a continuous physical map covering the entire ovine autosome inversion/insertion region was successfully constructed. The map confirmed the bovine sequence assembly for the same homologous region. The DNA sequences of 185 BAC-ends have been deposited into NCBI database with the access numbers HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826. Conclusions We have constructed a high-density BAC clone physical map for the ovine autosome inversion/insertion between the MHC class IIa and IIb. The entire ovine MHC region is now fully covered by a continuous BAC clone contig. The physical map we generated will facilitate MHC functional studies in the ovine, as well as the comparative MHC evolution in ruminants. PMID:22897909

High-resolution genetic mapping at the Bph15 locus for brown planthopper resistance in rice (Oryza sativa L.).

PubMed

Yang, Haiyuan; You, Aiqing; Yang, Zhifan; Zhang, Futie; He, Ruifeng; Zhu, Lili; He, Guangcun

2004-12-01

Resistance to the brown planthopper (BPH), Nilaparvata lugens Stal, a devastating sucking insect pest of rice, is an important breeding objective in rice improvement programs. Bph15, one of the 17 major BPH resistance genes so far identified in both cultivated and wild rice, has been identified in an introgression line, B5, and mapped on chromosome 4 flanked by restriction fragment length polymorphism markers C820 and S11182. In order to pave the way for positional cloning of this gene, we have developed a high-resolution genetic map of Bph15 by positioning 21 DNA markers in the target chromosomal region. Mapping was based on a PCR-based screening of 9,472 F(2) individuals derived from a cross between RI93, a selected recombinant inbred line of B5 bearing the resistance gene Bph15, and a susceptible variety, Taichung Native 1, in order to identify recombinant plants within the Bph15 region. Recombinant F(2) individuals with the Bph15 genotype were determined by phenotype evaluation. Analysis of recombination events in the Bph15 region delimited the gene locus to an interval between markers RG1 and RG2 that co-segregated with the M1 marker. A genomic library of B5 was screened using these markers, and bacterial artificial chromosome clones spanning the Bph15 chromosome region were obtained. An assay of the recombinants using the sub-clones of these clones in combination with sequence analysis delimited the Bph15 gene to a genomic segment of approximately 47 kb. This result should serve as the basis for eventual isolation of the Bph15 resistance gene.

Candidate gene database and transcript map for peach, a model species for fruit trees.

PubMed

Horn, Renate; Lecouls, Anne-Claire; Callahan, Ann; Dandekar, Abhaya; Garay, Lilibeth; McCord, Per; Howad, Werner; Chan, Helen; Verde, Ignazio; Main, Doreen; Jung, Sook; Georgi, Laura; Forrest, Sam; Mook, Jennifer; Zhebentyayeva, Tatyana; Yu, Yeisoo; Kim, Hye Ran; Jesudurai, Christopher; Sosinski, Bryon; Arús, Pere; Baird, Vance; Parfitt, Dan; Reighard, Gregory; Scorza, Ralph; Tomkins, Jeffrey; Wing, Rod; Abbott, Albert Glenn

2005-05-01

Peach (Prunus persica) is a model species for the Rosaceae, which includes a number of economically important fruit tree species. To develop an extensive Prunus expressed sequence tag (EST) database for identifying and cloning the genes important to fruit and tree development, we generated 9,984 high-quality ESTs from a peach cDNA library of developing fruit mesocarp. After assembly and annotation, a putative peach unigene set consisting of 3,842 ESTs was defined. Gene ontology (GO) classification was assigned based on the annotation of the single "best hit" match against the Swiss-Prot database. No significant homology could be found in the GenBank nr databases for 24.3% of the sequences. Using core markers from the general Prunus genetic map, we anchored bacterial artificial chromosome (BAC) clones on the genetic map, thereby providing a framework for the construction of a physical and transcript map. A transcript map was developed by hybridizing 1,236 ESTs from the putative peach unigene set and an additional 68 peach cDNA clones against the peach BAC library. Hybridizing ESTs to genetically anchored BACs immediately localized 11.2% of the ESTs on the genetic map. ESTs showed a clustering of expressed genes in defined regions of the linkage groups. [The data were built into a regularly updated Genome Database for Rosaceae (GDR), available at (http://www.genome.clemson.edu/gdr/).].

BACCardI--a tool for the validation of genomic assemblies, assisting genome finishing and intergenome comparison.

PubMed

Bartels, Daniela; Kespohl, Sebastian; Albaum, Stefan; Drüke, Tanja; Goesmann, Alexander; Herold, Julia; Kaiser, Olaf; Pühler, Alfred; Pfeiffer, Friedhelm; Raddatz, Günter; Stoye, Jens; Meyer, Folker; Schuster, Stephan C

2005-04-01

We provide the graphical tool BACCardI for the construction of virtual clone maps from standard assembler output files or BLAST based sequence comparisons. This new tool has been applied to numerous genome projects to solve various problems including (a) validation of whole genome shotgun assemblies, (b) support for contig ordering in the finishing phase of a genome project, and (c) intergenome comparison between related strains when only one of the strains has been sequenced and a large insert library is available for the other. The BACCardI software can seamlessly interact with various sequence assembly packages. Genomic assemblies generated from sequence information need to be validated by independent methods such as physical maps. The time-consuming task of building physical maps can be circumvented by virtual clone maps derived from read pair information of large insert libraries.

Physical Maps for Genome Analysis of Serotype A and D Strains of the Fungal Pathogen Cryptococcus neoformans

PubMed Central

Schein, Jacqueline E.; Tangen, Kristin L.; Chiu, Readman; Shin, Heesun; Lengeler, Klaus B.; MacDonald, William Kim; Bosdet, Ian; Heitman, Joseph; Jones, Steven J.M.; Marra, Marco A.; Kronstad, James W.

2002-01-01

The basidiomycete fungus Cryptococcus neoformans is an important opportunistic pathogen of humans that poses a significant threat to immunocompromised individuals. Isolates of C. neoformans are classified into serotypes (A, B, C, D, and AD) based on antigenic differences in the polysaccharide capsule that surrounds the fungal cells. Genomic and EST sequencing projects are underway for the serotype D strain JEC21 and the serotype A strain H99. As part of a genomics program for C. neoformans, we have constructed fingerprinted bacterial artificial chromosome (BAC) clone physical maps for strains H99 and JEC21 to support the genomic sequencing efforts and to provide an initial comparison of the two genomes. The BAC clones represented an estimated 10-fold redundant coverage of the genomes of each serotype and allowed the assembly of 20 contigs each for H99 and JEC21. We found that the genomes of the two strains are sufficiently distinct to prevent coassembly of the two maps when combined fingerprint data are used to construct contigs. Hybridization experiments placed 82 markers on the JEC21 map and 102 markers on the H99 map, enabling contigs to be linked with specific chromosomes identified by electrophoretic karyotyping. These markers revealed both extensive similarity in gene order (conservation of synteny) between JEC21 and H99 as well as examples of chromosomal rearrangements including inversions and translocations. Sequencing reads were generated from the ends of the BAC clones to allow correlation of genomic shotgun sequence data with physical map contigs. The BAC maps therefore represent a valuable resource for the generation, assembly, and finishing of the genomic sequence of both JEC21 and H99. The physical maps also serve as a link between map-based and sequence-based data, providing a powerful resource for continued genomic studies. [This paper is dedicated to the memory of Michael Smith, Founding Director of the Biotechnology Laboratory and the BC Cancer Agency Genome Sciences Centre. Supplemental material is available online at http://www.genome.org.] PMID:12213782

A maize map standard with sequenced core markers, grass genome reference points and 932 expressed sequence tagged sites (ESTs) in a 1736-locus map.

PubMed Central

Davis, G L; McMullen, M D; Baysdorfer, C; Musket, T; Grant, D; Staebell, M; Xu, G; Polacco, M; Koster, L; Melia-Hancock, S; Houchins, K; Chao, S; Coe, E H

1999-01-01

We have constructed a 1736-locus maize genome map containing1156 loci probed by cDNAs, 545 probed by random genomic clones, 16 by simple sequence repeats (SSRs), 14 by isozymes, and 5 by anonymous clones. Sequence information is available for 56% of the loci with 66% of the sequenced loci assigned functions. A total of 596 new ESTs were mapped from a B73 library of 5-wk-old shoots. The map contains 237 loci probed by barley, oat, wheat, rice, or tripsacum clones, which serve as grass genome reference points in comparisons between maize and other grass maps. Ninety core markers selected for low copy number, high polymorphism, and even spacing along the chromosome delineate the 100 bins on the map. The average bin size is 17 cM. Use of bin assignments enables comparison among different maize mapping populations and experiments including those involving cytogenetic stocks, mutants, or quantitative trait loci. Integration of nonmaize markers in the map extends the resources available for gene discovery beyond the boundaries of maize mapping information into the expanse of map, sequence, and phenotype information from other grass species. This map provides a foundation for numerous basic and applied investigations including studies of gene organization, gene and genome evolution, targeted cloning, and dissection of complex traits. PMID:10388831

A contiguous clone map over 3 Mb on the long arm of chromosome 11 across a balanced translocation associated with schizophrenia

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

Evans, K.L.; Shibasaki, Yoshiro; Devon, R.S.

1995-08-10

Forty-nine clones derived by microdissection of a schizophrenia-associated t(1;11)(q42.1;q14.3) breakpoint region have been assigned by somatic cell hybrid mapping to seven discrete intervals on the long arm of human chromosome 11. Eleven of the clones were shown to map to a small region immediately distal to the translocation breakpoint on 11q. A 3-Mb contiguous clone map of this region was established by isolation of corresponding YAC recombinants. The contig was oriented and shown to traverse the translocation breakpoint by FISH and microsatellite marker analysis. This contig will facilitate the isolation of candidate sequences whose expression may be affected by themore » translocation. 28 refs., 4 figs., 3 tabs.« less

In Silico Identification of Epitopes in Mycobacterium avium subsp. paratuberculosis Proteins That Were Upregulated under Stress Conditions

PubMed Central

Gurung, Ratna B.; Purdie, Auriol C.; Begg, Douglas J.

2012-01-01

Johne's disease in ruminants is caused by Mycobacterium avium subsp. paratuberculosis. Diagnosis of M. avium subsp. paratuberculosis infection is difficult, especially in the early stages. To date, ideal antigen candidates are not available for efficient immunization or immunodiagnosis. This study reports the in silico selection and subsequent analysis of epitopes of M. avium subsp. paratuberculosis proteins that were found to be upregulated under stress conditions as a means to identify immunogenic candidate proteins. Previous studies have reported differential regulation of proteins when M. avium subsp. paratuberculosis is exposed to stressors which induce a response similar to dormancy. Dormancy may be involved in evading host defense mechanisms, and the host may also mount an immune response against these proteins. Twenty-five M. avium subsp. paratuberculosis proteins that were previously identified as being upregulated under in vitro stress conditions were analyzed for B and T cell epitopes by use of the prediction tools at the Immune Epitope Database and Analysis Resource. Major histocompatibility complex class I T cell epitopes were predicted using an artificial neural network method, and class II T cell epitopes were predicted using the consensus method. Conformational B cell epitopes were predicted from the relevant three-dimensional structure template for each protein. Based on the greatest number of predicted epitopes, eight proteins (MAP2698c [encoded by desA2], MAP2312c [encoded by fadE19], MAP3651c [encoded by fadE3_2], MAP2872c [encoded by fabG5_2], MAP3523c [encoded by oxcA], MAP0187c [encoded by sodA], and the hypothetical proteins MAP3567 and MAP1168c) were identified as potential candidates for study of antibody- and cell-mediated immune responses within infected hosts. PMID:22496492

Molecular cloning, molecular evolution and gene expression of cDNAs encoding thyrotropin-releasing hormone receptor subtypes in a teleost, the sockeye salmon (Oncorhynchus nerka).

PubMed

Saito, Yuichi; Mekuchi, Miyuki; Kobayashi, Noriaki; Kimura, Makoto; Aoki, Yasuhiro; Masuda, Tomohiro; Azuma, Teruo; Fukami, Motohiro; Iigo, Masayuki; Yanagisawa, Tadashi

2011-11-01

Molecular cloning of thyrotropin-releasing hormone receptors (TRHR) was performed in a teleost, the sockeye salmon (Oncorhynchus nerka). Four different TRHR cDNAs were cloned and named TRHR1, TRHR2a, TRHR2b and TRHR3 based on their similarity to known TRHR subtypes in vertebrates. Important residues for TRH binding were conserved in deduced amino acid sequences of the three TRHR subtypes except for the TRHR2b. Seven transmembrane domains were predicted for TRHR1, TRHR2a and TRHR3 proteins but only five for TRHR2b which appears to be truncated. In silico database analysis identified putative TRHR sequences including invertebrate TRHR and reptilian, avian and mammalian TRHR3. Phylogenetic analyses predicted the molecular evolution of TRHR in vertebrates: from the common ancestral TRHR (i.e. invertebrate TRHR), the TRHR2 subtype diverged first and then TRHR1 and TRHR3 diverged. Reverse transcription-polymerase chain reaction analyses revealed TRHR1 transcripts in the brain (hypothalamus), retina, pituitary gland and large intestine; TRHR2a in the brain (telencephalon and hypothalamus); and TRHR3 in the brain (olfactory bulbs) and retina. Copyright © 2011 Elsevier Inc. All rights reserved.

Construction of a yeast artificial chromosome contig encompassing the chromosome 14 Alzheimer`s disease locus

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

Sharma, V.; Bonnycastle, L.; Poorkai, P.

1994-09-01

We have constructed a yeast artificial chromosome (YAC) contig of chromosome 14q24.3 which encompasses the chromosome 14 Alzheimer`s disease locus (AD3). Determined by linkage analysis of early-onset Alzheimer`s disease kindreds, this interval is bounded by the genetic markers D14S61-D14S63 and spans approximately 15 centimorgans. The contig consists of 29 markers and 74 YACs of which 57 are defined by one or more sequence tagged sites (STSs). The STS markers comprise 5 genes, 16 short tandem repeat polymorphisms and 8 cDNA clones. An additional number of genes, expressed sequence tags and cDNA fragments have been identified and localized to the contigmore » by hybridization and sequence analysis of anonymous clones isolated by cDNA direct selection techniques. A minimal contig of about 15 YACs averaging 0.5-1.5 megabase in length will span this interval and is, at first approximation, in rough agreement with the genetic map. For two regions of the contig, our coverage has relied on L1/THE fingerprint and Alu-PCR hybridization data of YACs provided by CEPH/Genethon. We are currently developing sequence tagged sites from these to confirm the overlaps revealed by the fingerprint data. Among the genes which map to the contig are transforming growth factor beta 3, c-fos, and heat shock protein 2A (HSPA2). C-fos is not a candidate gene for AD3 based on the sequence analysis of affected and unaffected individuals. HSPA2 maps to the proximal edge of the contig and Calmodulin 1, a candidate gene from 4q24.3, maps outside of the region. The YAC contig is a framework physical map from which cosmid or P1 clone contigs can be constructed. As more genes and cDNAs are mapped, a highly resolved transcription map will emerge, a necessary step towards positionally cloning the AD3 gene.« less

From gene networks to drugs: systems pharmacology approaches for AUD.

PubMed

Ferguson, Laura B; Harris, R Adron; Mayfield, Roy Dayne

2018-06-01

The alcohol research field has amassed an impressive number of gene expression datasets spanning key brain areas for addiction, species (humans as well as multiple animal models), and stages in the addiction cycle (binge/intoxication, withdrawal/negative effect, and preoccupation/anticipation). These data have improved our understanding of the molecular adaptations that eventually lead to dysregulation of brain function and the chronic, relapsing disorder of addiction. Identification of new medications to treat alcohol use disorder (AUD) will likely benefit from the integration of genetic, genomic, and behavioral information included in these important datasets. Systems pharmacology considers drug effects as the outcome of the complex network of interactions a drug has rather than a single drug-molecule interaction. Computational strategies based on this principle that integrate gene expression signatures of pharmaceuticals and disease states have shown promise for identifying treatments that ameliorate disease symptoms (called in silico gene mapping or connectivity mapping). In this review, we suggest that gene expression profiling for in silico mapping is critical to improve drug repurposing and discovery for AUD and other psychiatric illnesses. We highlight studies that successfully apply gene mapping computational approaches to identify or repurpose pharmaceutical treatments for psychiatric illnesses. Furthermore, we address important challenges that must be overcome to maximize the potential of these strategies to translate to the clinic and improve healthcare outcomes.

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

PubMed

Liszewska, Frantz; Gaganidze, Dali; Sirko, Agnieszka

2005-01-01

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

Positional cloning in maize (Zea mays subsp. mays, Poaceae)1

PubMed Central

Gallavotti, Andrea; Whipple, Clinton J.

2015-01-01

• Premise of the study: Positional (or map-based) cloning is a common approach to identify the molecular lesions causing mutant phenotypes. Despite its large and complex genome, positional cloning has been recently shown to be feasible in maize, opening up a diverse collection of mutants to molecular characterization. • Methods and Results: Here we outline a general protocol for positional cloning in maize. While the general strategy is similar to that used in other plant species, we focus on the unique resources and approaches that should be considered when applied to maize mutants. • Conclusions: Positional cloning approaches are appropriate for maize mutants and quantitative traits, opening up to molecular characterization the large array of genetic diversity in this agronomically important species. The cloning approach described should be broadly applicable to other species as more plant genomes become available. PMID:25606355

Energy-based culture medium design for biomanufacturing optimization: A case study in monoclonal antibody production by GS-NS0 cells.

PubMed

Quiroga-Campano, Ana L; Panoskaltsis, Nicki; Mantalaris, Athanasios

2018-03-02

Demand for high-value biologics, a rapidly growing pipeline, and pressure from competition, time-to-market and regulators, necessitate novel biomanufacturing approaches, including Quality by Design (QbD) principles and Process Analytical Technologies (PAT), to facilitate accelerated, efficient and effective process development platforms that ensure consistent product quality and reduced lot-to-lot variability. Herein, QbD and PAT principles were incorporated within an innovative in vitro-in silico integrated framework for upstream process development (UPD). The central component of the UPD framework is a mathematical model that predicts dynamic nutrient uptake and average intracellular ATP content, based on biochemical reaction networks, to quantify and characterize energy metabolism and its adaptive response, metabolic shifts, to maintain ATP homeostasis. The accuracy and flexibility of the model depends on critical cell type/product/clone-specific parameters, which are experimentally estimated. The integrated in vitro-in silico platform and the model's predictive capacity reduced burden, time and expense of experimentation resulting in optimal medium design compared to commercially available culture media (80% amino acid reduction) and a fed-batch feeding strategy that increased productivity by 129%. The framework represents a flexible and efficient tool that transforms, improves and accelerates conventional process development in biomanufacturing with wide applications, including stem cell-based therapies. Copyright © 2018. Published by Elsevier Inc.

A physical map of the bovine genome

PubMed Central

Snelling, Warren M; Chiu, Readman; Schein, Jacqueline E; Hobbs, Matthew; Abbey, Colette A; Adelson, David L; Aerts, Jan; Bennett, Gary L; Bosdet, Ian E; Boussaha, Mekki; Brauning, Rudiger; Caetano, Alexandre R; Costa, Marcos M; Crawford, Allan M; Dalrymple, Brian P; Eggen, André; Everts-van der Wind, Annelie; Floriot, Sandrine; Gautier, Mathieu; Gill, Clare A; Green, Ronnie D; Holt, Robert; Jann, Oliver; Jones, Steven JM; Kappes, Steven M; Keele, John W; de Jong, Pieter J; Larkin, Denis M; Lewin, Harris A; McEwan, John C; McKay, Stephanie; Marra, Marco A; Mathewson, Carrie A; Matukumalli, Lakshmi K; Moore, Stephen S; Murdoch, Brenda; Nicholas, Frank W; Osoegawa, Kazutoyo; Roy, Alice; Salih, Hanni; Schibler, Laurent; Schnabel, Robert D; Silveri, Licia; Skow, Loren C; Smith, Timothy PL; Sonstegard, Tad S; Taylor, Jeremy F; Tellam, Ross; Van Tassell, Curtis P; Williams, John L; Womack, James E; Wye, Natasja H; Yang, George; Zhao, Shaying

2007-01-01

Background Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project. Results A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly. Conclusion Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans. PMID:17697342

Cytokinin oxidase/dehydrogenase genes in barley and wheat: cloning and heterologous expression.

PubMed

Galuszka, Petr; Frébortová, Jitka; Werner, Tomás; Yamada, Mamoru; Strnad, Miroslav; Schmülling, Thomas; Frébort, Ivo

2004-10-01

The cloning of two novel genes that encode cytokinin oxidase/dehydrogenase (CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the genes codes for a functional enzyme, as its expression caused a cytokinin-deficient phenotype in the heterologous host plants. Additional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT-PCR and found to be organ-specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interestingly, this enzyme shows a pH optimum at 4.5 and a preference for cytokinin ribosides as substrates, which may indicate its vacuolar targeting. Different substrate specificities, and the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented.

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

PubMed

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

1995-06-01

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

Processing the Bouguer anomaly map of Biga and the surrounding area by the cellular neural network: application to the southwestern Marmara region

NASA Astrophysics Data System (ADS)

Aydogan, D.

2007-04-01

An image processing technique called the cellular neural network (CNN) approach is used in this study to locate geological features giving rise to gravity anomalies such as faults or the boundary of two geologic zones. CNN is a stochastic image processing technique based on template optimization using the neighborhood relationships of cells. These cells can be characterized by a functional block diagram that is typical of neural network theory. The functionality of CNN is described in its entirety by a number of small matrices (A, B and I) called the cloning template. CNN can also be considered to be a nonlinear convolution of these matrices. This template describes the strength of the nearest neighbor interconnections in the network. The recurrent perceptron learning algorithm (RPLA) is used in optimization of cloning template. The CNN and standard Canny algorithms were first tested on two sets of synthetic gravity data with the aim of checking the reliability of the proposed approach. The CNN method was compared with classical derivative techniques by applying the cross-correlation method (CC) to the same anomaly map as this latter approach can detect some features that are difficult to identify on the Bouguer anomaly maps. This approach was then applied to the Bouguer anomaly map of Biga and its surrounding area, in Turkey. Structural features in the area between Bandirma, Biga, Yenice and Gonen in the southwest Marmara region are investigated by applying the CNN and CC to the Bouguer anomaly map. Faults identified by these algorithms are generally in accordance with previously mapped surface faults. These examples show that the geologic boundaries can be detected from Bouguer anomaly maps using the cloning template approach. A visual evaluation of the outputs of the CNN and CC approaches is carried out, and the results are compared with each other. This approach provides quantitative solutions based on just a few assumptions, which makes the method more powerful than the classical methods.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei.

PubMed

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

2016-07-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes) or TINA-DNA (Twisted Intercalating Nucleic Acids). Gene targets can be specifically labelled with at least about 20 probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3d-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. Copyright © 2016. Published by Elsevier Inc.

Variations on a theme of Lander and Waterman

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

Speed, T.

1997-12-01

The original Lander and Waterman mathematical analysis was for fingerprinting random clones. Since that time, a number of variants of their theory have appeared, including ones which apply to mapping by anchoring random clones, and to non-random or directed clone mapping. The same theory is now widely used to devise random sequencing strategies. In this talk I will review these developments, and go on the discuss the theory required for directed sequencing strategies.

Comparative fine mapping of the Wax 1 (W1) locus in hexaploid wheat.

PubMed

Lu, Ping; Qin, Jinxia; Wang, Guoxin; Wang, Lili; Wang, Zhenzhong; Wu, Qiuhong; Xie, Jingzhong; Liang, Yong; Wang, Yong; Zhang, Deyun; Sun, Qixin; Liu, Zhiyong

2015-08-01

By applying comparative genomics analyses, a high-density genetic linkage map of the Wax 1 ( W1 ) locus was constructed as a framework for map-based cloning. Glaucousness is described as the scattering effect of visible light from wax deposited on the cuticle of plant aerial organs. In wheat, the wax on leaves and stems is mainly controlled by two sets of genes: glaucousness loci (W1 and W2) and non-glaucousness loci (Iw1 and Iw2). Bulked segregant analysis (BSA) and simple sequence repeat (SSR) mapping showed that Wax1 (W1) is located on chromosome arm 2BS between markers Xgwm210 and Xbarc35. By applying comparative genomics analyses, colinearity genomic regions of the W1 locus on wheat 2BS were identified in Brachypodium distachyon chromosome 5, rice chromosome 4 and sorghum chromosome 6, respectively. Four STS markers were developed using the Triticum aestivum cv. Chinese Spring 454 contig sequences and the International Wheat Genome Sequencing Consortium (IWGSC) survey sequences. W1 was mapped into a 0.93 cM genetic interval flanked by markers XWGGC3197 and XWGGC2484, which has synteny with genomic regions of 56.5 kb in Brachypodium, 390 kb in rice and 31.8 kb in sorghum. The fine genetic map can serve as a framework for chromosome landing, physical mapping and map-based cloning of the W1 in wheat.

Harnessing the sorghum genome sequence:development of a genome-wide microsattelite (SSR) resource for swift genetic mapping and map based cloning in sorghum

USDA-ARS?s Scientific Manuscript database

Sorghum is the second cereal crop to have a full genome completely sequenced (Nature (2009), 457:551). This achievement is widely recognized as a scientific milestone for grass genetics and genomics in general. However, the true worth of genetic information lies in translating the sequence informa...

Towards a transcription map spanning a 250 kb area within the DiGeorge syndrome chromosome region

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

Wong, W.; Emanuel, B.S.; Siegert, J.

1994-09-01

DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) are congenital anomalies affecting predominantly the thymus, parathyroid glands, heart and craniofacial development. Detection of 22q11.2 deletions in the majority of DGS and VCFS patients implicate 22q11 haploinsufficiency in the etiology of these disorders. The VCFS/DGS critical region lies within the proximal portion of a commonly deleted 1.2 Mb region in 22q11. A 250 kb cosmid contig covering this critical region and containing D22S74 (N25) has been established. From this contig, eleven cosmids with minimal overlap were biotinylated by nick translation, and hybridized to PCR-amplified cDNAs prepared from different tissues. The use ofmore » cDNAs from a variety of tissues increases the likelihood of identifying low abundance transcripts and tissue-specific expressed sequences. A DGCR-specific cDNA sublibrary consisting of 670 cDNA clones has been constructed. To date, 49 cDNA clones from this sub-library have been identified with single copy probes and cosmids containing putative CpG islands. Based on sequence analysis, 25 of the clones contain regions of homology to several cDNAs which map within the proximal contig. LAN is a novel partial cDNA isolated from a fetal brain library probed with one of the cosmids in the proximal contig. Using LAN as a probe, we have found 19 positive clones in the DGCR-specific cDNA sub-library (4 clones from fetal brain, 14 from adult skeletal muscle and one from fetal liver). Some of the LAN-positive clones extend the partial cDNA in the 5{prime} direction and will be useful in assembling a full length transcript. This resource will be used to develop a complete transcriptional map of the critical region in order to identify candidate gene(s) involved in the etiology of DGS/VCFS and to determine the relationship between the transcriptional and physical maps of 22q11.« less

Multidisciplinary insight into clonal expansion of HTLV-1-infected cells in adult T-cell leukemia via modeling by deterministic finite automata coupled with high-throughput sequencing.

PubMed

Farmanbar, Amir; Firouzi, Sanaz; Park, Sung-Joon; Nakai, Kenta; Uchimaru, Kaoru; Watanabe, Toshiki

2017-01-31

Clonal expansion of leukemic cells leads to onset of adult T-cell leukemia (ATL), an aggressive lymphoid malignancy with a very poor prognosis. Infection with human T-cell leukemia virus type-1 (HTLV-1) is the direct cause of ATL onset, and integration of HTLV-1 into the human genome is essential for clonal expansion of leukemic cells. Therefore, monitoring clonal expansion of HTLV-1-infected cells via isolation of integration sites assists in analyzing infected individuals from early infection to the final stage of ATL development. However, because of the complex nature of clonal expansion, the underlying mechanisms have yet to be clarified. Combining computational/mathematical modeling with experimental and clinical data of integration site-based clonality analysis derived from next generation sequencing technologies provides an appropriate strategy to achieve a better understanding of ATL development. As a comprehensively interdisciplinary project, this study combined three main aspects: wet laboratory experiments, in silico analysis and empirical modeling. We analyzed clinical samples from HTLV-1-infected individuals with a broad range of proviral loads using a high-throughput methodology that enables isolation of HTLV-1 integration sites and accurate measurement of the size of infected clones. We categorized clones into four size groups, "very small", "small", "big", and "very big", based on the patterns of clonal growth and observed clone sizes. We propose an empirical formal model based on deterministic finite state automata (DFA) analysis of real clinical samples to illustrate patterns of clonal expansion. Through the developed model, we have translated biological data of clonal expansion into the formal language of mathematics and represented the observed clonality data with DFA. Our data suggest that combining experimental data (absolute size of clones) with DFA can describe the clonality status of patients. This kind of modeling provides a basic understanding as well as a unique perspective for clarifying the mechanisms of clonal expansion in ATL.

Improving a Synechocystis-based photoautotrophic chassis through systematic genome mapping and validation of neutral sites

PubMed Central

Pinto, Filipe; Pacheco, Catarina C.; Oliveira, Paulo; Montagud, Arnau; Landels, Andrew; Couto, Narciso; Wright, Phillip C.; Urchueguía, Javier F.; Tamagnini, Paula

2015-01-01

The use of microorganisms as cell factories frequently requires extensive molecular manipulation. Therefore, the identification of genomic neutral sites for the stable integration of ectopic DNA is required to ensure a successful outcome. Here we describe the genome mapping and validation of five neutral sites in the chromosome of Synechocystis sp. PCC 6803, foreseeing the use of this cyanobacterium as a photoautotrophic chassis. To evaluate the neutrality of these loci, insertion/deletion mutants were produced, and to assess their functionality, a synthetic green fluorescent reporter module was introduced. The constructed integrative vectors include a BioBrick-compatible multiple cloning site insulated by transcription terminators, constituting robust cloning interfaces for synthetic biology approaches. Moreover, Synechocystis mutants (chassis) ready to receive purpose-built synthetic modules/circuits are also available. This work presents a systematic approach to map and validate chromosomal neutral sites in cyanobacteria, and that can be extended to other organisms. PMID:26490728

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

PubMed Central

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

2013-01-01

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

Combinatorial pooling enables selective sequencing of the barley gene space.

PubMed

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J

2013-04-01

For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

The Dynein Gene Family in Chlamydomonas Reinhardtii

PubMed Central

Porter, M. E.; Knott, J. A.; Myster, S. H.; Farlow, S. J.

1996-01-01

To correlate dynein heavy chain (Dhc) genes with flagellar mutations and gain insight into the function of specific dynein isoforms, we placed eight members of the Dhc gene family on the genetic map of Chlamydomonas. Using a PCR-based strategy, we cloned 11 Dhc genes from Chlamydomonas. Comparisons with other Dhc genes indicate that two clones correspond to genes encoding the alpha and beta heavy chains of the outer dynein arm. Alignment of the predicted amino acid sequences spanning the nucleotide binding site indicates that the remaining nine clones can be subdivided into three groups that are likely to include representatives of the inner-arm Dhc isoforms. Gene-specific probes reveal that each clone represents a single-copy gene that is expressed as a transcript of the appropriate size (>13 kb) sufficient to encode a high molecular weight Dhc polypeptide. The expression of all nine genes is upregulated in response to deflagellation, suggesting a role in axoneme assembly or motility. Restriction fragment length polymorphisms between divergent C. reinhardtii strains have been used to place each Dhc gene on the genetic map of Chlamydomonas. These studies lay the groundwork for correlating defects in different Dhc genes with specific flagellar mutations. PMID:8889521

Importance sampling with imperfect cloning for the computation of generalized Lyapunov exponents

NASA Astrophysics Data System (ADS)

Anteneodo, Celia; Camargo, Sabrina; Vallejos, Raúl O.

2017-12-01

We revisit the numerical calculation of generalized Lyapunov exponents, L (q ) , in deterministic dynamical systems. The standard method consists of adding noise to the dynamics in order to use importance sampling algorithms. Then L (q ) is obtained by taking the limit noise-amplitude → 0 after the calculation. We focus on a particular method that involves periodic cloning and pruning of a set of trajectories. However, instead of considering a noisy dynamics, we implement an imperfect (noisy) cloning. This alternative method is compared with the standard one and, when possible, with analytical results. As a workbench we use the asymmetric tent map, the standard map, and a system of coupled symplectic maps. The general conclusion of this study is that the imperfect-cloning method performs as well as the standard one, with the advantage of preserving the deterministic dynamics.

BAC-end sequence-based SNPs and Bin mapping for rapid integration of physical and genetic maps in apple.

PubMed

Han, Yuepeng; Chagné, David; Gasic, Ksenija; Rikkerink, Erik H A; Beever, Jonathan E; Gardiner, Susan E; Korban, Schuyler S

2009-03-01

A genome-wide BAC physical map of the apple, Malus x domestica Borkh., has been recently developed. Here, we report on integrating the physical and genetic maps of the apple using a SNP-based approach in conjunction with bin mapping. Briefly, BAC clones located at ends of BAC contigs were selected, and sequenced at both ends. The BAC end sequences (BESs) were used to identify candidate SNPs. Subsequently, these candidate SNPs were genetically mapped using a bin mapping strategy for the purpose of mapping the physical onto the genetic map. Using this approach, 52 (23%) out of 228 BESs tested were successfully exploited to develop SNPs. These SNPs anchored 51 contigs, spanning approximately 37 Mb in cumulative physical length, onto 14 linkage groups. The reliability of the integration of the physical and genetic maps using this SNP-based strategy is described, and the results confirm the feasibility of this approach to construct an integrated physical and genetic maps for apple.

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

Kimelman, Aya; Levy, Asaf; Sberro, Hila

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes our assays revealed novel toxins and restriction enzymes, and new classes of smallmore » non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator dnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.« less

CGI: Java Software for Mapping and Visualizing Data from Array-based Comparative Genomic Hybridization and Expression Profiling

PubMed Central

Gu, Joyce Xiuweu-Xu; Wei, Michael Yang; Rao, Pulivarthi H.; Lau, Ching C.; Behl, Sanjiv; Man, Tsz-Kwong

2007-01-01

With the increasing application of various genomic technologies in biomedical research, there is a need to integrate these data to correlate candidate genes/regions that are identified by different genomic platforms. Although there are tools that can analyze data from individual platforms, essential software for integration of genomic data is still lacking. Here, we present a novel Java-based program called CGI (Cytogenetics-Genomics Integrator) that matches the BAC clones from array-based comparative genomic hybridization (aCGH) to genes from RNA expression profiling datasets. The matching is computed via a fast, backend MySQL database containing UCSC Genome Browser annotations. This program also provides an easy-to-use graphical user interface for visualizing and summarizing the correlation of DNA copy number changes and RNA expression patterns from a set of experiments. In addition, CGI uses a Java applet to display the copy number values of a specific BAC clone in aCGH experiments side by side with the expression levels of genes that are mapped back to that BAC clone from the microarray experiments. The CGI program is built on top of extensible, reusable graphic components specifically designed for biologists. It is cross-platform compatible and the source code is freely available under the General Public License. PMID:19936083

CGI: Java software for mapping and visualizing data from array-based comparative genomic hybridization and expression profiling.

PubMed

Gu, Joyce Xiuweu-Xu; Wei, Michael Yang; Rao, Pulivarthi H; Lau, Ching C; Behl, Sanjiv; Man, Tsz-Kwong

2007-10-06

With the increasing application of various genomic technologies in biomedical research, there is a need to integrate these data to correlate candidate genes/regions that are identified by different genomic platforms. Although there are tools that can analyze data from individual platforms, essential software for integration of genomic data is still lacking. Here, we present a novel Java-based program called CGI (Cytogenetics-Genomics Integrator) that matches the BAC clones from array-based comparative genomic hybridization (aCGH) to genes from RNA expression profiling datasets. The matching is computed via a fast, backend MySQL database containing UCSC Genome Browser annotations. This program also provides an easy-to-use graphical user interface for visualizing and summarizing the correlation of DNA copy number changes and RNA expression patterns from a set of experiments. In addition, CGI uses a Java applet to display the copy number values of a specific BAC clone in aCGH experiments side by side with the expression levels of genes that are mapped back to that BAC clone from the microarray experiments. The CGI program is built on top of extensible, reusable graphic components specifically designed for biologists. It is cross-platform compatible and the source code is freely available under the General Public License.

Saturation of an Intra-Gene Pool Linkage Map: Towards a Unified Consensus Linkage Map for Fine Mapping and Synteny Analysis in Common Bean

PubMed Central

Galeano, Carlos H.; Fernandez, Andrea C.; Franco-Herrera, Natalia; Cichy, Karen A.; McClean, Phillip E.; Vanderleyden, Jos; Blair, Matthew W.

2011-01-01

Map-based cloning and fine mapping to find genes of interest and marker assisted selection (MAS) requires good genetic maps with reproducible markers. In this study, we saturated the linkage map of the intra-gene pool population of common bean DOR364×BAT477 (DB) by evaluating 2,706 molecular markers including SSR, SNP, and gene-based markers. On average the polymorphism rate was 7.7% due to the narrow genetic base between the parents. The DB linkage map consisted of 291 markers with a total map length of 1,788 cM. A consensus map was built using the core mapping populations derived from inter-gene pool crosses: DOR364×G19833 (DG) and BAT93×JALO EEP558 (BJ). The consensus map consisted of a total of 1,010 markers mapped, with a total map length of 2,041 cM across 11 linkage groups. On average, each linkage group on the consensus map contained 91 markers of which 83% were single copy markers. Finally, a synteny analysis was carried out using our highly saturated consensus maps compared with the soybean pseudo-chromosome assembly. A total of 772 marker sequences were compared with the soybean genome. A total of 44 syntenic blocks were identified. The linkage group Pv6 presented the most diverse pattern of synteny with seven syntenic blocks, and Pv9 showed the most consistent relations with soybean with just two syntenic blocks. Additionally, a co-linear analysis using common bean transcript map information against soybean coding sequences (CDS) revealed the relationship with 787 soybean genes. The common bean consensus map has allowed us to map a larger number of markers, to obtain a more complete coverage of the common bean genome. Our results, combined with synteny relationships provide tools to increase marker density in selected genomic regions to identify closely linked polymorphic markers for indirect selection, fine mapping or for positional cloning. PMID:22174773

Localization of causal locus in the genome of the brown macroalga Ectocarpus: NGS-based mapping and positional cloning approaches

PubMed Central

Billoud, Bernard; Jouanno, Émilie; Nehr, Zofia; Carton, Baptiste; Rolland, Élodie; Chenivesse, Sabine; Charrier, Bénédicte

2015-01-01

Mutagenesis is the only process by which unpredicted biological gene function can be identified. Despite that several macroalgal developmental mutants have been generated, their causal mutation was never identified, because experimental conditions were not gathered at that time. Today, progresses in macroalgal genomics and judicious choices of suitable genetic models make mutated gene identification possible. This article presents a comparative study of two methods aiming at identifying a genetic locus in the brown alga Ectocarpus siliculosus: positional cloning and Next-Generation Sequencing (NGS)-based mapping. Once necessary preliminary experimental tools were gathered, we tested both analyses on an Ectocarpus morphogenetic mutant. We show how a narrower localization results from the combination of the two methods. Advantages and drawbacks of these two approaches as well as potential transfer to other macroalgae are discussed. PMID:25745426

Cloning of Trametes versicolar genes induced by nitrogen starvation

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

Trudel, P.; Courchesne, D.; Roy, C.

1988-06-01

We have screened a genomic library of Trametes versicolar for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.

Bridging the gap between genome analysis and precision breeding in potato.

PubMed

Gebhardt, Christiane

2013-04-01

Efficiency and precision in plant breeding can be enhanced by using diagnostic DNA-based markers for the selection of superior cultivars. This technique has been applied to many crops, including potatoes. The first generation of diagnostic DNA-based markers useful in potato breeding were enabled by several developments: genetic linkage maps based on DNA polymorphisms, linkage mapping of qualitative and quantitative agronomic traits, cloning and functional analysis of genes for pathogen resistance and genes controlling plant metabolism, and association genetics in collections of tetraploid varieties and advanced breeding clones. Although these have led to significant improvements in potato genetics, the prediction of most, if not all, natural variation in agronomic traits by diagnostic markers ultimately requires the identification of the causal genes and their allelic variants. This objective will be facilitated by new genomic tools, such as genomic resequencing and comparative profiling of the proteome, transcriptome, and metabolome in combination with phenotyping genetic materials relevant for variety development. Copyright © 2012 Elsevier Ltd. All rights reserved.

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2.

PubMed

Han, Yonghua; Zhang, Zhonghua; Huang, Sanwen; Jin, Weiwei

2011-01-27

Integration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.). In this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed. Recombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.

Linkage map of the honey bee, Apis mellifera, based on RAPD markers

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

Hunt, G.J.; Page, R.E. Jr.

A linkage map was constructed for the honey bee based on the segregation of 365 random amplified polymorphic DNA (RAPD) markers in haploid male progeny of a single female bee. The X locus for sex determination and genes for black body color and malate dehydrogenase were mapped to separate linkage groups. RAPD markers were very efficient for mapping, with an average of about 2.8 loci mapped for each 10-nucleotide primer that was used in polymerase chain reactions. The mean interval size between markers on the map was 9.1 cM. The map covered 3110 cM of linked markers on 26 linkagemore » groups. We estimate the total genome size to be {approximately}3450 cM. The size of the map indicated a very high recombination rate for the honey bee. The relationship of physical to genetic distance was estimated at 52 kb/cM, suggesting that map-based cloning of genes will be feasible for this species. 71 refs., 6 figs., 1 tab.« less

Comparative genomics of Lupinus angustifolius gene-rich regions: BAC library exploration, genetic mapping and cytogenetics

PubMed Central

2013-01-01

Background The narrow-leafed lupin, Lupinus angustifolius L., is a grain legume species with a relatively compact genome. The species has 2n = 40 chromosomes and its genome size is 960 Mbp/1C. During the last decade, L. angustifolius genomic studies have achieved several milestones, such as molecular-marker development, linkage maps, and bacterial artificial chromosome (BAC) libraries. Here, these resources were integratively used to identify and sequence two gene-rich regions (GRRs) of the genome. Results The genome was screened with a probe representing the sequence of a microsatellite fragment length polymorphism (MFLP) marker linked to Phomopsis stem blight resistance. BAC clones selected by hybridization were subjected to restriction fingerprinting and contig assembly, and 232 BAC-ends were sequenced and annotated. BAC fluorescence in situ hybridization (BAC-FISH) identified eight single-locus clones. Based on physical mapping, cytogenetic localization, and BAC-end annotation, five clones were chosen for sequencing. Within the sequences of clones that hybridized in FISH to a single-locus, two large GRRs were identified. The GRRs showed strong and conserved synteny to Glycine max duplicated genome regions, illustrated by both identical gene order and parallel orientation. In contrast, in the clones with dispersed FISH signals, more than one-third of sequences were transposable elements. Sequenced, single-locus clones were used to develop 12 genetic markers, increasing the number of L. angustifolius chromosomes linked to appropriate linkage groups by five pairs. Conclusions In general, probes originating from MFLP sequences can assist genome screening and gene discovery. However, such probes are not useful for positional cloning, because they tend to hybridize to numerous loci. GRRs identified in L. angustifolius contained a low number of interspersed repeats and had a high level of synteny to the genome of the model legume G. max. Our results showed that not only was the gene nucleotide sequence conserved between soybean and lupin GRRs, but the order and orientation of particular genes in syntenic blocks was homologous, as well. These findings will be valuable to the forthcoming sequencing of the lupin genome. PMID:23379841

Physical and in silico approaches identify DNA-PK in a Tax DNA-damage response interactome

PubMed Central

Ramadan, Emad; Ward, Michael; Guo, Xin; Durkin, Sarah S; Sawyer, Adam; Vilela, Marcelo; Osgood, Christopher; Pothen, Alex; Semmes, Oliver J

2008-01-01

Background We have initiated an effort to exhaustively map interactions between HTLV-1 Tax and host cellular proteins. The resulting Tax interactome will have significant utility toward defining new and understanding known activities of this important viral protein. In addition, the completion of a full Tax interactome will also help shed light upon the functional consequences of these myriad Tax activities. The physical mapping process involved the affinity isolation of Tax complexes followed by sequence identification using tandem mass spectrometry. To date we have mapped 250 cellular components within this interactome. Here we present our approach to prioritizing these interactions via an in silico culling process. Results We first constructed an in silico Tax interactome comprised of 46 literature-confirmed protein-protein interactions. This number was then reduced to four Tax-interactions suspected to play a role in DNA damage response (Rad51, TOP1, Chk2, 53BP1). The first-neighbor and second-neighbor interactions of these four proteins were assembled from available human protein interaction databases. Through an analysis of betweenness and closeness centrality measures, and numbers of interactions, we ranked proteins in the first neighborhood. When this rank list was compared to the list of physical Tax-binding proteins, DNA-PK was the highest ranked protein common to both lists. An overlapping clustering of the Tax-specific second-neighborhood protein network showed DNA-PK to be one of three bridge proteins that link multiple clusters in the DNA damage response network. Conclusion The interaction of Tax with DNA-PK represents an important biological paradigm as suggested via consensus findings in vivo and in silico. We present this methodology as an approach to discovery and as a means of validating components of a consensus Tax interactome. PMID:18922151

Toward a Molecular Cytogenetic Map for Cultivated Sunflower (Helianthus annuus L.) by Landed BAC/BIBAC Clones

PubMed Central

Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

2013-01-01

Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (∼100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC- fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)−derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources. PMID:23316437

A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance

PubMed Central

Walter, Stephanie; Kahla, Amal; Arunachalam, Chanemoughasoundharam; Perochon, Alexandre; Khan, Mojibur R.; Scofield, Steven R.; Doohan, Fiona M.

2015-01-01

The mycotoxin deoxynivalenol (DON) acts as a disease virulence factor for Fusarium fungi, and tolerance of DON enhances wheat resistance to Fusarium head blight (FHB) disease. Two variants of an ATP-binding cassette (ABC) family C transporter gene were cloned from DON-treated wheat mRNA, namely TaABCC3.1 and TaABCC3.2. These represent two of three putative genes identified on chromosomes 3A, 3B, and 3D of the wheat genome sequence. Variant TaABCC3.1 represents the DON-responsive transcript previously associated with DON resistance in wheat. PCR-based mapping and in silico sequence analyses located TaABCC3.1 to the short arm of wheat chromosome 3B (not within the FHB resistance quantitative trait locus Fhb1). In silico analyses of microarray data indicated that TaABCC3 genes are expressed in reproductive tissue and roots, and in response to the DON producer Fusarium graminearum. Gene expression studies showed that TaABCC3.1 is activated as part of the early host response to DON and in response to the FHB defence hormone jasmonic acid. Virus-induced gene silencing (VIGS) confirmed that TaABCC3 genes contributed to DON tolerance. VIGS was performed using two independent viral construct applications: one specifically targeted TaABCC3.1 for silencing, while the other targeted this gene and the chromosome 3A homeologue. In both instances, VIGS resulted in more toxin-induced discoloration of spikelets, compared with the DON effects in non-silenced spikelets at 14 d after toxin treatment (≥2.2-fold increase, P<0.05). Silencing by both VIGS constructs enhanced head ripening, and especially so in DON-treated heads. VIGS of TaABCC3 genes also reduced the grain number by more than 28% (P<0.05), both with and without DON treatment, and the effects were greater for the construct that targeted the two homeologues. Hence, DON-responsive TaABCC3 genes warrant further study to determine their potential as disease resistance breeding targets and their function in grain formation and ripening. PMID:25732534

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

PubMed

Kaucka, Marketa; Zikmund, Tomas; Tesarova, Marketa; Gyllborg, Daniel; Hellander, Andreas; Jaros, Josef; Kaiser, Jozef; Petersen, Julian; Szarowska, Bara; Newton, Phillip T; Dyachuk, Vyacheslav; Li, Lei; Qian, Hong; Johansson, Anne-Sofie; Mishina, Yuji; Currie, Joshua D; Tanaka, Elly M; Erickson, Alek; Dudley, Andrew; Brismar, Hjalmar; Southam, Paul; Coen, Enrico; Chen, Min; Weinstein, Lee S; Hampl, Ales; Arenas, Ernest; Chagin, Andrei S; Fried, Kaj; Adameyko, Igor

2017-04-17

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

High-Throughput Gene Mapping in Caenorhabditis elegans

PubMed Central

Swan, Kathryn A.; Curtis, Damian E.; McKusick, Kathleen B.; Voinov, Alexander V.; Mapa, Felipa A.; Cancilla, Michael R.

2002-01-01

Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 ± 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18. [The sequence data described in this paper have been submitted to the NCBI dbSNP data library under accession nos. 4388625–4389689 and GenBank dbSTS under accession nos. 973810–974874. The following individuals and institutions kindly provided reagents, samples, or unpublished information as indicated in the paper: The C. elegans Sequencing Consortium and The Caenorhabditis Genetics Center.] PMID:12097347

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

PubMed

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

Improving a Synechocystis-based photoautotrophic chassis through systematic genome mapping and validation of neutral sites.

PubMed

Pinto, Filipe; Pacheco, Catarina C; Oliveira, Paulo; Montagud, Arnau; Landels, Andrew; Couto, Narciso; Wright, Phillip C; Urchueguía, Javier F; Tamagnini, Paula

2015-12-01

The use of microorganisms as cell factories frequently requires extensive molecular manipulation. Therefore, the identification of genomic neutral sites for the stable integration of ectopic DNA is required to ensure a successful outcome. Here we describe the genome mapping and validation of five neutral sites in the chromosome of Synechocystis sp. PCC 6803, foreseeing the use of this cyanobacterium as a photoautotrophic chassis. To evaluate the neutrality of these loci, insertion/deletion mutants were produced, and to assess their functionality, a synthetic green fluorescent reporter module was introduced. The constructed integrative vectors include a BioBrick-compatible multiple cloning site insulated by transcription terminators, constituting robust cloning interfaces for synthetic biology approaches. Moreover, Synechocystis mutants (chassis) ready to receive purpose-built synthetic modules/circuits are also available. This work presents a systematic approach to map and validate chromosomal neutral sites in cyanobacteria, and that can be extended to other organisms. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Cloning and Characterization of a Critical Regulator for Preharvest Sprouting in Wheat

PubMed Central

Liu, Shubing; Sehgal, Sunish K.; Li, Jiarui; Lin, Meng; Trick, Harold N.; Yu, Jianming; Gill, Bikram S.; Bai, Guihua

2013-01-01

Sprouting of grains in mature spikes before harvest is a major problem in wheat (Triticum aestivum) production worldwide. We cloned and characterized a gene underlying a wheat quantitative trait locus (QTL) on the short arm of chromosome 3A for preharvest sprouting (PHS) resistance in white wheat using comparative mapping and map-based cloning. This gene, designated TaPHS1, is a wheat homolog of a MOTHER OF FLOWERING TIME (TaMFT)-like gene. RNA interference-mediated knockdown of the gene confirmed that TaPHS1 positively regulates PHS resistance. We discovered two causal mutations in TaPHS1 that jointly altered PHS resistance in wheat. One GT-to-AT mutation generates a mis-splicing site, and the other A-to-T mutation creates a premature stop codon that results in a truncated nonfunctional transcript. Association analysis of a set of wheat cultivars validated the role of the two mutations on PHS resistance. The molecular characterization of TaPHS1 is significant for expediting breeding for PHS resistance to protect grain yield and quality in wheat production. PMID:23821595

Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

PubMed Central

Takahashi, Yuji; Shomura, Ayahiko; Sasaki, Takuji; Yano, Masahiro

2001-01-01

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of CK2α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype. PMID:11416158

Cloning

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

High Throughput T Epitope Mapping and Vaccine Development

PubMed Central

Li Pira, Giuseppina; Ivaldi, Federico; Moretti, Paolo; Manca, Fabrizio

2010-01-01

Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost. PMID:20617148

An 11-bp Insertion in Zea mays fatb Reduces the Palmitic Acid Content of Fatty Acids in Maize Grain

PubMed Central

Li, Qing; Yang, Xiaohong; Zheng, Debo; Warburton, Marilyn; Chai, Yuchao; Zhang, Pan; Guo, Yuqiu; Yan, Jianbing; Li, Jiansheng

2011-01-01

The ratio of saturated to unsaturated fatty acids in maize kernels strongly impacts human and livestock health, but is a complex trait that is difficult to select based on phenotype. Map-based cloning of quantitative trait loci (QTL) is a powerful but time-consuming method for the dissection of complex traits. Here, we combine linkage and association analyses to fine map QTL-Pal9, a QTL influencing levels of palmitic acid, an important class of saturated fatty acid. QTL-Pal9 was mapped to a 90-kb region, in which we identified a candidate gene, Zea mays fatb (Zmfatb), which encodes acyl-ACP thioesterase. An 11-bp insertion in the last exon of Zmfatb decreases palmitic acid content and concentration, leading to an optimization of the ratio of saturated to unsaturated fatty acids while having no effect on total oil content. We used three-dimensional structure analysis to explain the functional mechanism of the ZmFATB protein and confirmed the proposed model in vitro and in vivo. We measured the genetic effect of the functional site in 15 different genetic backgrounds and found a maximum change of 4.57 mg/g palmitic acid content, which accounts for ∼20–60% of the variation in the ratio of saturated to unsaturated fatty acids. A PCR-based marker for QTL-Pal9 was developed for marker-assisted selection of nutritionally healthier maize lines. The method presented here provides a new, efficient way to clone QTL, and the cloned palmitic acid QTL sheds lights on the genetic mechanism of oil biosynthesis and targeted maize molecular breeding. PMID:21931818

Map-based cloning and characterization of Zea mays male sterility33 (ZmMs33) gene, encoding a glycerol-3-phosphate acyltransferase.

PubMed

Xie, Ke; Wu, Suowei; Li, Ziwen; Zhou, Yan; Zhang, Danfeng; Dong, Zhenying; An, Xueli; Zhu, Taotao; Zhang, Simiao; Liu, Shuangshuang; Li, Jinping; Wan, Xiangyuan

2018-06-01

Map-based cloning of maize ms33 gene showed that ZmMs33 encodes a sn-2 glycerol-3-phosphate acyltransferase, the ortholog of rice OsGPAT3, and it is essential for male fertility in maize. Genetic male sterility has been widely studied for its biological significance and commercial value in hybrid seed production. Although many male-sterile mutants have been identified in maize (Zea mays L.), it is likely that most genes that cause male sterility are unknown. Here, we report a recessive genetic male-sterile mutant, male sterility33 (ms33), which displays small, pale yellow anthers, and complete male sterility. Using a map-based cloning approach, maize GRMZM2G070304 was identified as the ms33 gene (ZmMs33). ZmMs33 encodes a novel sn-2 glycerol-3-phosphate acyltransferase (GPAT) in maize. A functional complementation experiment showed that GRMZM2G070304 can rescue the male-sterile phenotype of the ms33-6029 mutant. GRMZM2G070304 was further confirmed to be the ms33 gene via targeted knockouts induced by the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system. ZmMs33 is preferentially expressed in the immature anther from the quartet to early-vacuolate microspore stages and in root tissues at the fifth leaf growth stage. Phylogenetic analysis indicated that ZmMs33 and OsGPAT3 are evolutionarily conserved for anther and pollen development in monocot species. This study reveals that the monocot-specific GPAT3 protein plays an important role in male fertility in maize, and ZmMs33 and mutants in this gene may have value in maize male-sterile line breeding and hybrid seed production.

Contribution of radiation hybrids to genome mapping in domestic animals.

PubMed

Faraut, T; de Givry, S; Hitte, C; Lahbib-Mansais, Y; Morisson, M; Milan, D; Schiex, T; Servin, B; Vignal, A; Galibert, F; Yerle, M

2009-01-01

Radiation hybrid mapping has emerged in the end of the 1990 s as a successful and complementary approach to map genomes, essentially because of its ability to bridge the gaps between genetic and clone-based physical maps, but also using comparative mapping approaches, between 'gene-rich' and 'gene-poor' maps. Since its early development in human, radiation hybrid mapping played a pivotal role in the process of mapping animal genomes, especially mammalian ones. We review here all the different steps involved in radiation hybrid mapping from the constitution of panels to the construction of maps. A description of its contribution to whole genome maps with a special emphasis on domestic animals will also be presented. Finally, current applications of radiation hybrid mapping in the context of whole genome assemblies will be described. (c) 2009 S. Karger AG, Basel.

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits

PubMed Central

2011-01-01

Background Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18) to duodecaploid (12X = 108). Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome. Results A switchgrass BAC library constructed by partial digestion of nuclear DNA with EcoRI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective). Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice OsBRI1 locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy. Conclusions The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy. PMID:21767393

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits.

PubMed

Saski, Christopher A; Li, Zhigang; Feltus, Frank A; Luo, Hong

2011-07-18

Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18) to duodecaploid (12X = 108). Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome. A switchgrass BAC library constructed by partial digestion of nuclear DNA with EcoRI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective). Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice OsBRI1 locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy. The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy.

Advances in Maize Genomics and Their Value for Enhancing Genetic Gains from Breeding

PubMed Central

Xu, Yunbi; Skinner, Debra J.; Wu, Huixia; Palacios-Rojas, Natalia; Araus, Jose Luis; Yan, Jianbing; Gao, Shibin; Warburton, Marilyn L.; Crouch, Jonathan H.

2009-01-01

Maize is an important crop for food, feed, forage, and fuel across tropical and temperate areas of the world. Diversity studies at genetic, molecular, and functional levels have revealed that, tropical maize germplasm, landraces, and wild relatives harbor a significantly wider range of genetic variation. Among all types of markers, SNP markers are increasingly the marker-of-choice for all genomics applications in maize breeding. Genetic mapping has been developed through conventional linkage mapping and more recently through linkage disequilibrium-based association analyses. Maize genome sequencing, initially focused on gene-rich regions, now aims for the availability of complete genome sequence. Conventional insertion mutation-based cloning has been complemented recently by EST- and map-based cloning. Transgenics and nutritional genomics are rapidly advancing fields targeting important agronomic traits including pest resistance and grain quality. Substantial advances have been made in methodologies for genomics-assisted breeding, enhancing progress in yield as well as abiotic and biotic stress resistances. Various genomic databases and informatics tools have been developed, among which MaizeGDB is the most developed and widely used by the maize research community. In the future, more emphasis should be given to the development of tools and strategic germplasm resources for more effective molecular breeding of tropical maize products. PMID:19688107

A 2.5-Mb contig constructed from Angus, Longhorn and horned Hereford DNA spanning the polled interval on bovine chromosome 1.

PubMed

Wunderlich, K R; Abbey, C A; Clayton, D R; Song, Y; Schein, J E; Georges, M; Coppieters, W; Adelson, D L; Taylor, J F; Davis, S L; Gill, C A

2006-12-01

The polled locus has been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. As an intermediate step in our efforts to identify the polled locus and the underlying causative mutation for the polled phenotype, we have constructed a BAC-based physical map of the interval containing the polled locus. Clones containing genes and markers in the critical interval were isolated from the TAMBT (constructed from Angus and Longhorn genomic DNA) and CHORI-240 (constructed from horned Hereford genomic DNA) BAC libraries and ordered based on fingerprinting and the presence or absence of 80 STS markers. A single contig spanning 2.5 Mb was assembled. Comparison of the physical order of STSs to the corresponding region of human chromosome 21 revealed the same order of genes within the polled critical interval. This contig of overlapping BAC clones from horned and polled breeds is a useful resource for SNP discovery and characterization of positional candidate genes.

Isolation and characterization of a novel human scFv inhibiting EGFR vIII expressing cancers.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Dariushnejad, Hassan; Hosseini, Mohammad Kazem

2016-12-01

EGFRvIII, a mutant form of epidermal growth factor receptor is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. This tumor specific antigen has emerged as a promising candidate for antibody based therapy of several cancers. The aim of the present study was isolation and characterization of a human single chain antibody against EGFRvIII as a promising target for cancer therapy. For this, a synthetic peptide corresponding to EGFRvIII protein was used for screening the naive human scFv phage library. Selection was performed using a novel screening strategy for enrichment of rare specific clones. After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, a clone with an amber mutation in VH CDR2 coding sequence showed higher reactivity. The mutation was corrected through site directed mutagenesis and then scFv fragment was expressed after subcloning into the bacterial expression vector. Expression in BL21 pLysS resulted in a highly soluble scFv appeared in soluble fraction of E. coli lysate. Bioinformatic in silico analysis between scFv and EGFRvIII sequences confirmed specific binding of desired scFv to EGFRvIII in CDR regions. The specific reactivity of the purified scFv with native EGFRvIII was confirmed by cell based ELISA and western blot. In conclusion, human anti- EGFRvIII scFv isolated from a scFv phage library displayed high reactivity with EGFRvIII. The scFv isolated in this study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

PubMed Central

Erickson, J. R.; Johnston, M.

1993-01-01

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning. PMID:8514124

In-silico QTL mapping of postpubertal mammary ductal development in the mouse uncovers potential human breast cancer risk loci

USDA-ARS?s Scientific Manuscript database

Genetic background plays a dominant role in mammary gland development and breast cancer (BrCa). Despite this, the role of genetics is only partially understood. This study used strain-dependent variation in an inbred mouse mapping panel, to identify quantitative trait loci (QTL) underlying structura...

Optimally cloned binary coherent states

NASA Astrophysics Data System (ADS)

Müller, C. R.; Leuchs, G.; Marquardt, Ch.; Andersen, U. L.

2017-10-01

Binary coherent state alphabets can be represented in a two-dimensional Hilbert space. We capitalize this formal connection between the otherwise distinct domains of qubits and continuous variable states to map binary phase-shift keyed coherent states onto the Bloch sphere and to derive their quantum-optimal clones. We analyze the Wigner function and the cumulants of the clones, and we conclude that optimal cloning of binary coherent states requires a nonlinearity above second order. We propose several practical and near-optimal cloning schemes and compare their cloning fidelity to the optimal cloner.

Construction of a map-based reference genome sequence for barley, Hordeum vulgare L.

PubMed Central

Beier, Sebastian; Himmelbach, Axel; Colmsee, Christian; Zhang, Xiao-Qi; Barrero, Roberto A.; Zhang, Qisen; Li, Lin; Bayer, Micha; Bolser, Daniel; Taudien, Stefan; Groth, Marco; Felder, Marius; Hastie, Alex; Šimková, Hana; Staňková, Helena; Vrána, Jan; Chan, Saki; Muñoz-Amatriaín, María; Ounit, Rachid; Wanamaker, Steve; Schmutzer, Thomas; Aliyeva-Schnorr, Lala; Grasso, Stefano; Tanskanen, Jaakko; Sampath, Dharanya; Heavens, Darren; Cao, Sujie; Chapman, Brett; Dai, Fei; Han, Yong; Li, Hua; Li, Xuan; Lin, Chongyun; McCooke, John K.; Tan, Cong; Wang, Songbo; Yin, Shuya; Zhou, Gaofeng; Poland, Jesse A.; Bellgard, Matthew I.; Houben, Andreas; Doležel, Jaroslav; Ayling, Sarah; Lonardi, Stefano; Langridge, Peter; Muehlbauer, Gary J.; Kersey, Paul; Clark, Matthew D.; Caccamo, Mario; Schulman, Alan H.; Platzer, Matthias; Close, Timothy J.; Hansson, Mats; Zhang, Guoping; Braumann, Ilka; Li, Chengdao; Waugh, Robbie; Scholz, Uwe; Stein, Nils; Mascher, Martin

2017-01-01

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. ‘Morex’ was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX). PMID:28448065

[Molecular cloning and characterization in silico of phospholipase A(2) transcript isolated from Lachesis muta peruvian snake venom].

PubMed

Jimenez, Karim L; Zavaleta, Amparo I; Izaguirre, Victor; Yarleque, Armando; Inga, Rosio R

2010-01-01

Isolate and characterize in silico gene phospholipase A(2) (PLA(2)) isolated from Lachesis muta venom of the Peruvian Amazon. Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13,976 kDa and 5.66 respectively. The aminoacid sequence was called Lm-PLA(2)-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA(2) from Lachesis stenophrys (93%) and other PLA(2) snake venoms and over 80% of other sPLA(2) family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA(2)-Peru grouped with other acidic [Asp(49)] sPLA(2) previously isolated from Bothriechis schlegelii venom showing 89 % nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA(2) group II that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. The nucleotide sequence corresponding to the first transcript of gene from PLA(2) cloned of Lachesis muta venom, snake from the Peruvian rainforest.

Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

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

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-02-01

The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNAmore » sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.« less

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket.

PubMed

Soin, Thomas; Iga, Masatoshi; Swevers, Luc; Rougé, Pierre; Janssen, Colin R; Smagghe, Guy

2009-08-01

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.

Whole-genome typing and characterization of blaVIM19-harbouring ST383 Klebsiella pneumoniae by PFGE, whole-genome mapping and WGS.

PubMed

Sabirova, Julia S; Xavier, Basil Britto; Coppens, Jasmine; Zarkotou, Olympia; Lammens, Christine; Janssens, Lore; Burggrave, Ronald; Wagner, Trevor; Goossens, Herman; Malhotra-Kumar, Surbhi

2016-06-01

We utilized whole-genome mapping (WGM) and WGS to characterize 12 clinical carbapenem-resistant Klebsiella pneumoniae strains (TGH1-TGH12). All strains were screened for carbapenemase genes by PCR, and typed by MLST, PFGE (XbaI) and WGM (AflII) (OpGen, USA). WGS (Illumina) was performed on TGH8 and TGH10. Reads were de novo assembled and annotated [SPAdes, Rapid Annotation Subsystem Technology (RAST)]. Contigs were aligned directly, and after in silico AflII restriction, with corresponding WGMs (MapSolver, OpGen; BioNumerics, Applied Maths). All 12 strains were ST383. Of the 12 strains, 11 were carbapenem resistant, 7 harboured blaKPC-2 and 11 harboured blaVIM-19. Varying the parameters for assigning WGM clusters showed that these were comparable to STs and to the eight PFGE types or subtypes (difference of three or more bands). A 95% similarity coefficient assigned all 12 WGMs to a single cluster, whereas a 99% similarity coefficient (or ≥10 unmatched-fragment difference) assigned the 12 WGMs to eight (sub)clusters. Based on a difference of three or more bands between PFGE profiles, the Simpson's diversity indices (SDIs) of WGM (0.94, Jackknife pseudo-values CI: 0.883-0.996) and PFGE (0.93, Jackknife pseudo-values CI: 0.828-1.000) were similar (P = 0.649). However, the discriminatory power of WGM was significantly higher (SDI: 0.94, Jackknife pseudo-values CI: 0.883-0.996) than that of PFGE profiles typed on a difference of seven or more bands (SDI: 0.53, Jackknife pseudo-values CI: 0.212-0.849) (P = 0.007). This study demonstrates the application of WGM to understanding the epidemiology of hospital-associated K. pneumoniae. Utilizing a combination of WGM and WGS, we also present here the first longitudinal genomic characterization of the highly dynamic carbapenem-resistant ST383 K. pneumoniae clone that is rapidly gaining importance in Europe. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Biotechnological Approaches in Plant Protection: Achievements, New Initiatives and Prospects

USDA-ARS?s Scientific Manuscript database

This chapter reviews the latest developments in the application of biotechnology to solve crop looses due to fungal disease. These include marker-assisted breeding, map-based cloning of genes, and development of transgenic plants. New areas of investigation which hold much promise are gene silenci...

Construction of a transfer vector for a clonal isolate of LdNPV

Treesearch

Shivanand T. Hiremath; Martha Fikes; Audrey Ichida

1991-01-01

Deoxyribonucleic acid from a clonal isolate of LdNPV (CI A2-1), obtained by in vivo cloning procedures, was used to construct genomic libraries in phage (lamda Gem 11) and cosmid (pHC79) vectors. Overlapping clones were selected to generate a restriction enzyme map. The restriction enzyme map, covering about 85% of the CI A2-1 genome, was determined...

Discovery of Anti-Hypertensive Oligopeptides from Adlay Based on In Silico Proteolysis and Virtual Screening

PubMed Central

Qiao, Liansheng; Li, Bin; Chen, Yankun; Li, Lingling; Chen, Xi; Wang, Lingzhi; Lu, Fang; Luo, Ganggang; Li, Gongyu; Zhang, Yanling

2016-01-01

Adlay (Coix larchryma-jobi L.) was the commonly used Traditional Chinese Medicine (TCM) with high content of seed storage protein. The hydrolyzed bioactive oligopeptides of adlay have been proven to be anti-hypertensive effective components. However, the structures and anti-hypertensive mechanism of bioactive oligopeptides from adlay were not clear. To discover the definite anti-hypertensive oligopeptides from adlay, in silico proteolysis and virtual screening were implemented to obtain potential oligopeptides, which were further identified by biochemistry assay and molecular dynamics simulation. In this paper, ten sequences of adlay prolamins were collected and in silico hydrolyzed to construct the oligopeptide library with 134 oligopeptides. This library was reverse screened by anti-hypertensive pharmacophore database, which was constructed by our research team and contained ten anti-hypertensive targets. Angiotensin-I converting enzyme (ACE) was identified as the main potential target for the anti-hypertensive activity of adlay oligopeptides. Three crystal structures of ACE were utilized for docking studies and 19 oligopeptides were finally identified with potential ACE inhibitory activity. According to mapping features and evaluation indexes of pharmacophore and docking, three oligopeptides were selected for biochemistry assay. An oligopeptide sequence, NPATY (IC50 = 61.88 ± 2.77 µM), was identified as the ACE inhibitor by reverse-phase high performance liquid chromatography (RP-HPLC) assay. Molecular dynamics simulation of NPATY was further utilized to analyze interactive bonds and key residues. ALA354 was identified as a key residue of ACE inhibitors. Hydrophobic effect of VAL518 and electrostatic effects of HIS383, HIS387, HIS513 and Zn2+ were also regarded as playing a key role in inhibiting ACE activities. This study provides a research strategy to explore the pharmacological mechanism of Traditional Chinese Medicine (TCM) proteins based on in silico proteolysis and virtual screening, which could be beneficial to reveal the pharmacological action of TCM proteins and provide new lead compounds for peptides-based drug design. PMID:27983650

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine

PubMed Central

2011-01-01

Background Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe. Results We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 in vitro SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 in silico SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for in silico and in vitro SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species. Conclusions Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers. PMID:21767361

Transposon-containing DNA cloning vector and uses thereof

DOEpatents

Berg, C.M.; Berg, D.E.; Wang, G.

1997-07-08

The present invention discloses a rapid method of restriction mapping, sequencing or localizing genetic features in a segment of deoxyribonucleic acid (DNA) that is up to 42 kb in size. The method in part comprises cloning of the DNA segment in a specialized cloning vector and then isolating nested deletions in either direction in vivo by intramolecular transposition into the cloned DNA. A plasmid has been prepared and disclosed. 4 figs.

Transposon-containing DNA cloning vector and uses thereof

DOEpatents

Berg, Claire M.; Berg, Douglas E.; Wang, Gan

1997-01-01

The present invention discloses a rapid method of restriction mapping, sequencing or localizing genetic features in a segment of deoxyribonucleic acid (DNA) that is up to 42 kb in size. The method in part comprises cloning of the DNA segment in a specialized cloning vector and then isolating nested deletions in either direction in vivo by intramolecular transposition into the cloned DNA. A plasmid has been prepared and disclosed.

IRS-PCR-based genetic mapping of the huntingtin interacting protein gene (HIP1) on mouse chromosome 5.

PubMed

Himmelbauer, H; Wedemeyer, N; Haaf, T; Wanker, E E; Schalkwyk, L C; Lehrach, H

1998-01-01

Huntington's disease (HD) is a devastating central nervous system disorder. Even though the gene responsible has been positionally cloned recently, its etiology has remained largely unclear. To investigate potential disease mechanisms, we conducted a search for binding partners of the HD-protein huntingtin. With the yeast two-hybrid system, one such interacting factor, the huntingtin interacting protein-1 (HIP-1), was identified (Wanker et al. 1997; Kalchman et al. 1997) and the human gene mapped to 7q11.2. In this paper we demonstrate the localization of the HIP1 mouse homologue (Hip1) into a previously identified region of human-mouse synteny on distal mouse Chromosome (Chr) 5, both employing an IRS-PCR-based mapping strategy and traditional fluorescent in situ hybridization (FISH) mapping.

Sensitivity of Small RNA-Based Detection of Plant Viruses.

PubMed

Santala, Johanna; Valkonen, Jari P T

2018-01-01

Plants recognize unrelated viruses by the antiviral defense system called RNA interference (RNAi). RNAi processes double-stranded viral RNA into small RNAs (sRNAs) of 21-24 nucleotides, the reassembly of which into longer strands in silico allows virus identification by comparison with the sequences available in databases. The aim of this study was to compare the virus detection sensitivity of sRNA-based virus diagnosis with the established virus species-specific polymerase chain reaction (PCR) approach. Viruses propagated in tobacco plants included three engineered, infectious clones of Potato virus A (PVA), each carrying a different marker gene, and an infectious clone of Potato virus Y (PVY). Total RNA (containing sRNA) was isolated and subjected to reverse-transcription real-time PCR (RT-RT-PCR) and sRNA deep-sequencing at different concentrations. RNA extracted from various crop plants was included in the reactions to normalize RNA concentrations. Targeted detection of selected viruses showed a similar threshold for the sRNA and reverse-transcription quantitative PCR (RT-qPCR) analyses. The detection limit for PVY and PVA by RT-qPCR in this study was 3 and 1.5 fg of viral RNA, respectively, in 50 ng of total RNA per PCR reaction. When knowledge was available about the viruses likely present in the samples, sRNA-based virus detection was 10 times more sensitive than RT-RT-PCR. The advantage of sRNA analysis is the detection of all tested viruses without the need for virus-specific primers or probes.

Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome

PubMed Central

2011-01-01

Background Flax (Linum usitatissimum L.) is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN) was initiated. We report here the first genome-wide physical map of flax and the generation and analysis of BAC-end sequences (BES) from 43,776 clones, providing initial insights into the genome. Results The physical map consists of 416 contigs spanning ~368 Mb, assembled from 32,025 fingerprints, representing roughly 54.5% to 99.4% of the estimated haploid genome (370-675 Mb). The N50 size of the contigs was estimated to be ~1,494 kb. The longest contig was ~5,562 kb comprising 437 clones. There were 96 contigs containing more than 100 clones. Approximately 54.6 Mb representing 8-14.8% of the genome was obtained from 80,337 BES. Annotation revealed that a large part of the genome consists of ribosomal DNA (~13.8%), followed by known transposable elements at 6.1%. Furthermore, ~7.4% of sequence was identified to harbour novel repeat elements. Homology searches against flax-ESTs and NCBI-ESTs suggested that ~5.6% of the transcriptome is unique to flax. A total of 4064 putative genomic SSRs were identified and are being developed as novel markers for their use in molecular breeding. Conclusion The first genome-wide physical map of flax constructed with BAC clones provides a framework for accessing target loci with economic importance for marker development and positional cloning. Analysis of the BES has provided insights into the uniqueness of the flax genome. Compared to other plant genomes, the proportion of rDNA was found to be very high whereas the proportion of known transposable elements was low. The SSRs identified from BES will be valuable in saturating existing linkage maps and for anchoring physical and genetic maps. The physical map and paired-end reads from BAC clones will also serve as scaffolds to build and validate the whole genome shotgun assembly. PMID:21554714

Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome.

PubMed

Ragupathy, Raja; Rathinavelu, Rajkumar; Cloutier, Sylvie

2011-05-09

Flax (Linum usitatissimum L.) is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN) was initiated. We report here the first genome-wide physical map of flax and the generation and analysis of BAC-end sequences (BES) from 43,776 clones, providing initial insights into the genome. The physical map consists of 416 contigs spanning ~368 Mb, assembled from 32,025 fingerprints, representing roughly 54.5% to 99.4% of the estimated haploid genome (370-675 Mb). The N50 size of the contigs was estimated to be ~1,494 kb. The longest contig was ~5,562 kb comprising 437 clones. There were 96 contigs containing more than 100 clones. Approximately 54.6 Mb representing 8-14.8% of the genome was obtained from 80,337 BES. Annotation revealed that a large part of the genome consists of ribosomal DNA (~13.8%), followed by known transposable elements at 6.1%. Furthermore, ~7.4% of sequence was identified to harbour novel repeat elements. Homology searches against flax-ESTs and NCBI-ESTs suggested that ~5.6% of the transcriptome is unique to flax. A total of 4064 putative genomic SSRs were identified and are being developed as novel markers for their use in molecular breeding. The first genome-wide physical map of flax constructed with BAC clones provides a framework for accessing target loci with economic importance for marker development and positional cloning. Analysis of the BES has provided insights into the uniqueness of the flax genome. Compared to other plant genomes, the proportion of rDNA was found to be very high whereas the proportion of known transposable elements was low. The SSRs identified from BES will be valuable in saturating existing linkage maps and for anchoring physical and genetic maps. The physical map and paired-end reads from BAC clones will also serve as scaffolds to build and validate the whole genome shotgun assembly.

Bacterial Artificial Chromosome Libraries for Mouse Sequencing and Functional Analysis

PubMed Central

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

2000-01-01

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

Identification of positional candidates for bovine placental genes responsible for early embryonic death during cloning-attempted pregnancy.

PubMed

Yamada, Takahisa; Muramatsu, Youji; Taniguchi, Yukio; Sasaki, Yoshiyuki

Our previous study detected 291 and 77 genes showing early embryonic death-associated elevation and reduction of expression, respectively, in the fetal placenta of the cow carrying somatic nuclear transfer-derived cloned embryo. In this study, we mapped the 10 genes showing the elevation and the 10 genes doing the reduction most significantly, using somatic cell hybrid and bovine draft genome sequence. We then compared the mapped positions for these genes with the genomic locations of bovine quantitative trait loci for still-birth and/or abortion. Among the mapped genes, peptidylglycine alpha-amidating monooxygenase (PAM), spectrin, beta, nonerythrocytic 1 (SPTBNI), and an unknown novel gene containing AU277832 expressed sequence tag were intriguing, in that the mapped positions were consistent with the genomic locations of bovine still-birth and/or abortion quantitative trait loci, and thus identified as positional candidates for bovine placental genes responsible for the early embryonic death during the pregnancy attempted by somatic nuclear transfer-derived cloning.

Ab initio multiple cloning simulations of pyrrole photodissociation: TKER spectra and velocity map imaging

DOE PAGES

Makhov, Dmitry V.; Saita, Kenichiro; Martinez, Todd J.; ...

2014-12-11

In this study, we report a detailed computational simulation of the photodissociation of pyrrole using the ab initio Multiple Cloning (AIMC) method implemented within MOLPRO. The efficiency of the AIMC implementation, employing train basis sets, linear approximation for matrix elements, and Ehrenfest configuration cloning, allows us to accumulate significant statistics. We calculate and analyze the total kinetic energy release (TKER) spectrum and Velocity Map Imaging (VMI) of pyrrole and compare the results directly with experimental measurements. Both the TKER spectrum and the structure of the velocity map image (VMI) are well reproduced. Previously, it has been assumed that the isotropicmore » component of the VMI arises from long time statistical dissociation. Instead, our simulations suggest that ultrafast dynamics contributes significantly to both low and high energy portions of the TKER spectrum.« less

Ab initio multiple cloning simulations of pyrrole photodissociation: TKER spectra and velocity map imaging

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

Makhov, Dmitry V.; Saita, Kenichiro; Martinez, Todd J.

In this study, we report a detailed computational simulation of the photodissociation of pyrrole using the ab initio Multiple Cloning (AIMC) method implemented within MOLPRO. The efficiency of the AIMC implementation, employing train basis sets, linear approximation for matrix elements, and Ehrenfest configuration cloning, allows us to accumulate significant statistics. We calculate and analyze the total kinetic energy release (TKER) spectrum and Velocity Map Imaging (VMI) of pyrrole and compare the results directly with experimental measurements. Both the TKER spectrum and the structure of the velocity map image (VMI) are well reproduced. Previously, it has been assumed that the isotropicmore » component of the VMI arises from long time statistical dissociation. Instead, our simulations suggest that ultrafast dynamics contributes significantly to both low and high energy portions of the TKER spectrum.« less

Novel in silico multivariate mapping of intrinsic and anticorrelated connectivity to neurocognitive functional maps supports the maturational hypothesis of ADHD.

PubMed

de Lacy, Nina; Kodish, Ian; Rachakonda, Srinivas; Calhoun, Vince D

2018-04-22

From childhood to adolescence, strengthened coupling in frontal, striatal and parieto-temporal regions associated with cognitive control, and increased anticorrelation between task-positive and task-negative circuits, subserve the reshaping of behavior. ADHD is a common condition peaking in adolescence and regressing in adulthood, with a wide variety of cognitive control deficits. Alternate hypotheses of ADHD emphasize lagging circuitry refinement versus categorical differences in network function. However, quantifying the individual circuit contributions to behavioral findings, and relative roles of maturational versus categorical effects, is challenging in vivo or in meta-analyses using task-based paradigms within the same pipeline, given the multiplicity of neurobehavioral functions implicated. To address this, we analyzed 46 positively-correlated and anticorrelated circuits in a multivariate model in resting-state data from 504 age- and gender-matched youth, and created a novel in silico method to map individual quantified effects to reverse inference maps of 8 neurocognitive functions consistently implicated in ADHD, as well as dopamine and hyperactivity. We identified only age- and gender-related effects in intrinsic connectivity, and found that maturational refinement of circuits in youth with ADHD occupied 3-10x more brain locations than in typical development, with the footprint, effect size and contribution of individual circuits varying substantially. Our analysis supports the maturational hypothesis of ADHD, suggesting lagging connectivity reorganization within specific subnetworks of fronto-parietal control, ventral attention, cingulo-opercular, temporo-limbic and cerebellar sub-networks contribute across neurocognitive findings present in this complex condition. We present the first analysis of anti-correlated connectivity in ADHD and suggest new directions for exploring residual and non-responsive symptoms. © 2018 Wiley Periodicals, Inc.

Identical substitutions in magnesium chelatase paralogs result in chlorophyll deficient soybean mutants

USDA-ARS?s Scientific Manuscript database

The soybean (Glycine max (L.) Merr.) chlorophyll deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a non-synonymous nucleotide substitution in the third exon of a Mg-chelat...

Parallel computing of physical maps--a comparative study in SIMD and MIMD parallelism.

PubMed

Bhandarkar, S M; Chirravuri, S; Arnold, J

1996-01-01

Ordering clones from a genomic library into physical maps of whole chromosomes presents a central computational problem in genetics. Chromosome reconstruction via clone ordering is usually isomorphic to the NP-complete Optimal Linear Arrangement problem. Parallel SIMD and MIMD algorithms for simulated annealing based on Markov chain distribution are proposed and applied to the problem of chromosome reconstruction via clone ordering. Perturbation methods and problem-specific annealing heuristics are proposed and described. The SIMD algorithms are implemented on a 2048 processor MasPar MP-2 system which is an SIMD 2-D toroidal mesh architecture whereas the MIMD algorithms are implemented on an 8 processor Intel iPSC/860 which is an MIMD hypercube architecture. A comparative analysis of the various SIMD and MIMD algorithms is presented in which the convergence, speedup, and scalability characteristics of the various algorithms are analyzed and discussed. On a fine-grained, massively parallel SIMD architecture with a low synchronization overhead such as the MasPar MP-2, a parallel simulated annealing algorithm based on multiple periodically interacting searches performs the best. For a coarse-grained MIMD architecture with high synchronization overhead such as the Intel iPSC/860, a parallel simulated annealing algorithm based on multiple independent searches yields the best results. In either case, distribution of clonal data across multiple processors is shown to exacerbate the tendency of the parallel simulated annealing algorithm to get trapped in a local optimum.

A combinatorial approach of comprehensive QTL-based comparative genome mapping and transcript profiling identified a seed weight-regulating candidate gene in chickpea

PubMed Central

Bajaj, Deepak; Upadhyaya, Hari D.; Khan, Yusuf; Das, Shouvik; Badoni, Saurabh; Shree, Tanima; Kumar, Vinod; Tripathi, Shailesh; Gowda, C. L. L.; Singh, Sube; Sharma, Shivali; Tyagi, Akhilesh K.; Chattopdhyay, Debasis; Parida, Swarup K.

2015-01-01

High experimental validation/genotyping success rate (94–96%) and intra-specific polymorphic potential (82–96%) of 1536 SNP and 472 SSR markers showing in silico polymorphism between desi ICC 4958 and kabuli ICC 12968 chickpea was obtained in a 190 mapping population (ICC 4958 × ICC 12968) and 92 diverse desi and kabuli genotypes. A high-density 2001 marker-based intra-specific genetic linkage map comprising of eight LGs constructed is comparatively much saturated (mean map-density: 0.94 cM) in contrast to existing intra-specific genetic maps in chickpea. Fifteen robust QTLs (PVE: 8.8–25.8% with LOD: 7.0–13.8) associated with pod and seed number/plant (PN and SN) and 100 seed weight (SW) were identified and mapped on 10 major genomic regions of eight LGs. One of 126.8 kb major genomic region harbouring a strong SW-associated robust QTL (Caq'SW1.1: 169.1–171.3 cM) has been delineated by integrating high-resolution QTL mapping with comprehensive marker-based comparative genome mapping and differential expression profiling. This identified one potential regulatory SNP (G/A) in the cis-acting element of candidate ERF (ethylene responsive factor) TF (transcription factor) gene governing seed weight in chickpea. The functionally relevant molecular tags identified have potential to be utilized for marker-assisted genetic improvement of chickpea. PMID:25786576

Linkage and homology analysis divides the eight genes for the small subunit of petunia ribulose 1,5-bisphosphate carboxylase into three gene families

PubMed Central

Dean, Caroline; van den Elzen, Peter; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Twenty-six λ phage clones with homology to coding sequences of the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase have been isolated from an EMBL3 λ phage bank of Petunia (Mitchell) DNA. Restriction mapping of the phage inserts shows that the clones were obtained from five nonoverlapping regions of petunia DNA that carry seven SSU genes. Comparison of the HindIII genomic fragments of petunia DNA with the HindIII restriction fragments of the isolated phage indicates that petunia nuclear DNA encodes eight SSU genes, seven of which are present in the phage clones. Two incomplete genes, which contain only the 3′ end of an SSU gene, were also found in the phage clones. We demonstrate that the eight SSU genes of petunia can be divided into three gene families based on homology to three petunia cDNA clones. Two gene families contain single SSU genes and the third contains six genes, four of which are closely linked within petunia nuclear DNA. Images PMID:16593584

In silico characterization and expression analysis of the multigene family encoding the Bowman-Birk protease inhibitor in soybean.

PubMed

de Almeida Barros, Beatriz; da Silva, Wiliane Garcia; Moreira, Maurilio Alves; de Barros, Everaldo Gonçalves

2012-01-01

The Bowman-Birk (BBI) protease inhibitors can be used as source of sulfur amino acids, can regulate endogenous protease activity during seed germination and during the defense response of plants to pathogens. In soybean this family has not been fully described. The goal of this work was to characterize in silico and analyze the expression of the members of this family in soybean. We identified 11 potential BBI genes in the soybean genome. In each one of them at least a characteristic BBI conserved domain was detected in addition to a potential signal peptide. The sequences have been positioned in the soybean physical map and the promoter regions were analyzed with respect to known regulatory elements. Elements related to seed-specific expression and also to response to biotic and abiotic stresses have been identified. Based on the in silico analysis and also on quantitative RT-PCR data it was concluded that BBI-A, BBI-CII and BBI-DII are expressed specifically in the seed. The expression profiles of these three genes are similar along seed development. Their expressions reach a maximum in the intermediate stages and decrease as the seed matures. The BBI-DII transcripts are the most abundant ones followed by those of BBI-A and BBI-CII.

Development of a YAC contig covering the minimal region of a CSNB1 locus in Xp11

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

Boycott, K.M.; Gratton, K.J.; Moore, B.J.

1994-09-01

X-linked congenital stationary night blindness (CSNB1) is an eye disorder that includes impairment of night vision, reduced visual acuity and, in some cases, myopia and congenital nystagmus. Electroretinography reveals a marked reduction of the b-wave in affected individuals suggesting that X-linked CSNB is due to a molecular defect in the bipolar layer of the retina. Based on our studies of a large four generation family with X-linked CSNB, a CSNB1 locus was mapped to a 4-5 cM region at Xp11.23-Xp11.22 bounded telomerically by DXS426 and centromerically by DXS988. Using a panel of radiation and conventional somatic cell hybrids, a detailedmore » map of new and published STSs has been generated for the minimal region of CSNB1. PCR primer pairs for STSs has been generated for the minimal region of CSNB1. PCR primer pairs for twenty-five STSs, including eleven end-clones, were used to isolate YAC clones from CEPH, mega-CEPH, and X chromosome-specific YAC libraries. In total, fifty-two YACs were characterized for STS overlaps and assembled to provide a minimum of 3 Mb of physical coverage in the region between DXS426 and DXS988. Five gaps proximal to SYP are still to be closed. Our physical map suggests the following gene order: Xpter-OTAL1-GF1-DXS1011E-MG81-HUMCRAS2P-SYP-Xcen. STS analysis of the YACs revealed three subregions of the physical map which appear to be particularly susceptible to internal deletions and end-clone analysis demonstrated chimerism in six of seventeen YACs. A physical map of Xp11.23-Xp11.22 will provide a resource for the isolation of candidate genes for the X-linked CSNB gene which maps to this region.« less

Chloroplast microsatellite markers for Artocarpus (Moraceae) developed from transcriptome sequences1

PubMed Central

Gardner, Elliot M.; Laricchia, Kristen M.; Murphy, Matthew; Ragone, Diane; Scheffler, Brian E.; Simpson, Sheron; Williams, Evelyn W.; Zerega, Nyree J. C.

2015-01-01

Premise of the study: Chloroplast microsatellite loci were characterized from transcriptomes of Artocarpus altilis (breadfruit) and A. camansi (breadnut). They were tested in A. odoratissimus (terap) and A. altilis and evaluated in silico for two congeners. Methods and Results: Fifteen simple sequence repeats (SSRs) were identified in chloroplast sequences from four Artocarpus transcriptome assemblies. The markers were evaluated using capillary electrophoresis in A. odoratissimus (105 accessions) and A. altilis (73). They were also evaluated in silico in A. altilis (10), A. camansi (6), and A. altilis × A. mariannensis (7) transcriptomes. All loci were polymorphic in at least one species, with all 15 polymorphic in A. camansi. Per species, average alleles per locus ranged between 2.2 and 2.5. Three loci had evidence of fragment-length homoplasy. Conclusions: These markers will complement existing nuclear markers by enabling confident identification of maternal and clone lines, which are often important in vegetatively propagated crops such as breadfruit. PMID:26421253

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest

PubMed Central

Ji, Hyeonso; Kim, Sung-Ryul; Kim, Yul-Ho; Suh, Jung-Pil; Park, Hyang-Mi; Sreenivasulu, Nese; Misra, Gopal; Kim, Suk-Man; Hechanova, Sherry Lou; Kim, Hakbum; Lee, Gang-Seob; Yoon, Ung-Han; Kim, Tae-Ho; Lim, Hyemin; Suh, Suk-Chul; Yang, Jungil; An, Gynheung; Jena, Kshirod K.

2016-01-01

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars. PMID:27682162

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest.

PubMed

Ji, Hyeonso; Kim, Sung-Ryul; Kim, Yul-Ho; Suh, Jung-Pil; Park, Hyang-Mi; Sreenivasulu, Nese; Misra, Gopal; Kim, Suk-Man; Hechanova, Sherry Lou; Kim, Hakbum; Lee, Gang-Seob; Yoon, Ung-Han; Kim, Tae-Ho; Lim, Hyemin; Suh, Suk-Chul; Yang, Jungil; An, Gynheung; Jena, Kshirod K

2016-09-29

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars.

Next-Generation Sequencing-Based Approaches for Mutation Mapping and Identification in Caenorhabditis elegans

PubMed Central

Doitsidou, Maria; Jarriault, Sophie; Poole, Richard J.

2016-01-01

The use of next-generation sequencing (NGS) has revolutionized the way phenotypic traits are assigned to genes. In this review, we describe NGS-based methods for mapping a mutation and identifying its molecular identity, with an emphasis on applications in Caenorhabditis elegans. In addition to an overview of the general principles and concepts, we discuss the main methods, provide practical and conceptual pointers, and guide the reader in the types of bioinformatics analyses that are required. Owing to the speed and the plummeting costs of NGS-based methods, mapping and cloning a mutation of interest has become straightforward, quick, and relatively easy. Removing this bottleneck previously associated with forward genetic screens has significantly advanced the use of genetics to probe fundamental biological processes in an unbiased manner. PMID:27729495

High-resolution mapping and sequence analysis of 597 cDNA clones transcribed from the 1 Mb region in human chromosome 4q16.3 containing Huntington disease gene

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

Hadano, S.; Ishida, Y.; Tomiyasu, H.

1994-09-01

To complete a transcription map of the 1 Mb region in human chromosome 4p16.3 containing the Huntington disease (HD) gene, the isolation of cDNA clones are being performed throughout. Our method relies on a direct screening of the cDNA libraries probed with single copy microclones from 3 YAC clones spanning 1 Mbp of the HD gene region. AC-DNAs were isolated by a preparative pulsed-field gel electrophoresis, amplified by both a single unique primer (SUP)-PCR and a linker ligation PCR, and 6 microclone-DNA libraries were generated. Then, 8,640 microclones from these libraries were independently amplified by PCR, and arrayed onto themore » membranes. 800-900 microclones that were not cross-hybridized with total human and yeast genomic DNA, TAC vector DNA, and ribosomal cDNA on a dot hybridization (putatively carrying single copy sequences) were pooled to make 9 probe pools. A total of {approximately}1.8x10{sup 7} plaques from the human brain cDNA libraries was screened with 9 pool-probes, and then 672 positive cDNA clones were obtained. So far, 597 cDNA clones were defined and arrayed onto a map of the 1 Mbp of the HD gene region by hybridization with HD region-specific cosmid contigs and YAC clones. Further characterization including a DNA sequencing and Northern blot analysis is currently underway.« less

A Fine Physical Map of the Rice Chromosome 4

PubMed Central

Zhao, Qiang; Zhang, Yu; Cheng, Zhukuan; Chen, Mingsheng; Wang, Shengyue; Feng, Qi; Huang, Yucheng; Li, Ying; Tang, Yesheng; Zhou, Bo; Chen, Zhehua; Yu, Shuliang; Zhu, Jingjie; Hu, Xin; Mu, Jie; Ying, Kai; Hao, Pei; Zhang, Lei; Lu, Yiqi; Zhang, Lei S.; Liu, Yilei; Yu, Zhen; Fan, Danlin; Weng, Qijun; Chen, Ling; Lu, Tingting; Liu, Xiaohui; Jia, Peixin; Sun, Tongguo; Wu, Yongrui; Zhang, Yujun; Lu, Ying; Li, Can; Wang, Rong; Lei, Haiyan; Li, Tao; Hu, Hao; Wu, Mei; Zhang, Runquan; Guan, Jianping; Zhu, Jia; Fu, Gang; Gu, Minghong; Hong, Guofan; Xue, Yongbiao; Wing, Rod; Jiang, Jiming; Han, Bin

2002-01-01

As part of an international effort to completely sequence the rice genome, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Oryza sativa japonica Nipponbare chromosome 4 through an integration of 114 sequenced BAC clones from a taxonomically related subspecies O. sativa indica Guangluai 4 and 182 RFLP and 407 expressed sequence tag (EST) markers with the fingerprinted data of the Nipponbare genome. The map consists of 11 contigs with a total length of 34.5 Mb covering 94% of the estimated chromosome size (36.8 Mb). BAC clones corresponding to telomeres, as well as to the centromere position, were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH). This gave rise to an estimated length ratio of 5.13 for the long arm and 2.9 for the short arm (on the basis of the physical map), which indicates that the short arm is a highly condensed one. The FISH analysis and physical mapping also showed that the short arm and the pericentromeric region of the long arm are rich in heterochromatin, which occupied 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence. To our knowledge, this map provides the first example of a rapid and reliable physical mapping on the basis of the integration of the data from two taxonomically related subspecies. [The following individuals and institutions kindly provided reagents, samples, or unpublished information as indicated in the paper: S. McCouch, T. Sasaki, and Monsanto.] PMID:11997348

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes.

PubMed

Staňková, Helena; Hastie, Alex R; Chan, Saki; Vrána, Jan; Tulpová, Zuzana; Kubaláková, Marie; Visendi, Paul; Hayashi, Satomi; Luo, Mingcheng; Batley, Jacqueline; Edwards, David; Doležel, Jaroslav; Šimková, Hana

2016-07-01

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

In silico evaluation of gadofosveset pharmacokinetics in different population groups using the Simcyp® simulator platform.

PubMed

Spanakis, Marios; Marias, Kostas

2014-12-01

Gadofosveset is a Gd-based contrast agent used for magnetic resonance imaging (MRI). Gadolinium kinetic distribution models are implemented in T1-weighted dynamic contrast-enhanced perfusion MRI for characterization of lesion sites in the body. Physiology changes in a disease state potentially can influence the pharmacokinetics of drugs and to this respect modify the distribution properties of contrast agents. This work focuses on the in silico modelling of pharmacokinetic properties of gadofosveset in different population groups through the application of physiologically-based pharmacokinetic models (PBPK) embedded in Simcyp® population pharmacokinetics platform. Physicochemical and pharmacokinetic properties of gadofosveset were introduced into Simcyp® simulator platform and a min-PBPK model was applied. In silico clinical trials were generated simulating the administration of the recommended dose for the contrast agent (i.v., 30 mg/kg) in population cohorts of healthy volunteers, obese, renal and liver impairment, and in a generated virtual oncology population. Results were evaluated regarding basic pharmacokinetic parameters of Cmax, AUC and systemic CL and differences were assessed through ANOVA and estimation of ratio of geometric mean between healthy volunteers and the other population groups. Simcyp® predicted a mean Cmax = 551.60 mg/l, a mean AUC = 4079.12 mg/L*h and a mean systemic CL = 0.56 L/h for the virtual population of healthy volunteers. Obese population showed a modulation in Cmax and CL, attributed to increased administered dose. In renal and liver impairment cohorts a significant modulation in Cmax, AUC and CL of gadofosveset is predicted. Oncology population exhibited statistical significant differences regarding AUC when compared with healthy volunteers. This work employed Simcyp® population pharmacokinetics platform in order to compute gadofosveset's pharmacokinetic profiles through PBPK models and in silico clinical trials and evaluate possible differences between population groups. The approach showed promising results that could provide new insights regarding administration of contrast agents in special population cohorts. In silico pharmacokinetics could further be used for evaluating of possible toxicity, interpretation of MRI PK image maps and development of novel contrast agents.

Refined mapping and YAC contig construction of the X-linked cleft palate and ankyloglossia locus (CPX) including the proximal X-Y homology breakpoint within Xq21.3

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

Forbes, S.A.; Brennan, L.; Richardson, M.

1996-01-01

The gene for X-linked cleft palate (CPX) has previously been mapped in an Icelandic kindred between the unordered proximal markers DXS1002/DXS349/DXS95 and the distal marker DXYS1X, which maps to the proximal end of the X-Y homology region in Xq21.3. Using six sequence-tagged sites (STSs) within the region, a total of 91 yeast artificial chromosome (YAC) clones were isolated and overlapped in a single contig that spans approximately 3.1 Mb between DXS1002 and DXYS1X. The order of microsatellite and STS markers in this was established as DXS1002-DXS1168-DXS349-DXS95-DXS364-DXS1196-DXS472-DXS1217-DXYS1X. A long-range restriction map of this region was created using eight nonchimeric, overlapping YACmore » clones. Analysis of newly positioned polymorphic markers in recombinant individuals from the Icelandic family has enabled us to identify DXS1196 and DXS1217 as the flanking markers for CPX. The maximum physical distance containing the CPX gene has been estimated to be 2.0 Mb, which is spanned by a minimum set of five nonchimeric YAC clones. In addition, YAC end clone and STS analyses have pinpointed the location of the proximal boundary of the X-Y homology region within the map. 40 refs., 2 figs., 2 tabs.« less

Molecular mapping within the mouse albino-deletion complex.

PubMed Central

Johnson, D K; Hand, R E; Rinchik, E M

1989-01-01

Induced germ-line deletion mutations in the mouse provide a malleable experimental system for in-depth molecular and functional analysis of large segments of the mammalian genome. To obtain an initial bank of molecular probes for the region of mouse chromosome 7 associated with the albino-deletion complex, random anonymous DNA clones, derived from a library constructed from flow-sorted chromosomes, were screened on DNAs from Mus musculus-Mus spretus F1 hybrids carrying large, multilocus, lethal albino deletions. Clones falling within a given deletion interval can easily be recognized because hybridization bands that represent restriction fragment length polymorphisms specific for the mutant (deleted) chromosome inherited from the M. musculus parent will be absent. Among 72 informative clones used as probes, one, which defines the locus D7OR1, mapped within two deletions that are 6-11 centimorgans in length. Submapping of this anonymous clone across a panel of 27 smaller deletions localized D7OR1 distal to a chromosomal subregion important for survival of the preimplantation embryo, proximal to globin [beta-chain (Hbb)], and near the shaker-1 (sh-1) locus. The results of these deletion-mapping experiments were also confirmed by standard three-point linkage analysis. This strategy for selection and rapid mapping of anonymous DNA probes to chromosomal segments corresponding to germ-line deletion mutations should contribute to the generation of more detailed physical and functional maps of genomic regions associated with mutant developmental phenotypes. Images PMID:2813427

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion.

PubMed

Santillán, Orlando; Ramírez-Romero, Miguel A; Dávila, Guillermo

2017-06-25

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Identification and characterization of novel and potent transcription promoters of Francisella tularensis.

PubMed

Zaide, Galia; Grosfeld, Haim; Ehrlich, Sharon; Zvi, Anat; Cohen, Ofer; Shafferman, Avigdor

2011-03-01

Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli σ(70)-related promoter motifs. These motifs include the -10 (TATAAT) and -35 [TTGA(C/T)A] domains and an AT-rich region upstream from -35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.

Species-Level Phylogeny and Polyploid Relationships in Hordeum (Poaceae) Inferred by Next-Generation Sequencing and In Silico Cloning of Multiple Nuclear Loci.

PubMed

Brassac, Jonathan; Blattner, Frank R

2015-09-01

Polyploidization is an important speciation mechanism in the barley genus Hordeum. To analyze evolutionary changes after allopolyploidization, knowledge of parental relationships is essential. One chloroplast and 12 nuclear single-copy loci were amplified by polymerase chain reaction (PCR) in all Hordeum plus six out-group species. Amplicons from each of 96 individuals were pooled, sheared, labeled with individual-specific barcodes and sequenced in a single run on a 454 platform. Reference sequences were obtained by cloning and Sanger sequencing of all loci for nine supplementary individuals. The 454 reads were assembled into contigs representing the 13 loci and, for polyploids, also homoeologues. Phylogenetic analyses were conducted for all loci separately and for a concatenated data matrix of all loci. For diploid taxa, a Bayesian concordance analysis and a coalescent-based dated species tree was inferred from all gene trees. Chloroplast matK was used to determine the maternal parent in allopolyploid taxa. The relative performance of different multilocus analyses in the presence of incomplete lineage sorting and hybridization was also assessed. The resulting multilocus phylogeny reveals for the first time species phylogeny and progenitor-derivative relationships of all di- and polyploid Hordeum taxa within a single analysis. Our study proves that it is possible to obtain a multilocus species-level phylogeny for di- and polyploid taxa by combining PCR with next-generation sequencing, without cloning and without creating a heavy load of sequence data. © The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.

In Silico Identification of Candidate Genes for Fertility Restoration in Cytoplasmic Male Sterile Perennial Ryegrass (Lolium perenne L.)

PubMed Central

Sykes, Timothy; Yates, Steven; Nagy, Istvan; Asp, Torben; Small, Ian

2017-01-01

Perennial ryegrass (Lolium perenne L.) is widely used for forage production in both permanent and temporary grassland systems. To increase yields in perennial ryegrass, recent breeding efforts have been focused on strategies to more efficiently exploit heterosis by hybrid breeding. Cytoplasmic male sterility (CMS) is a widely applied mechanism to control pollination for commercial hybrid seed production and although CMS systems have been identified in perennial ryegrass, they are yet to be fully characterized. Here, we present a bioinformatics pipeline for efficient identification of candidate restorer of fertility (Rf) genes for CMS. From a high-quality draft of the perennial ryegrass genome, 373 pentatricopeptide repeat (PPR) genes were identified and classified, further identifying 25 restorer of fertility-like PPR (RFL) genes through a combination of DNA sequence clustering and comparison to known Rf genes. This extensive gene family was targeted as the majority of Rf genes in higher plants are RFL genes. These RFL genes were further investigated by phylogenetic analyses, identifying three groups of perennial ryegrass RFLs. These three groups likely represent genomic regions of active RFL generation and identify the probable location of perennial ryegrass PPR-Rf genes. This pipeline allows for the identification of candidate PPR-Rf genes from genomic sequence data and can be used in any plant species. Functional markers for PPR-Rf genes will facilitate map-based cloning of Rf genes and enable the use of CMS as an efficient tool to control pollination for hybrid crop production. PMID:26951780

Direct isolation of differentially expressed genes from a specific chromosome region of common wheat: application of the amplified fragment length polymorphism-based mRNA fingerprinting (AMF) method in combination with a deletion line of wheat.

PubMed

Kojima, T; Habu, Y; Iida, S; Ogihara, Y

2000-05-01

The amplified restriction fragment length polymorphism (AFLP)-based mRNA fingerprinting (AMF) method makes it possible systematically and conveniently to identify differentially expressed cDNAs with high reproducibility. We have applied the AMF method to the cloning of the Q gene of common wheat, which is located on the long arm of chromosome 5A and pleiotropically controls the spike morphology and the threshing character of seeds. Using the AMF method, we compared the fingerprints of mRNA samples extracted from the young spikes of Triticum aestivum cv. Chinese Spring (CS) carrying the Q gene to those of a chromosome deletion line of CS, namely, q5, which lacks 15% of 5AL including the Q gene. Approximately 12,200 fragments were produced after PCR with 256 primer combinations. Of these, 92 fragments were differentially expressed between CS and q5. Northern and Southern analyses showed that 16 fragments gave specific or relatively stronger transcript signals in CS, and these clones were present in single copy or in low copy numbers in the wheat genome. Four clones were genetically mapped to the region deleted in q5. Subsequently, one clone, pTaQ22, was mapped at the same locus as the Q gene, indicating that pTaQ22 corresponds to the Q gene or is tightly linked to it. DNA sequence data showed that pTaQ22 had no homology to any known genes, thus suggesting a novel function for this gene in flower morphogenesis. This AMF method might provide a straightforward method for isolating genes in the hexaploid background of common wheat.

A physical map, including a BAC/PAC clone contig, of the Williams-Beuren syndrome--deletion region at 7q11.23.

PubMed

Peoples, R; Franke, Y; Wang, Y K; Pérez-Jurado, L; Paperna, T; Cisco, M; Francke, U

2000-01-01

Williams-Beuren syndrome (WBS) is a developmental disorder caused by haploinsufficiency for genes in a 2-cM region of chromosome band 7q11.23. With the exception of vascular stenoses due to deletion of the elastin gene, the various features of WBS have not yet been attributed to specific genes. Although >/=16 genes have been identified within the WBS deletion, completion of a physical map of the region has been difficult because of the large duplicated regions flanking the deletion. We present a physical map of the WBS deletion and flanking regions, based on assembly of a bacterial artificial chromosome/P1-derived artificial chromosome contig, analysis of high-throughput genome-sequence data, and long-range restriction mapping of genomic and cloned DNA by pulsed-field gel electrophoresis. Our map encompasses 3 Mb, including 1.6 Mb within the deletion. Two large duplicons, flanking the deletion, of >/=320 kb contain unique sequence elements from the internal border regions of the deletion, such as sequences from GTF2I (telomeric) and FKBP6 (centromeric). A third copy of this duplicon exists in inverted orientation distal to the telomeric flanking one. These duplicons show stronger sequence conservation with regard to each other than to the presumptive ancestral loci within the common deletion region. Sequence elements originating from beyond 7q11.23 are also present in these duplicons. Although the duplicons are not present in mice, the order of the single-copy genes in the conserved syntenic region of mouse chromosome 5 is inverted relative to the human map. A model is presented for a mechanism of WBS-deletion formation, based on the orientation of duplicons' components relative to each other and to the ancestral elements within the deletion region.

A high-resolution genetic, physical, and comparative gene map of the doublefoot (Dbf) region of mouse chromosome 1 and the region of conserved synteny on human chromosome 2q35.

PubMed

Hayes, C; Rump, A; Cadman, M R; Harrison, M; Evans, E P; Lyon, M F; Morriss-Kay, G M; Rosenthal, A; Brown, S D

2001-12-01

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0.4-cM (+/-0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.

Map-based cloning and characterization of a brown planthopper resistance gene BPH26 from Oryza sativa L. ssp. indica cultivar ADR52.

PubMed

Tamura, Yasumori; Hattori, Makoto; Yoshioka, Hirofumi; Yoshioka, Miki; Takahashi, Akira; Wu, Jianzhong; Sentoku, Naoki; Yasui, Hideshi

2014-07-29

The brown planthopper (BPH) is the most serious insect pest of rice in Asia. The indica rice cultivar ADR52 carries two BPH resistance genes, BPH26 (brown planthopper resistance 26) and BPH25. Map-based cloning of BPH26 revealed that BPH26 encodes a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein. BPH26 mediated sucking inhibition in the phloem sieve element. BPH26 was identical to BPH2 on the basis of DNA sequence analysis and feeding ability of the BPH2-virulent biotype of BPH. BPH2 was widely incorporated in elite rice cultivars and was well-cultivated in many Asian countries as a favorable gene resource in rice breeding against BPH. However, BPH2 was rendered ineffective by a virulent biotype of BPH in rice fields in Asia. In this study, we suggest that BPH2 can be reused by combining with other BPH resistance genes, such as BPH25, to ensure durable resistance to BPH.

Map-based Cloning and Characterization of a Brown Planthopper Resistance Gene BPH26 from Oryza sativa L. ssp. indica Cultivar ADR52

PubMed Central

Tamura, Yasumori; Hattori, Makoto; Yoshioka, Hirofumi; Yoshioka, Miki; Takahashi, Akira; Wu, Jianzhong; Sentoku, Naoki; Yasui, Hideshi

2014-01-01

The brown planthopper (BPH) is the most serious insect pest of rice in Asia. The indica rice cultivar ADR52 carries two BPH resistance genes, BPH26 (BROWN PLANTHOPPER RESISTANCE 26) and BPH25. Map-based cloning of BPH26 revealed that BPH26 encodes a coiled-coil-nucleotide-binding-site–leucine-rich repeat (CC–NBS–LRR) protein. BPH26 mediated sucking inhibition in the phloem sieve element. BPH26 was identical to BPH2 on the basis of DNA sequence analysis and feeding ability of the BPH2-virulent biotype of BPH. BPH2 was widely incorporated in elite rice cultivars and was well-cultivated in many Asian countries as a favorable gene resource in rice breeding against BPH. However, BPH2 was rendered ineffective by a virulent biotype of BPH in rice fields in Asia. In this study, we suggest that BPH2 can be reused by combining with other BPH resistance genes, such as BPH25, to ensure durable resistance to BPH. PMID:25076167

Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives.

PubMed

Wang, Chao; Shi, Xue; Liu, Lin; Li, Haiyan; Ammiraju, Jetty S S; Kudrna, David A; Xiong, Wentao; Wang, Hao; Dai, Zhaozhao; Zheng, Yonglian; Lai, Jinsheng; Jin, Weiwei; Messing, Joachim; Bennetzen, Jeffrey L; Wing, Rod A; Luo, Meizhong

2013-11-01

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.

ADME-Space: a new tool for medicinal chemists to explore ADME properties.

PubMed

Bocci, Giovanni; Carosati, Emanuele; Vayer, Philippe; Arrault, Alban; Lozano, Sylvain; Cruciani, Gabriele

2017-07-25

We introduce a new chemical space for drugs and drug-like molecules, exclusively based on their in silico ADME behaviour. This ADME-Space is based on self-organizing map (SOM) applied to 26,000 molecules. Twenty accurate QSPR models, describing important ADME properties, were developed and, successively, used as new molecular descriptors not related to molecular structure. Applications include permeability, active transport, metabolism and bioavailability studies, but the method can be even used to discuss drug-drug interactions (DDIs) or it can be extended to additional ADME properties. Thus, the ADME-Space opens a new framework for the multi-parametric data analysis in drug discovery where all ADME behaviours of molecules are condensed in one map: it allows medicinal chemists to simultaneously monitor several ADME properties, to rapidly select optimal ADME profiles, retrieve warning on potential ADME problems and DDIs or select proper in vitro experiments.

Identification of expressed sequences in the coffee genome potentially associated with somatic embryogenesis.

PubMed

Silva, A T; Paiva, L V; Andrade, A C; Barduche, D

2013-05-21

Brazil possesses the most modern and productive coffee growing farms in the world, but technological development is desired to cope with the increasing world demand. One way to increase Brazilian coffee growing productivity is wide scale production of clones with superior genotypes, which can be obtained with in vitro propagation technique, or from tissue culture. These procedures can generate thousands of clones. However, the methodologies for in vitro cultivation are genotype-dependent, which leads to an almost empirical development of specific protocols for each species. Therefore, molecular markers linked to the biochemical events of somatic embryogenesis would greatly facilitate the development of such protocols. In this context, sequences potentially involved in embryogenesis processes in the coffee plant were identified in silico from libraries generated by the Brazilian Coffee Genome Project. Through these in silico analyses, we identified 15 EST-contigs related to the embryogenesis process. Among these, 5 EST-contigs (3605, 9850, 13686, 17240, and 17265) could readily be associated with plant embryogenesis. Sequence analysis of EST-contig 3605, 9850, and 17265 revealed similarity to a polygalacturonase, to a cysteine-proteinase, and to an allergenine, respectively. Results also show that EST-contig 17265 sequences presented similarity to an expansin. Finally, analysis of EST-contig 17240 revealed similarity to a protein of unknown function, but it grouped in the similarity dendrogram with the WUSCHEL transcription factor. The data suggest that these EST-contigs are related to the embryogenic process and have potential as molecular markers to increase methodological efficiency in obtaining coffee plant embryogenic materials.

Design and synthesis of multiple antigenic peptides and their application for dengue diagnosis.

PubMed

Rai, Reeta; Dubey, Sameer; Santosh, K V; Biswas, Ashutosh; Mehrotra, Vinit; Rao, D N

2017-09-01

Major difficulty in development of dengue diagnostics is availability of suitable antigens. To overcome this, we made an attempt to develop a peptide based diagnosis which offers significant advantage over other methods. With the help of in silico methods, two epitopes were selected from envelope protein and three from NS1 protein of dengue virus. These were synthesized in combination as three multiple antigenic peptides (MAPs). We have tested 157 dengue positive sera confirmed for NS1 antigen. MAP1 showed 96.81% sera positive for IgM and 68.15% positive for IgG. MAP2 detected 94.90% IgM and 59.23% IgG positive sera. MAP3 also detected 96.17% IgM and 59.87% IgG positive sera. To the best of our knowledge this is the first study describing the use of synthetic multiple antigenic peptides for the diagnosis of dengue infection. This study describes MAPs as a promising tool for the use in serodiagnosis of dengue. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Characterization of a microdissection library from human chromosome region 3p14

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

Bardenheuer, W.; Szymanski, S.; Lux, A.

1994-01-15

Structural alterations in human chromosome region 3p14-p23 resulting in the inactivation of one or more tumor suppressor genes are thought to play a pathogenic role in small cell lung cancer, renal cell carcinoma, and other human neoplasms. To identify putative tumor suppressor genes, 428 recombinant clones from a microdissection library specific for human chromosome region 3p14 were isolated and characterized. Ninety-six of these (22.5%) were human single-copy DNA sequences, 57 of which were unique sequence clones. Forty-four of these were mapped to the microdissected region using a cell hybrid mapping panel. Within this mapping panel, four probes detected two newmore » chromosome breakpoints that were previously indistinguishable from the translocation breakpoint t(3;8) in 3p14.2 in hereditary renal cell carcinoma. One probe maps to the homozygously deleted region of the small cell lung cancer cell line U2020. In addition, microdissection clones have been shown to be suitable for isolation of yeast artificial chromosomes. 52 refs., 3 figs., 2 tabs.« less

Large-Scale SNP Discovery and Genotyping for Constructing a High-Density Genetic Map of Tea Plant Using Specific-Locus Amplified Fragment Sequencing (SLAF-seq)

PubMed Central

Ma, Chun-Lei; Jin, Ji-Qiang; Li, Chun-Fang; Wang, Rong-Kai; Zheng, Hong-Kun; Yao, Ming-Zhe; Chen, Liang

2015-01-01

Genetic maps are important tools in plant genomics and breeding. The present study reports the large-scale discovery of single nucleotide polymorphisms (SNPs) for genetic map construction in tea plant. We developed a total of 6,042 valid SNP markers using specific-locus amplified fragment sequencing (SLAF-seq), and subsequently mapped them into the previous framework map. The final map contained 6,448 molecular markers, distributing on fifteen linkage groups corresponding to the number of tea plant chromosomes. The total map length was 3,965 cM, with an average inter-locus distance of 1.0 cM. This map is the first SNP-based reference map of tea plant, as well as the most saturated one developed to date. The SNP markers and map resources generated in this study provide a wealth of genetic information that can serve as a foundation for downstream genetic analyses, such as the fine mapping of quantitative trait loci (QTL), map-based cloning, marker-assisted selection, and anchoring of scaffolds to facilitate the process of whole genome sequencing projects for tea plant. PMID:26035838

Miniprimer PCR, a New Lens for Viewing the Microbial World▿ †

PubMed Central

Isenbarger, Thomas A.; Finney, Michael; Ríos-Velázquez, Carlos; Handelsman, Jo; Ruvkun, Gary

2008-01-01

Molecular methods based on the 16S rRNA gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. Here we show that a new PCR method using an engineered polymerase and 10-nucleotide “miniprimers” expands the scope of detectable sequences beyond those detected by standard methods using longer primers and Taq polymerase. After testing the method in silico to identify divergent ribosomal genes in previously cloned environmental sequences, we applied the method to soil and microbial mat samples, which revealed novel 16S rRNA gene sequences that would not have been detected with standard primers. Deeply divergent sequences were discovered with high frequency and included representatives that define two new division-level taxa, designated CR1 and CR2, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity. PMID:18083877

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage

PubMed Central

Kaucka, Marketa; Zikmund, Tomas; Tesarova, Marketa; Gyllborg, Daniel; Hellander, Andreas; Jaros, Josef; Kaiser, Jozef; Petersen, Julian; Szarowska, Bara; Newton, Phillip T; Dyachuk, Vyacheslav; Li, Lei; Qian, Hong; Johansson, Anne-Sofie; Mishina, Yuji; Currie, Joshua D; Tanaka, Elly M; Erickson, Alek; Dudley, Andrew; Brismar, Hjalmar; Southam, Paul; Coen, Enrico; Chen, Min; Weinstein, Lee S; Hampl, Ales; Arenas, Ernest; Chagin, Andrei S; Fried, Kaj; Adameyko, Igor

2017-01-01

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale. DOI: http://dx.doi.org/10.7554/eLife.25902.001 PMID:28414273

Genetic Mapping

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Mapping, fine mapping, and molecular dissection of quantitative trait Loci in domestic animals.

PubMed

Georges, Michel

2007-01-01

Artificial selection has created myriad breeds of domestic animals, each characterized by unique phenotypes pertaining to behavior, morphology, physiology, and disease. Most domestic animal populations share features with isolated founder populations, making them well suited for positional cloning. Genome sequences are now available for most domestic species, and with them a panoply of tools including high-density single-nucleotide polymorphism panels. As a result, domestic animal populations are becoming invaluable resources for studying the molecular architecture of complex traits and of adaptation. Here we review recent progress and issues in the positional identification of genes underlying complex traits in domestic animals. As many phenotypes studied in animals are quantitative, we focus on mapping, fine mapping, and cloning of quantitative trait loci.

Interval mapping of high growth (hg), a major locus that increases weight gain in mice

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

Horvat, S.; Medrano, J.F.

1995-04-01

The high growth locus (hg) causes a major increase in weight gain and body size in mice. As a first step to map-based cloning of hg, we developed a genetic map of the hg-containing region using interval mapping of 403 F{sub 2} from a C57BL/6J-hghg x CAST/EiJ cross. The maximum likelihood position of hg was at the chromosome 10 marker D10Mit41 (LOD = 24.8) in the F{sub 2} females and 1.5 cM distal to D10Mit41 (LOD = 9.56) in the F{sub 2} males with corresponding LOD 2 support intervals of 3.7 and 5.4 cM, respectively. The peak LOD scores weremore » significantly higher than the estimated empirical threshold LOD values. The localization of hg by interval mapping was supported by a test cross of F{sub 2} mice recombinant between the LOD 2 support interval and the flanking marker. The interval mapping and test-cross indicate that hg is not allelic with candidate genes Igf1 or decorin (Dcn), a gene that was mapped close to hg in this study. The hg inheritance was recessive in females, although we could not reject recessive or additive inheritance in males. Possible causes for sex differences in peak LOD scores and for the distortion of transmission ratios observed in F{sub 2} males are discussed. The genetic map of the hg region will facilitate further fine mapping and cloning of hg, and allow searches for a homologous quantitative trait locus affecting growth in humans and domestic animals. 48 refs., 3 figs., 3 tabs.« less

In silico fragment-based drug design.

PubMed

Konteatis, Zenon D

2010-11-01

In silico fragment-based drug design (FBDD) is a relatively new approach inspired by the success of the biophysical fragment-based drug discovery field. Here, we review the progress made by this approach in the last decade and showcase how it complements and expands the capabilities of biophysical FBDD and structure-based drug design to generate diverse, efficient drug candidates. Advancements in several areas of research that have enabled the development of in silico FBDD and some applications in drug discovery projects are reviewed. The reader is introduced to various computational methods that are used for in silico FBDD, the fragment library composition for this technique, special applications used to identify binding sites on the surface of proteins and how to assess the druggability of these sites. In addition, the reader will gain insight into the proper application of this approach from examples of successful programs. In silico FBDD captures a much larger chemical space than high-throughput screening and biophysical FBDD increasing the probability of developing more diverse, patentable and efficient molecules that can become oral drugs. The application of in silico FBDD holds great promise for historically challenging targets such as protein-protein interactions. Future advances in force fields, scoring functions and automated methods for determining synthetic accessibility will all aid in delivering more successes with in silico FBDD.

Identification of downy mildew resistance gene candidates by positional cloning in maize (Zea mays subsp. mays; Poaceae)1

PubMed Central

Kim, Jae Yoon; Moon, Jun-Cheol; Kim, Hyo Chul; Shin, Seungho; Song, Kitae; Kim, Kyung-Hee; Lee, Byung-Moo

2017-01-01

Premise of the study: Positional cloning in combination with phenotyping is a general approach to identify disease-resistance gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combined strategy to improve the identification of disease-resistance gene candidates. Methods and Results: Downy mildew (DM)–resistant maize was selected from five cultivars using a spreader row technique. Positional cloning and bioinformatics tools were used to identify the DM-resistance quantitative trait locus marker (bnlg1702) and 47 protein-coding gene annotations. Eventually, five DM-resistance gene candidates, including bZIP34, Bak1, and Ppr, were identified by quantitative reverse-transcription PCR (RT-PCR) without fine mapping of the bnlg1702 locus. Conclusions: The combined protocol with the spreader row technique, quantitative trait locus positional cloning, and quantitative RT-PCR was effective for identifying DM-resistance candidate genes. This cloning approach may be applied to other whole-genome-sequenced crops or resistance to other diseases. PMID:28224059

Local activation time sampling density for atrial tachycardia contact mapping: how much is enough?

PubMed

Williams, Steven E; Harrison, James L; Chubb, Henry; Whitaker, John; Kiedrowicz, Radek; Rinaldi, Christopher A; Cooklin, Michael; Wright, Matthew; Niederer, Steven; O'Neill, Mark D

2018-02-01

Local activation time (LAT) mapping forms the cornerstone of atrial tachycardia diagnosis. Although anatomic and positional accuracy of electroanatomic mapping (EAM) systems have been validated, the effect of electrode sampling density on LAT map reconstruction is not known. Here, we study the effect of chamber geometry and activation complexity on optimal LAT sampling density using a combined in silico and in vivo approach. In vivo 21 atrial tachycardia maps were studied in three groups: (1) focal activation, (2) macro-re-entry, and (3) localized re-entry. In silico activation was simulated on a 4×4cm atrial monolayer, sampled randomly at 0.25-10 points/cm2 and used to re-interpolate LAT maps. Activation patterns were studied in the geometrically simple porcine right atrium (RA) and complex human left atrium (LA). Activation complexity was introduced into the porcine RA by incomplete inter-caval linear ablation. In all cases, optimal sampling density was defined as the highest density resulting in minimal further error reduction in the re-interpolated maps. Optimal sampling densities for LA tachycardias were 0.67 ± 0.17 points/cm2 (focal activation), 1.05 ± 0.32 points/cm2 (macro-re-entry) and 1.23 ± 0.26 points/cm2 (localized re-entry), P = 0.0031. Increasing activation complexity was associated with increased optimal sampling density both in silico (focal activation 1.09 ± 0.14 points/cm2; re-entry 1.44 ± 0.49 points/cm2; spiral-wave 1.50 ± 0.34 points/cm2, P < 0.0001) and in vivo (porcine RA pre-ablation 0.45 ± 0.13 vs. post-ablation 0.78 ± 0.17 points/cm2, P = 0.0008). Increasing chamber geometry was also associated with increased optimal sampling density (0.61 ± 0.22 points/cm2 vs. 1.0 ± 0.34 points/cm2, P = 0.0015). Optimal sampling densities can be identified to maximize diagnostic yield of LAT maps. Greater sampling density is required to correctly reveal complex activation and represent activation across complex geometries. Overall, the optimal sampling density for LAT map interpolation defined in this study was ∼1.0-1.5 points/cm2. Published on behalf of the European Society of Cardiology

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

PubMed Central

2009-01-01

Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes. PMID:19656416

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome.

PubMed

Hamberger, Björn; Hall, Dawn; Yuen, Mack; Oddy, Claire; Hamberger, Britta; Keeling, Christopher I; Ritland, Carol; Ritland, Kermit; Bohlmann, Jörg

2009-08-06

Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes.

Construction and characterization of a bacterial artificial chromosome library for hexaploid wheat line 92R137

USDA-ARS?s Scientific Manuscript database

For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sub libraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was compos...

Validation and Implementation of Marker-Assisted Selection (MAS) for PVY Resistance (Ryadg gene) in a Tetraploid Potato Breeding Program

USDA-ARS?s Scientific Manuscript database

The gene Ryadg from S. tuberosum ssp. andigena provides extreme resistance to PVY. This gene has been mapped to chromosome XI and linked PCR-based DNA markers have been identified. Advanced tetraploid russeted potato clones developed by the U.S. Pacific Northwest Potato Breeding Program with Ryadg P...

An atlas of B-cell clonal distribution in the human body.

PubMed

Meng, Wenzhao; Zhang, Bochao; Schwartz, Gregory W; Rosenfeld, Aaron M; Ren, Daqiu; Thome, Joseph J C; Carpenter, Dustin J; Matsuoka, Nobuhide; Lerner, Harvey; Friedman, Amy L; Granot, Tomer; Farber, Donna L; Shlomchik, Mark J; Hershberg, Uri; Luning Prak, Eline T

2017-09-01

B-cell responses result in clonal expansion, and can occur in a variety of tissues. To define how B-cell clones are distributed in the body, we sequenced 933,427 B-cell clonal lineages and mapped them to eight different anatomic compartments in six human organ donors. We show that large B-cell clones partition into two broad networks-one spans the blood, bone marrow, spleen and lung, while the other is restricted to tissues within the gastrointestinal (GI) tract (jejunum, ileum and colon). Notably, GI tract clones display extensive sharing of sequence variants among different portions of the tract and have higher frequencies of somatic hypermutation, suggesting extensive and serial rounds of clonal expansion and selection. Our findings provide an anatomic atlas of B-cell clonal lineages, their properties and tissue connections. This resource serves as a foundation for studies of tissue-based immunity, including vaccine responses, infections, autoimmunity and cancer.

The chimeric mapping problem: algorithmic strategies and performance evaluation on synthetic genomic data.

PubMed

Greenberg, D; Istrail, S

1994-09-01

The Human Genome Project requires better software for the creation of physical maps of chromosomes. Current mapping techniques involve breaking large segments of DNA into smaller, more-manageable pieces, gathering information on all the small pieces, and then constructing a map of the original large piece from the information about the small pieces. Unfortunately, in the process of breaking up the DNA some information is lost and noise of various types is introduced; in particular, the order of the pieces is not preserved. Thus, the map maker must solve a combinatorial problem in order to reconstruct the map. Good software is indispensable for quick, accurate reconstruction. The reconstruction is complicated by various experimental errors. A major source of difficulty--which seems to be inherent to the recombination technology--is the presence of chimeric DNA clones. It is fairly common for two disjoint DNA pieces to form a chimera, i.e., a fusion of two pieces which appears as a single piece. Attempts to order chimera will fail unless they are algorithmically divided into their constituent pieces. Despite consensus within the genomic mapping community of the critical importance of correcting chimerism, algorithms for solving the chimeric clone problem have received only passing attention in the literature. Based on a model proposed by Lander (1992a, b) this paper presents the first algorithms for analyzing chimerism. We construct physical maps in the presence of chimerism by creating optimization functions which have minimizations which correlate with map quality. Despite the fact that these optimization functions are invariably NP-complete our algorithms are guaranteed to produce solutions which are close to the optimum. The practical import of using these algorithms depends on the strength of the correlation of the function to the map quality as well as on the accuracy of the approximations. We employ two fundamentally different optimization functions as a means of avoiding biases likely to decorrelate the solutions from the desired map. Experiments on simulated data show that both our algorithm which minimizes the number of chimeric fragments in a solution and our algorithm which minimizes the maximum number of fragments per clone in a solution do, in fact, correlate to high quality solutions. Furthermore, tests on simulated data using parameters set to mimic real experiments show that that the algorithms have the potential to find high quality solutions with real data. We plan to test our software against real data from the Whitehead Institute and from Los Alamos Genomic Research Center in the near future.

A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa.

PubMed Central

Choi, Hong-Kyu; Kim, Dongjin; Uhm, Taesik; Limpens, Eric; Lim, Hyunju; Mun, Jeong-Hwan; Kalo, Peter; Penmetsa, R Varma; Seres, Andrea; Kulikova, Olga; Roe, Bruce A; Bisseling, Ton; Kiss, Gyorgy B; Cook, Douglas R

2004-01-01

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F(2) population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map. PMID:15082563

Development and characterization of BAC-end sequence derived SSRs, and their incorporation into a new higher density genetic map for cultivated peanut (Arachis hypogaea L.)

PubMed Central

2012-01-01

Background Cultivated peanut (Arachis hypogaea L.) is an important crop worldwide, valued for its edible oil and digestible protein. It has a very narrow genetic base that may well derive from a relatively recent single polyploidization event. Accordingly molecular markers have low levels of polymorphism and the number of polymorphic molecular markers available for cultivated peanut is still limiting. Results Here, we report a large set of BAC-end sequences (BES), use them for developing SSR (BES-SSR) markers, and apply them in genetic linkage mapping. The majority of BESs had no detectable homology to known genes (49.5%) followed by sequences with similarity to known genes (44.3%), and miscellaneous sequences (6.2%) such as transposable element, retroelement, and organelle sequences. A total of 1,424 SSRs were identified from 36,435 BESs. Among these identified SSRs, dinucleotide (47.4%) and trinucleotide (37.1%) SSRs were predominant. The new set of 1,152 SSRs as well as about 4,000 published or unpublished SSRs were screened against two parents of a mapping population, generating 385 polymorphic loci. A genetic linkage map was constructed, consisting of 318 loci onto 21 linkage groups and covering a total of 1,674.4 cM, with an average distance of 5.3 cM between adjacent loci. Two markers related to resistance gene homologs (RGH) were mapped to two different groups, thus anchoring 1 RGH-BAC contig and 1 singleton. Conclusions The SSRs mined from BESs will be of use in further molecular analysis of the peanut genome, providing a novel set of markers, genetically anchoring BAC clones, and incorporating gene sequences into a linkage map. This will aid in the identification of markers linked to genes of interest and map-based cloning. PMID:22260238

Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon.

PubMed

Song, Beng-Kah; Nadarajah, Kalaivani; Romanov, Michael N; Ratnam, Wickneswari

2005-01-01

The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.

Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library.

PubMed

Buzatti, Andréia; Fernandez, Arnielis Diaz; Arenal, Amilcar; Pereira, Erlán; Monteiro, Alda Lucia Gomes; Molento, Marcelo Beltrão

2018-05-24

The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.

Molecular mapping of chromosomes 17 and X

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

Barker, D.F.

1991-01-15

Progress toward the construction of high density genetic maps of chromosomes 17 and X has been made by isolating and characterizing a relatively large set of polymorphic probes for each chromosome and using these probes to construct genetic maps. We have mapped the same polymorphic probes against a series of chromosome breakpoints on X and 17. The probes could be assigned to over 30 physical intervals on the X chromosome and 7 intervals on 17. In many cases, this process resulted in improved characterization of the relative locations of the breakpoints with respect to each other and the definition ofmore » new physical intervals. The strategy for isolation of the polymorphic clones utilized chromosome specific libraries of 1--15 kb segments from each of the two chromosomes. From these libraries, clones were screened for those detecting restriction fragment length polymorphisms. The markers were further characterized, the chromosomal assignments confirmed and in most cases segments of the original probes were subcloned into plasmids to produce probes with improved signal to noise ratios for use in the genetic marker studies. The linkage studies utilize the CEPH reference families and other well-characterized families in our collection which have been used for genetic disease linkage work. Preliminary maps and maps of portions of specific regions of 17 and X are provided. We have nearly completed a map of the 1 megabase Mycoplasma arthritidis genome by applying these techniques to a lambda phage library of its genome. We have found bit mapping to be an efficient means to organize a contiguous set of overlapping clones from a larger genome.« less

Molecular mapping of chromosomes 17 and X. Progress report

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

Barker, D.F.

1991-01-15

Progress toward the construction of high density genetic maps of chromosomes 17 and X has been made by isolating and characterizing a relatively large set of polymorphic probes for each chromosome and using these probes to construct genetic maps. We have mapped the same polymorphic probes against a series of chromosome breakpoints on X and 17. The probes could be assigned to over 30 physical intervals on the X chromosome and 7 intervals on 17. In many cases, this process resulted in improved characterization of the relative locations of the breakpoints with respect to each other and the definition ofmore » new physical intervals. The strategy for isolation of the polymorphic clones utilized chromosome specific libraries of 1--15 kb segments from each of the two chromosomes. From these libraries, clones were screened for those detecting restriction fragment length polymorphisms. The markers were further characterized, the chromosomal assignments confirmed and in most cases segments of the original probes were subcloned into plasmids to produce probes with improved signal to noise ratios for use in the genetic marker studies. The linkage studies utilize the CEPH reference families and other well-characterized families in our collection which have been used for genetic disease linkage work. Preliminary maps and maps of portions of specific regions of 17 and X are provided. We have nearly completed a map of the 1 megabase Mycoplasma arthritidis genome by applying these techniques to a lambda phage library of its genome. We have found bit mapping to be an efficient means to organize a contiguous set of overlapping@ clones from a larger genome.« less

Identification of a mouse synaptic glycoprotein gene in cultured neurons.

PubMed

Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

2005-10-01

Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

In silico and in vivo studies of truncated forms of flagellin (FliC) of enteroaggregative Escherichia coli fused to FimH from uropathogenic Escherichia coli as a vaccine candidate against urinary tract infections.

PubMed

Savar, Nastaran Sadat; Jahanian-Najafabadi, Ali; Mahdavi, Mehdi; Shokrgozar, Mohammad Ali; Jafari, Anis; Bouzari, Saeid

2014-04-10

The new generation of vaccines against infectious diseases is based on recombinant fusion proteins. Flagellin (FliC) of enteroaggregative Escherichia coli (EAEC) could be considered as a potent adjuvant in designing new vaccines. However, because of its large size, incorporation of this protein with a vaccine antigen might negatively influence recognition of the vaccine epitopes by the immune system. Designing the truncated forms of FliC, capable of inducing innate immune response, enhances the immune responses to the target antigen. We have previously shown that two truncated forms of FliC are able to induce Interleukine-8 production in HT-29 epithelial cell line. In this study we designed recombinant vaccine against urinary tract infections (UTIs) using truncated forms of FliC and type 1 fimbrial FimH adhesin from uropathogenic Escherichia coli (UPEC) and studied their in silico interactions with Toll-like receptor 5 (TLR-5) via docking protocols. The best fusion protein was subjected to cloning and expression. The ability of the recombinant vaccine and the truncated forms in inducing immune responses was investigated. Our results showed that truncated forms are capable of inducing Th1 (forms A and B) and Th2 (form A) responses and fusion vaccine induced strong cellular and humoral immune responses. Copyright © 2014 Elsevier B.V. All rights reserved.

The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection.

PubMed

Sandra, Koen; Steenbeke, Mieke; Vandenheede, Isabel; Vanhoenacker, Gerd; Sandra, Pat

2017-11-10

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development. Copyright © 2017 Elsevier B.V. All rights reserved.

SNP discovery by high-throughput sequencing in soybean

PubMed Central

2010-01-01

Background With the advance of new massively parallel genotyping technologies, quantitative trait loci (QTL) fine mapping and map-based cloning become more achievable in identifying genes for important and complex traits. Development of high-density genetic markers in the QTL regions of specific mapping populations is essential for fine-mapping and map-based cloning of economically important genes. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation existing between any diverse genotypes that are usually used for QTL mapping studies. The massively parallel sequencing technologies (Roche GS/454, Illumina GA/Solexa, and ABI/SOLiD), have been widely applied to identify genome-wide sequence variations. However, it is still remains unclear whether sequence data at a low sequencing depth are enough to detect the variations existing in any QTL regions of interest in a crop genome, and how to prepare sequencing samples for a complex genome such as soybean. Therefore, with the aims of identifying SNP markers in a cost effective way for fine-mapping several QTL regions, and testing the validation rate of the putative SNPs predicted with Solexa short sequence reads at a low sequencing depth, we evaluated a pooled DNA fragment reduced representation library and SNP detection methods applied to short read sequences generated by Solexa high-throughput sequencing technology. Results A total of 39,022 putative SNPs were identified by the Illumina/Solexa sequencing system using a reduced representation DNA library of two parental lines of a mapping population. The validation rates of these putative SNPs predicted with low and high stringency were 72% and 85%, respectively. One hundred sixty four SNP markers resulted from the validation of putative SNPs and have been selectively chosen to target a known QTL, thereby increasing the marker density of the targeted region to one marker per 42 K bp. Conclusions We have demonstrated how to quickly identify large numbers of SNPs for fine mapping of QTL regions by applying massively parallel sequencing combined with genome complexity reduction techniques. This SNP discovery approach is more efficient for targeting multiple QTL regions in a same genetic population, which can be applied to other crops. PMID:20701770

A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid.

PubMed

Indarte, Martín; Lazza, Cristian M; Assis, Diego; Caffini, Néstor O; Juliano, María A; Avilés, Francesc X; Daura, Xavier; López, Laura M I; Trejo, Sebastián A

2017-02-01

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M -1  cm -1 . MpBBI inhibits strongly trypsin with K i in the 10 -10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

USDA-ARS?s Scientific Manuscript database

Background: BAC-based physical maps provide for sequencing across an entire genome or selected sub-genome regions of biological interest. Using the minimum tiling path as a guide, it is possible to select specific BAC clones from prioritized genome sections such as a genetically defined QTL interv...

A TRIM insertion in the promoter of Ms2 causes male sterility in wheat

USDA-ARS?s Scientific Manuscript database

The male sterile ms2 mutant has been known for 40 years and has become extremely important in the commercial production of wheat. However, the gene responsible for this phenotype has remained unknown. We here report the map-based-cloning of the Ms2 gene. The Ms2 locus is remarkable in several ways ...

Construction of a High-Density Genetic Map from RNA-Seq Data for an Arabidopsis Bay-0 × Shahdara RIL Population

PubMed Central

Serin, Elise A. R.; Snoek, L. B.; Nijveen, Harm; Willems, Leo A. J.; Jiménez-Gómez, Jose M.; Hilhorst, Henk W. M.; Ligterink, Wilco

2017-01-01

High-density genetic maps are essential for high resolution mapping of quantitative traits. Here, we present a new genetic map for an Arabidopsis Bayreuth × Shahdara recombinant inbred line (RIL) population, built on RNA-seq data. RNA-seq analysis on 160 RILs of this population identified 30,049 single-nucleotide polymorphisms (SNPs) covering the whole genome. Based on a 100-kbp window SNP binning method, 1059 bin-markers were identified, physically anchored on the genome. The total length of the RNA-seq genetic map spans 471.70 centimorgans (cM) with an average marker distance of 0.45 cM and a maximum marker distance of 4.81 cM. This high resolution genotyping revealed new recombination breakpoints in the population. To highlight the advantages of such high-density map, we compared it to two publicly available genetic maps for the same population, comprising 69 PCR-based markers and 497 gene expression markers derived from microarray data, respectively. In this study, we show that SNP markers can effectively be derived from RNA-seq data. The new RNA-seq map closes many existing gaps in marker coverage, saturating the previously available genetic maps. Quantitative trait locus (QTL) analysis for published phenotypes using the available genetic maps showed increased QTL mapping resolution and reduced QTL confidence interval using the RNA-seq map. The new high-density map is a valuable resource that facilitates the identification of candidate genes and map-based cloning approaches. PMID:29259624

Construction of an SNP-based high-density linkage map for flax (Linum usitatissimum L.) using specific length amplified fragment sequencing (SLAF-seq) technology.

PubMed

Yi, Liuxi; Gao, Fengyun; Siqin, Bateer; Zhou, Yu; Li, Qiang; Zhao, Xiaoqing; Jia, Xiaoyun; Zhang, Hui

2017-01-01

Flax is an important crop for oil and fiber, however, no high-density genetic maps have been reported for this species. Specific length amplified fragment sequencing (SLAF-seq) is a high-resolution strategy for large scale de novo discovery and genotyping of single nucleotide polymorphisms. In this study, SLAF-seq was employed to develop SNP markers in an F2 population to construct a high-density genetic map for flax. In total, 196.29 million paired-end reads were obtained. The average sequencing depth was 25.08 in male parent, 32.17 in the female parent, and 9.64 in each F2 progeny. In total, 389,288 polymorphic SLAFs were detected, from which 260,380 polymorphic SNPs were developed. After filtering, 4,638 SNPs were found suitable for genetic map construction. The final genetic map included 4,145 SNP markers on 15 linkage groups and was 2,632.94 cM in length, with an average distance of 0.64 cM between adjacent markers. To our knowledge, this map is the densest SNP-based genetic map for flax. The SNP markers and genetic map reported in here will serve as a foundation for the fine mapping of quantitative trait loci (QTLs), map-based gene cloning and marker assisted selection (MAS) for flax.

Insights into the structure-function relationship of brown plant hopper resistance protein, Bph14 of rice plant: a computational structural biology approach.

PubMed

Gupta, Manoj Kumar; Vadde, Ramakrishna; Donde, Ravindra; Gouda, Gayatri; Kumar, Jitendra; Nayak, Subhashree; Jena, Mayabini; Behera, Lambodar

2018-05-02

Brown plant hopper (BPH) is one of the major destructive insect pests of rice, causing severe yield loss. Thirty-two BPH resistance genes have been identified in cultivated and wild species of rice Although, molecular mechanism of rice plant resistance against BPH studied through map-based cloning, due to non-existence of NMR/crystal structures of Bph14 protein, recognition of leucine-rich repeat (LRR) domain and its interaction with different ligands are poorly understood. Thus, in the present study, in silico approach was adopted to predict three-dimensional structure of LRR domain of Bph14 using comparative modelling approach followed by interaction study with jasmonic and salicylic acids. LRR domain along with LRR-jasmonic and salicylic acid complexes were subjected to dynamic simulation using GROMACS, individually, for energy minimisation and refinement of the structure. Final binding energy of jasmonic and salicylic acid with LRR domain was calculated using MM/PBSA. Free-energy landscape analysis revealed that overall stability of LRR domain of Bph14 is not much affected after forming complex with jasmonic and salicylic acid. MM/PBSA analysis revealed that binding affinities of LRR domain towards salicylic acid is higher as compared to jasmonic acid. Interaction study of LRR domain with salicylic acid and jasmonic acid reveals that THR987 of LRR form hydrogen bond with both complexes. Thus, THR987 plays active role in the Bph14 and phytochemical interaction for inducing resistance in rice plant against BPH. In future, Bph14 gene and phytochemicals could be used in BPH management and development of novel resistant varieties for increasing rice yield.

SNP Discovery and Linkage Map Construction in Cultivated Tomato

PubMed Central

Shirasawa, Kenta; Isobe, Sachiko; Hirakawa, Hideki; Asamizu, Erika; Fukuoka, Hiroyuki; Just, Daniel; Rothan, Christophe; Sasamoto, Shigemi; Fujishiro, Tsunakazu; Kishida, Yoshie; Kohara, Mitsuyo; Tsuruoka, Hisano; Wada, Tsuyuko; Nakamura, Yasukazu; Sato, Shusei; Tabata, Satoshi

2010-01-01

Few intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. Single nucleotide polymorphisms (SNPs), the most abundant source of genomic variation, are the most promising source of polymorphisms for the construction of linkage maps for closely related intraspecific lines. In this study, we developed SNP markers based on expressed sequence tags for the construction of intraspecific linkage maps in tomato. Out of the 5607 SNP positions detected through in silico analysis, 1536 were selected for high-throughput genotyping of two mapping populations derived from crosses between ‘Micro-Tom’ and either ‘Ailsa Craig’ or ‘M82’. A total of 1137 markers, including 793 out of the 1338 successfully genotyped SNPs, along with 344 simple sequence repeat and intronic polymorphism markers, were mapped onto two linkage maps, which covered 1467.8 and 1422.7 cM, respectively. The SNP markers developed were then screened against cultivated tomato lines in order to estimate the transferability of these SNPs to other breeding materials. The molecular markers and linkage maps represent a milestone in the genomics and genetics, and are the first step toward molecular breeding of cultivated tomato. Information on the DNA markers, linkage maps, and SNP genotypes for these tomato lines is available at http://www.kazusa.or.jp/tomato/. PMID:21044984

Ultra-high density intra-specific genetic linkage maps accelerate identification of functionally relevant molecular tags governing important agronomic traits in chickpea

PubMed Central

Kujur, Alice; Upadhyaya, Hari D.; Shree, Tanima; Bajaj, Deepak; Das, Shouvik; Saxena, Maneesha S.; Badoni, Saurabh; Kumar, Vinod; Tripathi, Shailesh; Gowda, C. L. L.; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K.; Parida, Swarup K.

2015-01-01

We discovered 26785 and 16573 high-quality SNPs differentiating two parental genotypes of a RIL mapping population using reference desi and kabuli genome-based GBS assay. Of these, 3625 and 2177 SNPs have been integrated into eight desi and kabuli chromosomes, respectively in order to construct ultra-high density (0.20–0.37 cM) intra-specific chickpea genetic linkage maps. One of these constructed high-resolution genetic map has potential to identify 33 major genomic regions harbouring 35 robust QTLs (PVE: 17.9–39.7%) associated with three agronomic traits, which were mapped within <1 cM mean marker intervals on desi chromosomes. The extended LD (linkage disequilibrium) decay (~15 cM) in chromosomes of genetic maps have encouraged us to use a rapid integrated approach (comparative QTL mapping, QTL-region specific haplotype/LD-based trait association analysis, expression profiling and gene haplotype-based association mapping) rather than a traditional QTL map-based cloning method to narrow-down one major seed weight (SW) robust QTL region. It delineated favourable natural allelic variants and superior haplotype-containing one seed-specific candidate embryo defective gene regulating SW in chickpea. The ultra-high-resolution genetic maps, QTLs/genes and alleles/haplotypes-related genomic information generated and integrated strategy for rapid QTL/gene identification developed have potential to expedite genomics-assisted breeding applications in crop plants, including chickpea for their genetic enhancement. PMID:25942004

Are we there yet for rice disease control

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes play an important role in fighting against plant pathogens. For the past two decades, significant efforts have been directed to map and clone R genes. Most of the cloned plant R genes encode proteins with leucine rich repeat and nucleotide binding sites (NLR), their cellul...

Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression.

PubMed Central

Haussmann, C; Rohdich, F; Lottspeich, F; Eberhardt, S; Scheuring, J; Mackamul, S; Bacher, A

1997-01-01

The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome. PMID:9006053

A cloned DNA segment from the telomeric region of human chromosome 4p is not detectably rearranged in Huntington disease patients

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

Pritchard, C.; Casher, D.; Myers, R.M.

1990-09-01

Genetic linkage studies have mapped the Huntington disease (HD) mutation to the distal region of the short arm of human chromosome 4. Analysis of recombination events in this region has produced contradictory locations for HD. One possible location is in the region distal to the D4S90 marker, which is located within 300 kilobases of the telomere. Other crossover events predict a more centromeric position for HD. Here the authors analyze the telomeric region of 4p in detail. Cloned DNA segments were derived from this region by utilizing a radiation-induced somatic cell hybrid as a source of DNA combined with preparativemore » pulsed-field gel electrophoresis to enrich for the telmoeric fraction. Additional DNA was obtained by using the cloned segments as multiple start points for cosmid walks. This strategy proved to be an effective method for cloning 250 kilobases of DNA in the region telomeric to D4S90. Hybridization analysis with the cloned DNA did not provide any evidence for the presence of rearrangements of 100 base pairs or greater in the DNA of individuals affected with HD. They also found no charge in the size or structure of the 4p telomere in these samples.« less

A Physical Map, Including a BAC/PAC Clone Contig, of the Williams-Beuren Syndrome–Deletion Region at 7q11.23

PubMed Central

Peoples, Risa; Franke, Yvonne; Wang, Yu-Ker; Pérez-Jurado, Luis; Paperna, Tamar; Cisco, Michael; Francke, Uta

2000-01-01

Summary Williams-Beuren syndrome (WBS) is a developmental disorder caused by haploinsufficiency for genes in a 2-cM region of chromosome band 7q11.23. With the exception of vascular stenoses due to deletion of the elastin gene, the various features of WBS have not yet been attributed to specific genes. Although ⩾16 genes have been identified within the WBS deletion, completion of a physical map of the region has been difficult because of the large duplicated regions flanking the deletion. We present a physical map of the WBS deletion and flanking regions, based on assembly of a bacterial artificial chromosome/P1-derived artificial chromosome contig, analysis of high-throughput genome-sequence data, and long-range restriction mapping of genomic and cloned DNA by pulsed-field gel electrophoresis. Our map encompasses 3 Mb, including 1.6 Mb within the deletion. Two large duplicons, flanking the deletion, of ⩾320 kb contain unique sequence elements from the internal border regions of the deletion, such as sequences from GTF2I (telomeric) and FKBP6 (centromeric). A third copy of this duplicon exists in inverted orientation distal to the telomeric flanking one. These duplicons show stronger sequence conservation with regard to each other than to the presumptive ancestral loci within the common deletion region. Sequence elements originating from beyond 7q11.23 are also present in these duplicons. Although the duplicons are not present in mice, the order of the single-copy genes in the conserved syntenic region of mouse chromosome 5 is inverted relative to the human map. A model is presented for a mechanism of WBS-deletion formation, based on the orientation of duplicons' components relative to each other and to the ancestral elements within the deletion region. PMID:10631136

Long-range restriction map of human chromosome 22q11-22q12 between the lambda immunoglobulin locus and the Ewing sarcoma breakpoint

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

McDermid, H.E.; Budarf, M.L.; Emanuel, B.S.

1993-11-01

A long-range restriction map of the region between the immunoglobulin lambda locus and the Ewing sarcoma breakpoint has been constructed using the rare-cutting enzymes NotI, NruI, AscI, and BsiWI. The map spans approximately 11,000 kb and represents about one-fifth of the long arm of chromosome 22. Thirty-nine markers, including seven NotI junction clones as well as numerous genes and anonymous sequences, were mapped to the region with a somatic cell hybrid panel. These probes were then used to produce the map. The seven NotI junction clones each identified a possible CpG island. The breakpoints of the RAJ5 hybrid and themore » Ewing sarcoma t(11;22) were also localized in the resulting map. This physical map will be useful in studying chromosomal rearrangements in the region, as well as providing the details to examine the fidelity of the YAC and cosmid contigs currently under construction. Comparisons of this physical map to genetic and radiation hybrid maps are discussed. 52 refs., 7 figs., 3 tabs.« less

Evidence for different origin of sex chromosomes in snakes, birds, and mammals and step-wise differentiation of snake sex chromosomes

PubMed Central

Matsubara, Kazumi; Tarui, Hiroshi; Toriba, Michihisa; Yamada, Kazuhiko; Nishida-Umehara, Chizuko; Agata, Kiyokazu; Matsuda, Yoichi

2006-01-01

All snake species exhibit genetic sex determination with the ZZ/ZW type of sex chromosomes. To investigate the origin and evolution of snake sex chromosomes, we constructed, by FISH, a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 109 cDNA clones. Eleven of the 109 clones were localized to the Z chromosome. All human and chicken homologues of the snake Z-linked genes were located on autosomes, suggesting that the sex chromosomes of snakes, mammals, and birds were all derived from different autosomal pairs of the common ancestor. We mapped the 11 Z-linked genes of E. quadrivirgata to chromosomes of two other species, the Burmese python (Python molurus bivittatus) and the habu (Trimeresurus flavoviridis), to investigate the process of W chromosome differentiation. All and 3 of the 11 clones were localized to both the Z and W chromosomes in P. molurus and E. quadrivirgata, respectively, whereas no cDNA clones were mapped to the W chromosome in T. flavoviridis. Comparative mapping revealed that the sex chromosomes are only slightly differentiated in P. molurus, whereas they are fully differentiated in T. flavoviridis, and E. quadrivirgata is at a transitional stage of sex-chromosome differentiation. The differentiation of sex chromosomes was probably initiated from the distal region on the short arm of the protosex chromosome of the common ancestor, and then deletion and heterochromatization progressed on the sex-specific chromosome from the phylogenetically primitive boids to the more advanced viperids. PMID:17110446

Evolving phenotypic networks in silico.

PubMed

François, Paul

2014-11-01

Evolved gene networks are constrained by natural selection. Their structures and functions are consequently far from being random, as exemplified by the multiple instances of parallel/convergent evolution. One can thus ask if features of actual gene networks can be recovered from evolutionary first principles. I review a method for in silico evolution of small models of gene networks aiming at performing predefined biological functions. I summarize the current implementation of the algorithm, insisting on the construction of a proper "fitness" function. I illustrate the approach on three examples: biochemical adaptation, ligand discrimination and vertebrate segmentation (somitogenesis). While the structure of the evolved networks is variable, dynamics of our evolved networks are usually constrained and present many similar features to actual gene networks, including properties that were not explicitly selected for. In silico evolution can thus be used to predict biological behaviours without a detailed knowledge of the mapping between genotype and phenotype. Copyright © 2014 The Author. Published by Elsevier Ltd.. All rights reserved.

Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus.

PubMed

Darai, G; Delius, H; Clarke, J; Apfel, H; Schnitzler, P; Flügel, R M

1985-10-30

A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.

Decoding Crucial LncRNAs Implicated in Neurogenesis and Neurological Disorders.

PubMed

Ayana, R; Singh, Shailja; Pati, Soumya

2017-04-15

Unraveling transcriptional heterogeneity and the labyrinthine nature of neurodevelopment can probe insights into neuropsychiatric disorders. It is noteworthy that adult neurogenesis is restricted to the subventricular and subgranular zones of the brain. Recent studies suggest long non-coding RNAs (lncRNAs) as an avant-garde class of regulators implicated in neurodevelopment. But, paucity exists in the knowledge regarding lncRNAs in neurogenesis and their associations with neurodevelopmental defects. To address this, we extensively reviewed the existing literature databases as well as performed relevant in-silico analysis. We utilized Allen Brain Atlas (ABA) differential search module and generated a catalogue of ∼30,000 transcripts specific to the neurogenic zones, including coding and non-coding transcripts. To explore the existing lncRNAs reported in neurogenesis, we performed extensive literature mining and identified 392 lncRNAs. These degenerate lncRNAs were mapped onto the ABA transcript list leading to detection of 20 lncRNAs specific to neurogenic zones (Dentate gyrus/Lateral ventricle), among which 10 showed associations to several neurodevelopmental disorders following in-silico mapping onto brain disease databases like Simons Foundation Autism Research Initiative, AutDB, and lncRNADisease. Notably, using ABA correlation module, we could establish lncRNA-to-mRNA coexpression networks for the above 10 candidate lncRNAs. Finally, pathway prediction revealed physical, biochemical, or regulatory interactions for nine lncRNAs. In addition, ABA differential search also revealed 54 novel significant lncRNAs from the null set (∼30,000). Conclusively, this review represents an updated catalogue of lncRNAs in neurogenesis and neurological diseases, and overviews the field of OMICs-based data analysis for understanding lncRNome-based regulation in neurodevelopment.

The NB-LRR gene Pm60 confers powdery mildew resistance in wheat.

PubMed

Zou, Shenghao; Wang, Huan; Li, Yiwen; Kong, Zhaosheng; Tang, Dingzhong

2018-04-01

Powdery mildew is one of the most devastating diseases of wheat. To date, few powdery mildew resistance genes have been cloned from wheat due to the size and complexity of the wheat genome. Triticum urartu is the progenitor of the A genome of wheat and is an important source for powdery mildew resistance genes. Using molecular markers designed from scaffolds of the sequenced T. urartu accession and standard map-based cloning, a powdery mildew resistance locus was mapped to a 356-kb region, which contains two nucleotide-binding and leucine-rich repeat domain (NB-LRR) protein-encoding genes. Virus-induced gene silencing, single-cell transient expression, and stable transformation assays demonstrated that one of these two genes, designated Pm60, confers resistance to powdery mildew. Overexpression of full-length Pm60 and two allelic variants in Nicotiana benthamiana leaves induced hypersensitive cell death response, but expression of the coiled-coil domain alone was insufficient to induce hypersensitive response. Yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation imaging assays showed that Pm60 protein interacts with its neighboring NB-containing protein, suggesting that they might be functionally related. The identification and cloning of this novel wheat powdery mildew resistance gene will facilitate breeding for disease resistance in wheat. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents.

PubMed

Richie, Daryl L; Thompson, Katherine V; Studer, Christian; Prindle, Vivian C; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Jürg; Schuierer, Sven; Hartmann, Nicole; Movva, N Rao; Tallarico, John A; Ryder, Neil S; Hoepfner, Dominic

2013-05-01

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.

Optimizing the Targeting of Mouse Parvovirus 1 to Murine Melanoma Selects for Recombinant Genomes and Novel Mutations in the Viral Capsid Gene

PubMed Central

Marr, Matthew; D’Abramo, Anthony; Agbandje-McKenna, Mavis; Cotmore, Susan; Tattersall, Peter

2018-01-01

Combining virus-enhanced immunogenicity with direct delivery of immunomodulatory molecules would represent a novel treatment modality for melanoma, and would require development of new viral vectors capable of targeting melanoma cells preferentially. Here we explore the use of rodent protoparvoviruses targeting cells of the murine melanoma model B16F10. An uncloned stock of mouse parvovirus 1 (MPV1) showed some efficacy, which was substantially enhanced following serial passage in the target cell. Molecular cloning of the genes of both starter and selected virus pools revealed considerable sequence diversity. Chimera analysis mapped the majority of the improved infectivity to the product of the major coat protein gene, VP2, in which linked blocks of amino acid changes and one or other of two apparently spontaneous mutations were selected. Intragenic chimeras showed that these represented separable components, both contributing to enhanced infection. Comparison of biochemical parameters of infection by clonal viruses indicated that the enhancement due to changes in VP2 operates after the virus has bound to the cell surface and penetrated into the cell. Construction of an in silico homology model for MPV1 allowed placement of these changes within the capsid shell, and revealed aspects of the capsid involved in infection initiation that had not been previously recognized. PMID:29385689

Construction of Reference Chromosome-Scale Pseudomolecules for Potato: Integrating the Potato Genome with Genetic and Physical Maps

PubMed Central

Sharma, Sanjeev Kumar; Bolser, Daniel; de Boer, Jan; Sønderkær, Mads; Amoros, Walter; Carboni, Martin Federico; D’Ambrosio, Juan Martín; de la Cruz, German; Di Genova, Alex; Douches, David S.; Eguiluz, Maria; Guo, Xiao; Guzman, Frank; Hackett, Christine A.; Hamilton, John P.; Li, Guangcun; Li, Ying; Lozano, Roberto; Maass, Alejandro; Marshall, David; Martinez, Diana; McLean, Karen; Mejía, Nilo; Milne, Linda; Munive, Susan; Nagy, Istvan; Ponce, Olga; Ramirez, Manuel; Simon, Reinhard; Thomson, Susan J.; Torres, Yerisf; Waugh, Robbie; Zhang, Zhonghua; Huang, Sanwen; Visser, Richard G. F.; Bachem, Christian W. B.; Sagredo, Boris; Feingold, Sergio E.; Orjeda, Gisella; Veilleux, Richard E.; Bonierbale, Merideth; Jacobs, Jeanne M. E.; Milbourne, Dan; Martin, David Michael Alan; Bryan, Glenn J.

2013-01-01

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker−based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal “pseudomolecules”. PMID:24062527

Molecular Mapping of Restriction-Site Associated DNA Markers In Allotetraploid Upland Cotton.

PubMed

Wang, Yangkun; Ning, Zhiyuan; Hu, Yan; Chen, Jiedan; Zhao, Rui; Chen, Hong; Ai, Nijiang; Guo, Wangzhen; Zhang, Tianzhen

2015-01-01

Upland cotton (Gossypium hirsutum L., 2n = 52, AADD) is an allotetraploid, therefore the discovery of single nucleotide polymorphism (SNP) markers is difficult. The recent emergence of genome complexity reduction technologies based on the next-generation sequencing (NGS) platform has greatly expedited SNP discovery in crops with highly repetitive and complex genomes. Here we applied restriction-site associated DNA (RAD) sequencing technology for de novo SNP discovery in allotetraploid cotton. We identified 21,109 SNPs between the two parents and used these for genotyping of 161 recombinant inbred lines (RILs). Finally, a high dense linkage map comprising 4,153 loci over 3500-cM was developed based on the previous result. Using this map quantitative trait locus (QTLs) conferring fiber strength and Verticillium Wilt (VW) resistance were mapped to a more accurate region in comparison to the 1576-cM interval determined using the simple sequence repeat (SSR) genetic map. This suggests that the newly constructed map has more power and resolution than the previous SSR map. It will pave the way for the rapid identification of the marker-assisted selection in cotton breeding and cloning of QTL of interest traits.

A cytological-physical map of 22q11

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

Lindsay, E.A.; Rizzu, P.; Gaddini, L.

Our laboratory is involved in the construction of a cytological-physical map of 22q11 and isolation of expressed sequences from the region involved in DiGeorge syndrome (DGS) and Velo-Cardio-Facial syndrome (VCFS). One of the goals of the mapping is an understanding of the molecular mechanisms which generate the 22q11 microdeletions observed with high frequency in DGS and VCFS. Our of over 60 deleted patients studied in our laboratory, all but one were deleted for two loci approximately 1-2 Mb apart. There is evidence from patients with balanced and unbalanced translocations that deletion of the whole region is not necessary for determinationmore » of the clinical phenotype. Therefore, it is possible that deletion breakpoints occur as a consequence of structural characteristics of the DNA that predispose to rearrangements. A striking characteristic of the 22q11 region is the abundance of low copy repeat sequences. It is reasonable to think that recombination between these repeats may lead to microdeletions. However, a direct demonstration of such mechanism is not available yet. The presence of repeats makes standard physical mapping techniques based on hybridization or STS mapping often difficult to interpret. For example, we have found clones positive for the same STS that are located in different positions within 22q11. For this reason we have used high resolution cytological mapping as a supporting technique for map validation. We present the current status map which includes known polymorphic and non-polymorphic loci, newly isolated clones and chromosomal deletion breakpoints. The map extends from the loci D22S9/D22S24 to TOP1P2. Extended chromatin hybridization experiments visually demonstrate the presence of at least two repeat islands flanking (or at) the region where chromosomal breakpoints of the commonly deleted region occur.« less

Whole-Genome Sequencing and Assembly with High-Throughput, Short-Read Technologies

PubMed Central

Sundquist, Andreas; Ronaghi, Mostafa; Tang, Haixu; Pevzner, Pavel; Batzoglou, Serafim

2007-01-01

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology. PMID:17534434

In silico toxicology protocols.

PubMed

Myatt, Glenn J; Ahlberg, Ernst; Akahori, Yumi; Allen, David; Amberg, Alexander; Anger, Lennart T; Aptula, Aynur; Auerbach, Scott; Beilke, Lisa; Bellion, Phillip; Benigni, Romualdo; Bercu, Joel; Booth, Ewan D; Bower, Dave; Brigo, Alessandro; Burden, Natalie; Cammerer, Zoryana; Cronin, Mark T D; Cross, Kevin P; Custer, Laura; Dettwiler, Magdalena; Dobo, Krista; Ford, Kevin A; Fortin, Marie C; Gad-McDonald, Samantha E; Gellatly, Nichola; Gervais, Véronique; Glover, Kyle P; Glowienke, Susanne; Van Gompel, Jacky; Gutsell, Steve; Hardy, Barry; Harvey, James S; Hillegass, Jedd; Honma, Masamitsu; Hsieh, Jui-Hua; Hsu, Chia-Wen; Hughes, Kathy; Johnson, Candice; Jolly, Robert; Jones, David; Kemper, Ray; Kenyon, Michelle O; Kim, Marlene T; Kruhlak, Naomi L; Kulkarni, Sunil A; Kümmerer, Klaus; Leavitt, Penny; Majer, Bernhard; Masten, Scott; Miller, Scott; Moser, Janet; Mumtaz, Moiz; Muster, Wolfgang; Neilson, Louise; Oprea, Tudor I; Patlewicz, Grace; Paulino, Alexandre; Lo Piparo, Elena; Powley, Mark; Quigley, Donald P; Reddy, M Vijayaraj; Richarz, Andrea-Nicole; Ruiz, Patricia; Schilter, Benoit; Serafimova, Rositsa; Simpson, Wendy; Stavitskaya, Lidiya; Stidl, Reinhard; Suarez-Rodriguez, Diana; Szabo, David T; Teasdale, Andrew; Trejo-Martin, Alejandra; Valentin, Jean-Pierre; Vuorinen, Anna; Wall, Brian A; Watts, Pete; White, Angela T; Wichard, Joerg; Witt, Kristine L; Woolley, Adam; Woolley, David; Zwickl, Craig; Hasselgren, Catrin

2018-07-01

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Exploiting induced variation to dissect quantitative traits in barley.

PubMed

Druka, Arnis; Franckowiak, Jerome; Lundqvist, Udda; Bonar, Nicola; Alexander, Jill; Guzy-Wrobelska, Justyna; Ramsay, Luke; Druka, Ilze; Grant, Iain; Macaulay, Malcolm; Vendramin, Vera; Shahinnia, Fahimeh; Radovic, Slobodanka; Houston, Kelly; Harrap, David; Cardle, Linda; Marshall, David; Morgante, Michele; Stein, Nils; Waugh, Robbie

2010-04-01

The identification of genes underlying complex quantitative traits such as grain yield by means of conventional genetic analysis (positional cloning) requires the development of several large mapping populations. However, it is possible that phenotypically related, but more extreme, allelic variants generated by mutational studies could provide a means for more efficient cloning of QTLs (quantitative trait loci). In barley (Hordeum vulgare), with the development of high-throughput genome analysis tools, efficient genome-wide identification of genetic loci harbouring mutant alleles has recently become possible. Genotypic data from NILs (near-isogenic lines) that carry induced or natural variants of genes that control aspects of plant development can be compared with the location of QTLs to potentially identify candidate genes for development--related traits such as grain yield. As yield itself can be divided into a number of allometric component traits such as tillers per plant, kernels per spike and kernel size, mutant alleles that both affect these traits and are located within the confidence intervals for major yield QTLs may represent extreme variants of the underlying genes. In addition, the development of detailed comparative genomic models based on the alignment of a high-density barley gene map with the rice and sorghum physical maps, has enabled an informed prioritization of 'known function' genes as candidates for both QTLs and induced mutant genes.

Genetic and physical mapping of homologues of the virus resistance gene Rx1 and the cyst nematode resistance gene Gpa2 in potato.

PubMed

Bakker, E; Butterbach, P; Rouppe van der Voort, J; van der Vossen, E; van Vliet, J; Bakker, J; Goverse, A

2003-05-01

Nine resistance gene homologues (RGHs) were identified in two diploid potato clones (SH and RH), with a specific primer pair based on conserved motifs in the LRR domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene Rx1. A modified AFLP method was used to facilitate the genetic mapping of the RGHs in the four haplotypes under investigation. All nine RGHs appeared to be located in the Gpa2/ Rx1 cluster on chromosome XII. Construction of a physical map using bacterial artificial chromosome (BAC) clones for both the Solanum tuberosum ssp. tuberosum and the S. tuberosum ssp. andigena haplotype of SH showed that the RGHs are located within a stretch of less than 200 kb. Sequence analysis of the RGHs revealed that they are highly similar (93 to 95%) to Gpa2 and Rx1. The sequence identities among all RGHs range from 85 to 100%. Two pairs of RGHs are identical, or nearly so (100 and 99.9%), with each member located in a different genotype. Southern-blot analysis on genomic DNA revealed no evidence for additional homologues outside the Gpa2/ Rx1 cluster on chromosome XII.

Comparison of the chromosome maps around a resistance hot spot on chromosome 5 of potato and tomato using BAC-FISH painting.

PubMed

Achenbach, Ute C; Tang, Xiaomin; Ballvora, Agim; de Jong, Hans; Gebhardt, Christiane

2010-02-01

Potato chromosome 5 harbours numerous genes for important qualitative and quantitative traits, such as resistance to the root cyst nematode Globodera pallida and the late blight fungus, Phytophthora infestans. The genes make up part of a "hot spot" for resistances to various pathogens covering a genetic map length of 3 cM between markers GP21 and GP179. We established the physical size and position of this region on chromosome 5 in potato and tomato using fluorescence in situ hybridization (FISH) on pachytene chromosomes. Five potato bacterial artificial chromosome (BAC) clones with the genetically anchored markers GP21, R1-contig (proximal end), CosA, GP179, and StPto were selected, labeled with different fluorophores, and hybridized in a five-colour FISH experiment. Our results showed the location of the BAC clones in the middle of the long arm of chromosome 5 in both potato and tomato. Based on chromosome measurements, we estimate the physical size of the GP21-GP179 interval at 0.85 Mb and 1.2 Mb in potato and tomato, respectively. The GP21-GP179 interval is part of a genome segment known to have inverted map positions between potato and tomato.

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

Kato, Go

We consider the situation where s replicas of a qubit with an unknown state and its orthogonal k replicas are given as an input, and we try to make c clones of the qubit with the unknown state. As a function of s, k, and c, we obtain the optimal fidelity between the qubit with an unknown state and the clone by explicitly giving a completely positive trace-preserving (CPTP) map that represents a cloning machine. We discuss dependency of the fidelity on the values of the parameters s, k, and c.

Construction of a general human chromosome jumping library, with application to cystic fibrosis

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

Collins, F.S.; Drumm, M.L.; Cole, J.L.

1987-02-27

In many genetic disorders, the responsible gene and its protein product are unknown. The technique known as reverse genetics, in which chromosomal map positions and genetically linked DNA markers are used to identify and clone such genes, is complicated by the fact that the molecular distances from the closest DNA markers to the gene itself are often too large to traverse by standard cloning techniques. To address this situation, a general human chromosome jumping library was constructed that allows the cloning of DNA sequences approximately 100 kilobases away from any starting point in genomic DNA. As an illustration of itsmore » usefulness, this library was searched for a jumping clone, starting at the met oncogene, which is a marker tightly linked to the cystic fibrosis gene that is located on human chromosome 7. Mapping of the new genomic fragment by pulsed field gel electrophoresis confirmed that it resides on chromosome 7 within 240 kilobases downstream of the met gene. The use of chromosome jumping should be applicable to any genetic locus for which a closely linked DNA marker is available.« less

Optical mapping and its potential for large-scale sequencing projects.

PubMed

Aston, C; Mishra, B; Schwartz, D C

1999-07-01

Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.

Large-scale virtual screening on public cloud resources with Apache Spark.

PubMed

Capuccini, Marco; Ahmed, Laeeq; Schaal, Wesley; Laure, Erwin; Spjuth, Ola

2017-01-01

Structure-based virtual screening is an in-silico method to screen a target receptor against a virtual molecular library. Applying docking-based screening to large molecular libraries can be computationally expensive, however it constitutes a trivially parallelizable task. Most of the available parallel implementations are based on message passing interface, relying on low failure rate hardware and fast network connection. Google's MapReduce revolutionized large-scale analysis, enabling the processing of massive datasets on commodity hardware and cloud resources, providing transparent scalability and fault tolerance at the software level. Open source implementations of MapReduce include Apache Hadoop and the more recent Apache Spark. We developed a method to run existing docking-based screening software on distributed cloud resources, utilizing the MapReduce approach. We benchmarked our method, which is implemented in Apache Spark, docking a publicly available target receptor against [Formula: see text]2.2 M compounds. The performance experiments show a good parallel efficiency (87%) when running in a public cloud environment. Our method enables parallel Structure-based virtual screening on public cloud resources or commodity computer clusters. The degree of scalability that we achieve allows for trying out our method on relatively small libraries first and then to scale to larger libraries. Our implementation is named Spark-VS and it is freely available as open source from GitHub (https://github.com/mcapuccini/spark-vs).Graphical abstract.

Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.

PubMed

Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Whittington, Richard J

2014-03-15

Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research. Copyright © 2013 Elsevier B.V. All rights reserved.

In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

PubMed Central

Lepoittevin, Camille; Frigerio, Jean-Marc; Garnier-Géré, Pauline; Salin, Franck; Cervera, María-Teresa; Vornam, Barbara; Harvengt, Luc; Plomion, Christophe

2010-01-01

Background There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C). Methodology/Principal Findings A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). Conclusions/Significance This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome. PMID:20543950

Machine learning-, rule- and pharmacophore-based classification on the inhibition of P-glycoprotein and NorA.

PubMed

Ngo, T-D; Tran, T-D; Le, M-T; Thai, K-M

2016-09-01

The efflux pumps P-glycoprotein (P-gp) in humans and NorA in Staphylococcus aureus are of great interest for medicinal chemists because of their important roles in multidrug resistance (MDR). The high polyspecificity as well as the unavailability of high-resolution X-ray crystal structures of these transmembrane proteins lead us to combining ligand-based approaches, which in the case of this study were machine learning, perceptual mapping and pharmacophore modelling. For P-gp inhibitory activity, individual models were developed using different machine learning algorithms and subsequently combined into an ensemble model which showed a good discrimination between inhibitors and noninhibitors (acctrain-diverse = 84%; accinternal-test = 92% and accexternal-test = 100%). For ligand promiscuity between P-gp and NorA, perceptual maps and pharmacophore models were generated for the detection of rules and features. Based on these in silico tools, hit compounds for reversing MDR were discovered from the in-house and DrugBank databases through virtual screening in an attempt to restore drug sensitivity in cancer cells and bacteria.

Molecular cloning and chromosomal localization of a pseudogene related to the human Acyl-CoA binding protein/diazepam binding inhibitor

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

Gersuk, V.H.; Rose, T.M.; Todaro, G.J.

The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5{prime} ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-relatedmore » sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1. 33 refs., 3 figs., 1 tab.« less

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

Fiurasek, Jaromir; Cerf, Nicolas J.

We investigate the asymmetric Gaussian cloning of coherent states which produces M copies from N input replicas in such a way that the fidelity of each copy may be different. We show that the optimal asymmetric Gaussian cloning can be performed with a single phase-insensitive amplifier and an array of beam splitters. We obtain a simple analytical expression characterizing the set of optimal asymmetric Gaussian cloning machines and prove the optimality of these cloners using the formalism of Gaussian completely positive maps and semidefinite programming techniques. We also present an alternative implementation of the asymmetric cloning machine where the phase-insensitivemore » amplifier is replaced with a beam splitter, heterodyne detector, and feedforward.« less

Universality of clone dynamics during tissue development

NASA Astrophysics Data System (ADS)

Rulands, Steffen; Lescroart, Fabienne; Chabab, Samira; Hindley, Christopher J.; Prior, Nicole; Sznurkowska, Magdalena K.; Huch, Meritxell; Philpott, Anna; Blanpain, Cedric; Simons, Benjamin D.

2018-05-01

The emergence of complex organs is driven by the coordinated proliferation, migration and differentiation of precursor cells. The fate behaviour of these cells is reflected in the time evolution of their progeny, termed clones, which serve as a key experimental observable. In adult tissues, where cell dynamics is constrained by the condition of homeostasis, clonal tracing studies based on transgenic animal models have advanced our understanding of cell fate behaviour and its dysregulation in disease1,2. But what can be learnt from clonal dynamics in development, where the spatial cohesiveness of clones is impaired by tissue deformations during tissue growth? Drawing on the results of clonal tracing studies, we show that, despite the complexity of organ development, clonal dynamics may converge to a critical state characterized by universal scaling behaviour of clone sizes. By mapping clonal dynamics onto a generalization of the classical theory of aerosols, we elucidate the origin and range of scaling behaviours and show how the identification of universal scaling dependences may allow lineage-specific information to be distilled from experiments. Our study shows the emergence of core concepts of statistical physics in an unexpected context, identifying cellular systems as a laboratory to study non-equilibrium statistical physics.

Inversions and Gene Order Shuffling in Anopheles gambiae and A. funestus

NASA Astrophysics Data System (ADS)

Sharakhov, Igor V.; Serazin, Andrew C.; Grushko, Olga G.; Dana, Ali; Lobo, Neil; Hillenmeyer, Maureen E.; Westerman, Richard; Romero-Severson, Jeanne; Costantini, Carlo; Sagnon, N'Fale; Collins, Frank H.; Besansky, Nora J.

2002-10-01

In tropical Africa, Anopheles funestus is one of the three most important malaria vectors. We physically mapped 157 A. funestus complementary DNAs (cDNAs) to the polytene chromosomes of this species. Sequences of the cDNAs were mapped in silico to the A. gambiae genome as part of a comparative genomic study of synteny, gene order, and sequence conservation between A. funestus and A. gambiae. These species are in the same subgenus and diverged about as recently as humans and chimpanzees. Despite nearly perfect preservation of synteny, we found substantial shuffling of gene order along corresponding chromosome arms. Since the divergence of these species, at least 70 chromosomal inversions have been fixed, the highest rate of rearrangement of any eukaryote studied to date. The high incidence of paracentric inversions and limited colinearity suggests that locating genes in one anopheline species based on gene order in another may be limited to closely related taxa.

The Use of Multidimensional Image-Based Analysis to Accurately Monitor Cell Growth in 3D Bioreactor Culture

PubMed Central

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells. PMID:22028809

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture.

PubMed

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.

A consensus genetic map of cowpea [Vigna unguiculata (L) Walp.] and synteny based on EST-derived SNPs.

PubMed

Muchero, Wellington; Diop, Ndeye N; Bhat, Prasanna R; Fenton, Raymond D; Wanamaker, Steve; Pottorff, Marti; Hearne, Sarah; Cisse, Ndiaga; Fatokun, Christian; Ehlers, Jeffrey D; Roberts, Philip A; Close, Timothy J

2009-10-27

Consensus genetic linkage maps provide a genomic framework for quantitative trait loci identification, map-based cloning, assessment of genetic diversity, association mapping, and applied breeding in marker-assisted selection schemes. Among "orphan crops" with limited genomic resources such as cowpea [Vigna unguiculata (L.) Walp.] (2n = 2x = 22), the use of transcript-derived SNPs in genetic maps provides opportunities for automated genotyping and estimation of genome structure based on synteny analysis. Here, we report the development and validation of a high-throughput EST-derived SNP assay for cowpea, its application in consensus map building, and determination of synteny to reference genomes. SNP mining from 183,118 ESTs sequenced from 17 cDNA libraries yielded approximately 10,000 high-confidence SNPs from which an Illumina 1,536-SNP GoldenGate genotyping array was developed and applied to 741 recombinant inbred lines from six mapping populations. Approximately 90% of the SNPs were technically successful, providing 1,375 dependable markers. Of these, 928 were incorporated into a consensus genetic map spanning 680 cM with 11 linkage groups and an average marker distance of 0.73 cM. Comparison of this cowpea genetic map to reference legumes, soybean (Glycine max) and Medicago truncatula, revealed extensive macrosynteny encompassing 85 and 82%, respectively, of the cowpea map. Regions of soybean genome duplication were evident relative to the simpler diploid cowpea. Comparison with Arabidopsis revealed extensive genomic rearrangement with some conserved microsynteny. These results support evolutionary closeness between cowpea and soybean and identify regions for synteny-based functional genomics studies in legumes.

Characterization of Three Maize Bacterial Artificial Chromosome Libraries toward Anchoring of the Physical Map to the Genetic Map Using High-Density Bacterial Artificial Chromosome Filter Hybridization1

PubMed Central

Yim, Young-Sun; Davis, Georgia L.; Duru, Ngozi A.; Musket, Theresa A.; Linton, Eric W.; Messing, Joachim W.; McMullen, Michael D.; Soderlund, Carol A.; Polacco, Mary L.; Gardiner, Jack M.; Coe, Edward H.

2002-01-01

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage λ. The results indicate that the libraries are of high quality with low contamination by organellar and λ-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6× coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 × Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 ± 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction. PMID:12481051

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

PubMed Central

Subramaniam, R; Reinold, S; Molitor, E K; Douglas, C J

1993-01-01

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues. PMID:8108506

Translating genome wide association study results to associations among common diseases: in silico study with an electronic medical record.

PubMed

Anand, Vibha; Rosenman, Marc B; Downs, Stephen M

2013-09-01

To develop a map of disease associations exclusively using two publicly available genetic sources: the catalog of single nucleotide polymorphisms (SNPs) from the HapMap, and the catalog of Genome Wide Association Studies (GWAS) from the NHGRI, and to evaluate it with a large, long-standing electronic medical record (EMR). A computational model, In Silico Bayesian Integration of GWAS (IsBIG), was developed to learn associations among diseases using a Bayesian network (BN) framework, using only genetic data. The IsBIG model (I-Model) was re-trained using data from our EMR (M-Model). Separately, another clinical model (C-Model) was learned from this training dataset. The I-Model was compared with both the M-Model and the C-Model for power to discriminate a disease given other diseases using a test dataset from our EMR. Area under receiver operator characteristics curve was used as a performance measure. Direct associations between diseases in the I-Model were also searched in the PubMed database and in classes of the Human Disease Network (HDN). On the basis of genetic information alone, the I-Model linked a third of diseases from our EMR. When compared to the M-Model, the I-Model predicted diseases given other diseases with 94% specificity, 33% sensitivity, and 80% positive predictive value. The I-Model contained 117 direct associations between diseases. Of those associations, 20 (17%) were absent from the searches of the PubMed database; one of these was present in the C-Model. Of the direct associations in the I-Model, 7 (35%) were absent from disease classes of HDN. Using only publicly available genetic sources we have mapped associations in GWAS to a human disease map using an in silico approach. Furthermore, we have validated this disease map using phenotypic data from our EMR. Models predicting disease associations on the basis of known genetic associations alone are specific but not sensitive. Genetic data, as it currently exists, can only explain a fraction of the risk of a disease. Our approach makes a quantitative statement about disease variation that can be explained in an EMR on the basis of genetic associations described in the GWAS. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Construction of the first genetic linkage map of Japanese gentian (Gentianaceae)

PubMed Central

2012-01-01

Background Japanese gentians (Gentiana triflora and Gentiana scabra) are amongst the most popular floricultural plants in Japan. However, genomic resources for Japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. In this study, we developed a genetic linkage map to improve breeding programs of Japanese gentians. Results Enriched simple sequence repeat (SSR) libraries from a G. triflora double haploid line yielded almost 20,000 clones using 454 pyrosequencing technology, 6.7% of which could be used to design SSR markers. To increase the number of molecular markers, we identified three putative long terminal repeat (LTR) sequences using the recently developed inter-primer binding site (iPBS) method. We also developed retrotransposon microsatellite amplified polymorphism (REMAP) markers combining retrotransposon and inter-simple sequence repeat (ISSR) markers. In addition to SSR and REMAP markers, modified amplified fragment length polymorphism (AFLP) and random amplification polymorphic DNA (RAPD) markers were developed. Using 93 BC1 progeny from G. scabra backcrossed with a G. triflora double haploid line, 19 linkage groups were constructed with a total of 263 markers (97 SSR, 97 AFLP, 39 RAPD, and 30 REMAP markers). One phenotypic trait (stem color) and 10 functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility. Conclusions This is the first reported genetic linkage map for Japanese gentians and for any species belonging to the family Gentianaceae. As demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. Our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species. PMID:23186361

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the fucosyltransferase multigene family.

PubMed

Peterson, Nathan A; Anderson, Tavis K; Yoshino, Timothy P

2013-01-01

Fucosylated glycans of the parasitic flatworm Schistosoma mansoni play key roles in its development and immunobiology. In the present study we used a genome-wide homology-based bioinformatics approach to search for genes that contribute to fucosylated glycan expression in S. mansoni, specifically the α2-, α3-, and α6-fucosyltransferases (FucTs), which transfer L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. We identified and in silico characterized several novel schistosome FucT homologs, including six α3-FucTs and six α6-FucTs, as well as two protein O-FucTs that catalyze the unrelated transfer of L-fucose to serine and threonine residues of epidermal growth factor- and thrombospondin-type repeats. No α2-FucTs were observed. Primary sequence analyses identified key conserved FucT motifs as well as characteristic transmembrane domains, consistent with their putative roles as fucosyltransferases. Most genes exhibit alternative splicing, with multiple transcript variants generated. A phylogenetic analysis demonstrated that schistosome α3- and α6-FucTs form monophyletic clades within their respective gene families, suggesting multiple gene duplications following the separation of the schistosome lineage from the main evolutionary tree. Quantitative decreases in steady-state transcript levels of some FucTs during early larval development suggest a possible mechanism for differential expression of fucosylated glycans in schistosomes. This study systematically identifies the complete repertoire of FucT homologs in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, developmental expression, and evolutionary history.

Sdt97: A Point Mutation in the 5′ Untranslated Region Confers Semidwarfism in Rice

PubMed Central

Tong, Jiping; Han, Zhengshu; Han, Aonan; Liu, Xuejun; Zhang, Shiyong; Fu, Binying; Hu, Jun; Su, Jingping; Li, Shaoqing; Wang, Shengjun; Zhu, Yingguo

2016-01-01

Semidwarfism is an important agronomic trait in rice breeding programs. The semidwarf mutant gene Sdt97 was previously described. However, the molecular mechanism underlying the mutant is yet to be elucidated. In this study, we identified the mutant gene by a map-based cloning method. Using a residual heterozygous line (RHL) population, Sdt97 was mapped to the long arm of chromosome 6 in the interval of nearly 60 kb between STS marker N6 and SNP marker N16 within the PAC clone P0453H04. Sequencing of the candidate genes in the target region revealed that a base transversion from G to C occurred in the 5′ untranslated region of Sdt97. qRT-PCR results confirmed that the transversion induced an obvious change in the expression pattern of Sdt97 at different growth and developmental stages. Plants transgenic for Sdt97 resulted in the restoration of semidwarfism of the mutant phenotype, or displayed a greater dwarf phenotype than the mutant. Our results indicate that a point mutation in the 5′ untranslated region of Sdt97 confers semidwarfism in rice. Functional analysis of Sdt97 will open a new field of study for rice semidwarfism, and also expand our knowledge of the molecular mechanism of semidwarfism in rice. PMID:27172200

Genotypic variation in sulfur assimilation and metabolism of onion (Allium cepa L.) III. Characterization of sulfite reductase

USDA-ARS?s Scientific Manuscript database

Genomic and cDNA sequences corresponding to a ferredoxin-sulfite reductase (SiR) have been cloned from bulb onion (Allium cepa L.) and the expression of the gene and activity of the enzyme characterised with respect to sulfur (S) supply. Cloning, mapping and expression studies revealed that onion ha...

Accumulation of multiple mutations in linezolid-resistant Staphylococcus epidermidis causing bloodstream infections; in silico analysis of L3 amino acid substitutions that might confer high-level linezolid resistance.

PubMed

Ikonomidis, Alexandros; Grapsa, Anastasia; Pavlioglou, Charikleia; Demiri, Antonia; Batarli, Alexandra; Panopoulou, Maria

2016-12-01

Fifty-six Staphylococcus epidermidis clinical isolates, showing high-level linezolid resistance and causing bacteremia in critically ill patients, were studied. All isolates belonged to ST22 clone and carried the T2504A and C2534T mutations in gene coding for 23SrRNA as well as the C189A, G208A, C209T and G384C missense mutations in L3 protein which resulted in Asp159Tyr, Gly152Asp and Leu94Val substitutions. Other silent mutations were also detected in genes coding for ribosomal proteins L3 and L22. In silico analysis of missense mutations showed that although L3 protein retained the sequence of secondary motifs, the tertiary structure was influenced. The observed alteration in L3 protein folding provides an indication on the putative role of L3-coding gene mutations in high-level linezolid resistance. Furthermore, linezolid pressure in health care settings where linezolid consumption is of high rates might lead to the selection of resistant mutants possessing L3 mutations that might confer high-level linezolid resistance.

Analysis of BAC-end sequences (BESs) and development of BES-SSR markers for genetic mapping and hybrid purity assessment in pigeonpea (Cajanus spp.)

PubMed Central

2011-01-01

Background Pigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea. Results A set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme. Conclusion In summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea. PMID:21447154

Genomic clones for human cholinesterase

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

Kott, M.; Venta, P.J.; Larsen, J.

1987-05-01

A human genomic library was prepared from peripheral white blood cells from a single donor by inserting an MboI partial digest into BamHI poly-linker sites of EMBL3. This library was screened using an oligolabeled human cholinesterase cDNA probe over 700 bp long. The latter probe was obtained from a human basal ganglia cDNA library. Of approximately 2 million clones screened with high stringency conditions several positive clones were identified; two have been plaque purified. One of these clones has been partially mapped using restriction enzymes known to cut within the coded region of the cDNA for human serum cholinesterase. Hybridizationmore » of the fragments and their sizes are as expected if the genomic clone is cholinesterase. Sequencing of the DNA fragments in M13 is in progress to verify the identify of the clone and the location of introns.« less

Polylox barcoding reveals haematopoietic stem cell fates realized in vivo

PubMed Central

Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

2017-01-01

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping1 has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites2, viral barcodes3, and strategies based on transposons4 and CRISPR/Cas9 genome editing5; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system6,7. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs8–10. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure. PMID:28813413

Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

PubMed

Pei, Weike; Feyerabend, Thorsten B; Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

2017-08-24

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

Protein modeling and molecular dynamics simulation of SlWRKY4 protein cloned from drought tolerant tomato (Solanum habrochaites) line EC520061.

PubMed

Karkute, Suhas G; Easwaran, Murugesh; Gujjar, Ranjit Singh; Piramanayagam, Shanmughavel; Singh, Major

2015-10-01

WRKY genes are members of one of the largest families of plant transcription factors and play an important role in response to biotic and abiotic stresses, and overall growth and development. Understanding the interaction of WRKY proteins with other proteins/ligands in plant cells is of utmost importance to develop plants having tolerance to biotic and abiotic stresses. The SlWRKY4 gene was cloned from a drought tolerant wild species of tomato (Solanum habrochaites) and the secondary structure and 3D modeling of this protein were predicted using Schrödinger Suite-Prime. Predicted structures were also subjected to plot against Ramachandran's conformation, and the modeled structure was minimized using Macromodel. Finally, the minimized structure was simulated in the water environment to check the protein stability. The behavior of the modeled structure was well-simulated and analyzed through RMSD and RMSF of the protein. The present work provides the modeled 3D structure of SlWRKY4 that will help in understanding the mechanism of gene regulation by further in silico interaction studies.

MSD-MAP: A Network-Based Systems Biology Platform for Predicting Disease-Metabolite Links.

PubMed

Wathieu, Henri; Issa, Naiem T; Mohandoss, Manisha; Byers, Stephen W; Dakshanamurthy, Sivanesan

2017-01-01

Cancer-associated metabolites result from cell-wide mechanisms of dysregulation. The field of metabolomics has sought to identify these aberrant metabolites as disease biomarkers, clues to understanding disease mechanisms, or even as therapeutic agents. This study was undertaken to reliably predict metabolites associated with colorectal, esophageal, and prostate cancers. Metabolite and disease biological action networks were compared in a computational platform called MSD-MAP (Multi Scale Disease-Metabolite Association Platform). Using differential gene expression analysis with patient-based RNAseq data from The Cancer Genome Atlas, genes up- or down-regulated in cancer compared to normal tissue were identified. Relational databases were used to map biological entities including pathways, functions, and interacting proteins, to those differential disease genes. Similar relational maps were built for metabolites, stemming from known and in silico predicted metabolite-protein associations. The hypergeometric test was used to find statistically significant relationships between disease and metabolite biological signatures at each tier, and metabolites were assessed for multi-scale association with each cancer. Metabolite networks were also directly associated with various other diseases using a disease functional perturbation database. Our platform recapitulated metabolite-disease links that have been empirically verified in the scientific literature, with network-based mapping of jointly-associated biological activity also matching known disease mechanisms. This was true for colorectal, esophageal, and prostate cancers, using metabolite action networks stemming from both predicted and known functional protein associations. By employing systems biology concepts, MSD-MAP reliably predicted known cancermetabolite links, and may serve as a predictive tool to streamline conventional metabolomic profiling methodologies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4

PubMed Central

Hong, Kar Wai; Chan, Kok-Gan

2015-01-01

Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL) appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity. PMID:26290785

Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli

NASA Astrophysics Data System (ADS)

Küpper, Hans; Keller, Walter; Kurz, Christina; Forss, Sonja; Schaller, Heinz

1981-02-01

Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage λ PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

Isolation and characterization of two overlapping cosmid clones from the 4q35 region, near the facioscapulohumeral muscular dystrophy locus

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

Deidda, G.; Grisanti, P.; Vigneti, E.

1994-09-01

The gene for facioscapulohumeral muscular dystrophy (FSHD) has been localized by linkage analysis to the 4q35 region. The most telomeric p13E-11 prove has been shown to detect 4q35 DNA rearrangements in both sporadic and familial cases of the disease. With the aim of constructing a detailed physical map of the 4q35 region and searching for the mutant gene, we used p13E-11 probe to isolate cosmid clones from a human genomic library in a pCos-EMBL 2 vector. Two positive clones were isolated, clones 3 and 5, which partially overlap and carry human genomic inserts of 42 and 45 kb, respectively. Themore » cosmids share a common region containing the p13E-11 region and a stretch of KpnI units consisting of 3.2 kb tandemly repeated sequences (about 10). The restriction maps were constructed using the following enzymes: Bam HI, BgIII, Eco RI, EcoRV, KpnI and Sfi I. Clone 3 extends 4 kb upstream of C5 and stops within the Kpn repeats. Clone 5 extends 4 kb downstream from the Kpn repeats and it presents an additional EcoRI site. Clone 5 contains a stretch of Kpn sequences of nearly 32 kb, corresponding to 10 Kpn repeats; clone 3 contains a stretch of 29 kb corresponding to 9 Kpn repeats, as determined by PFGE analysis of partial digestion of the clones. Clone 5 seems to contain the entire Eco RI region prone to rearrangements in FSHD patients. From clone 5 several subclones were obtained, from the Kpn region and from the region spanning from the last Kpn repeat to the cloning site. No single copy sequences were detected. Subclones from the 3{prime} end region contain beta-satellite or Sau3A-like sequences. In situ hybridization with the whole C5 cosmid shows hybridization signals at the tip of chromosome 4 (4q35) and chromosome 10 (10q26), in the pericentromeric region of chromosome 1 (1q12) and in the p12 region of the acrocentric chromosomes (chr. 21, 22, 13, 14, 15).« less

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts.

PubMed

Hirozane-Kishikawa, Tomoko; Shiraki, Toshiyuki; Waki, Kazunori; Nakamura, Mari; Arakawa, Takahiro; Kawai, Jun; Fagiolini, Michela; Hensch, Takao K; Hayashizaki, Yoshihide; Carninci, Piero

2003-09-01

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.

Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics.

PubMed

Prien, Justin M; Prater, Bradley D; Qin, Qiang; Cockrill, Steven L

2010-02-15

Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.

Development of genomic resources for the narrow-leafed lupin (Lupinus angustifolius): construction of a bacterial artificial chromosome (BAC) library and BAC-end sequencing

PubMed Central

2011-01-01

Background Lupinus angustifolius L, also known as narrow-leafed lupin (NLL), is becoming an important grain legume crop that is valuable for sustainable farming and is becoming recognised as a potential human health food. Recent interest is being directed at NLL to improve grain production, disease and pest management and health benefits of the grain. However, studies have been hindered by a lack of extensive genomic resources for the species. Results A NLL BAC library was constructed consisting of 111,360 clones with an average insert size of 99.7 Kbp from cv Tanjil. The library has approximately 12 × genome coverage. Both ends of 9600 randomly selected BAC clones were sequenced to generate 13985 BAC end-sequences (BESs), covering approximately 1% of the NLL genome. These BESs permitted a preliminary characterisation of the NLL genome such as organisation and composition, with the BESs having approximately 39% G:C content, 16.6% repetitive DNA and 5.4% putative gene-encoding regions. From the BESs 9966 simple sequence repeat (SSR) motifs were identified and some of these are shown to be potential markers. Conclusions The NLL BAC library and BAC-end sequences are powerful resources for genetic and genomic research on lupin. These resources will provide a robust platform for future high-resolution mapping, map-based cloning, comparative genomics and assembly of whole-genome sequencing data for the species. PMID:22014081

Progressive myoclonus epilepsy EPM1 locus maps to a 175-kb interval in distal 21q

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

Virtaneva, K.; Miao, J.; Traeskelin, A.L.

1996-06-01

The EPM1 locus responsible for progressive myoclonus epilepsy of Unverricht-Lundborg type (MIM 254800) maps to a region in distal chromosome 21q where positional cloning has been hampered by the lack of physical and genetic mapping resolution. We here report the use of a recently constituted contig of cosmid, BAC, and P1 clones that allowed new polymorphic markers to be positioned. These were typed in 53 unrelated disease families from an isolated Finnish population in which a putative single ancestral EPM1 mutation has segregated for an estimated 100 generations. By thus exploiting historical recombinations in haplotype analysis, EPM1 could be assignedmore » to the {approximately}175-kb interval between the markers D21S2040 and D21S1259. 26 refs., 2 figs., 4 tabs.« less

In silico simulation and in vitro evaluation of an elastomeric scaffold using ultrasonic shear wave imaging

NASA Astrophysics Data System (ADS)

Yu, Jiao; Nie, Erwei; Zhu, Yanying; Hong, Yi

2018-03-01

Biodegradable elastomeric scaffolds for soft tissue repair represent a growing area of biomaterials research. Mechanical strength is one of the key factors to consider in the evaluation of candidate materials and the designs for tissue scaffolds. It is desirable to develop non-invasive evaluation methods of the mechanical property of scaffolds which would provide options for monitoring temporal mechanical property changes in situ. In this paper, we conduct in silico simulation and in vitro evaluation of an elastomeric scaffold using a novel ultrasonic shear wave imaging (USWI). The scaffold is fabricated from a biodegradable elastomer, poly(carbonate urethane) urea using salt leaching method. A numerical simulation is performed to test the robustness of the developed inversion algorithm for the elasticity map reconstruction which will be implemented in the phantom experiment. The generation and propagation of shear waves in a homogeneous tissue-mimicking medium with a circular scaffold inclusion is simulated and the elasticity map is well reconstructed. A PVA phantom experiment is performed to test the ability of USWI combined with the inversion algorithm to non-invasively characterize the mechanical property of a porous, biodegradable elastomeric scaffold. The elastic properties of the tested scaffold can be easily differentiated from the surrounding medium in the reconstructed image. The ability of the developed method to identify the edge of the scaffold and characterize the elasticity distribution is demonstrated. Preliminary results in this pilot study support the idea of applying the USWI based method for non-invasive elasticity characterization of tissue scaffolds.

Physical mapping of a large plant genome using global high-information-content-fingerprinting: the distal region of the wheat ancestor Aegilops tauschii chromosome 3DS

PubMed Central

2010-01-01

Background Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat. Results We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs. Conclusions The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes. PMID:20553621

Contig Maps and Genomic Sequencing Identify Candidate Genes in the Usher 1C Locus

PubMed Central

Higgins, Michael J.; Day, Colleen D.; Smilinich, Nancy J.; Ni, L.; Cooper, Paul R.; Nowak, Norma J.; Davies, Chris; de Jong, Pieter J.; Hejtmancik, Fielding; Evans, Glen A.; Smith, Richard J.H.; Shows, Thomas B.

1998-01-01

Usher syndrome 1C (USH1C) is a congenital condition manifesting profound hearing loss, the absence of vestibular function, and eventual retinal degeneration. The USH1C locus has been mapped genetically to a 2- to 3-cM interval in 11p14–15.1 between D11S899 and D11S861. In an effort to identify the USH1C disease gene we have isolated the region between these markers in yeast artificial chromosomes (YACs) using a combination of STS content mapping and Alu–PCR hybridization. The YAC contig is ∼3.5 Mb and has located several other loci within this interval, resulting in the order CEN-LDHA-SAA1-TPH-D11S1310-(D11S1888/KCNC1)-MYOD1-D11S902D11S921-D11S1890-TEL. Subsequent haplotyping and homozygosity analysis refined the location of the disease gene to a 400-kb interval between D11S902 and D11S1890 with all affected individuals being homozygous for the internal marker D11S921. To facilitate gene identification, the critical region has been converted into P1 artificial chromosome (PAC) clones using sequence-tagged sites (STSs) mapped to the YAC contig, Alu–PCR products generated from the YACs, and PAC end probes. A contig of >50 PAC clones has been assembled between D11S1310 and D11S1890, confirming the order of markers used in haplotyping. Three PAC clones representing nearly two-thirds of the USH1C critical region have been sequenced. PowerBLAST analysis identified six clusters of expressed sequence tags (ESTs), two known genes (BIR,SUR1) mapped previously to this region, and a previously characterized but unmapped gene NEFA (DNA binding/EF hand/acidic amino-acid-rich). GRAIL analysis identified 11 CpG islands and 73 exons of excellent quality. These data allowed the construction of a transcription map for the USH1C critical region, consisting of three known genes and six or more novel transcripts. Based on their map location, these loci represent candidate disease loci for USH1C. The NEFA gene was assessed as the USH1C locus by the sequencing of an amplified NEFA cDNA from an USH1C patient; however, no mutations were detected. [The sequence data described in this paper have been submitted to GenBank under accession numbers AC000406–AC000407.] PMID:9445488

Comparative mapping of DNA probes derived from the V{sub k} immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization

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

Arnold, N.; Wienberg, J.; Ermert, K.

Fluorescence in situ hybridization (FISH) of cosmid clones of human V{sub K} gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the K locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo Pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore,more » be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V{sub K}I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V{sub K} region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution. 18 refs., 2 figs.« less

AFLP-based genetic mapping of the “bud-flowering” trait in heather (Calluna vulgaris)

PubMed Central

2013-01-01

Background Calluna vulgaris is one of the most important landscaping plants produced in Germany. Its enormous economic success is due to the prolonged flower attractiveness of mutants in flower morphology, the so-called bud-bloomers. In this study, we present the first genetic linkage map of C. vulgaris in which we mapped a locus of the economically highly desired trait “flower type”. Results The map was constructed in JoinMap 4.1. using 535 AFLP markers from a single mapping population. A large fraction (40%) of markers showed distorted segregation. To test the effect of segregation distortion on linkage estimation, these markers were sorted regarding their segregation ratio and added in groups to the data set. The plausibility of group formation was evaluated by comparison of the “two-way pseudo-testcross” and the “integrated” mapping approach. Furthermore, regression mapping was compared to the multipoint-likelihood algorithm. The majority of maps constructed by different combinations of these methods consisted of eight linkage groups corresponding to the chromosome number of C. vulgaris. Conclusions All maps confirmed the independent inheritance of the most important horticultural traits “flower type”, “flower colour”, and “leaf colour”. An AFLP marker for the most important breeding target “flower type” was identified. The presented genetic map of C. vulgaris can now serve as a basis for further molecular marker selection and map-based cloning of the candidate gene encoding the unique flower architecture of C. vulgaris bud-bloomers. PMID:23915059

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

Feyereisen-Koener, J.M.

Double-stranded cDNA was prepared from infectious hematopoietic necrosis virus mRNA and cloned into the plasmid vector pUC8. A coprotein (G-protein) of infectious hematopoietic necrosis virus was selected by hybridization to a /sup 32/P-labeled probe. The restriction map and nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined using this full-length cDNA clone.

DNA sequences and composition from 12 BAC clones-derived MUSB SSR markers mapped to cotton (Gossypium Hirsutum L. x G. Barbadense L.)chromosomes 11 and 21

USDA-ARS?s Scientific Manuscript database

To discover resistance (R) and/or pathogen-induced (PR) genes involved in disease response, 12 bacterial artificial chromosome (BAC) clones from cv. Acala Maxxa (G. hirsutum) were sequenced at the Clemson University, Genomics Institute, Clemson, SC. These BACs derived MUSB single sequence repeat (SS...

Genome survey and high-density genetic map construction provide genomic and genetic resources for the Pacific White Shrimp Litopenaeus vannamei

PubMed Central

Yu, Yang; Zhang, Xiaojun; Yuan, Jianbo; Li, Fuhua; Chen, Xiaohan; Zhao, Yongzhen; Huang, Long; Zheng, Hongkun; Xiang, Jianhai

2015-01-01

The Pacific white shrimp Litopenaeus vannamei is the dominant crustacean species in global seafood mariculture. Understanding the genome and genetic architecture is useful for deciphering complex traits and accelerating the breeding program in shrimp. In this study, a genome survey was conducted and a high-density linkage map was constructed using a next-generation sequencing approach. The genome survey was used to identify preliminary genome characteristics and to generate a rough reference for linkage map construction. De novo SNP discovery resulted in 25,140 polymorphic markers. A total of 6,359 high-quality markers were selected for linkage map construction based on marker coverage among individuals and read depths. For the linkage map, a total of 6,146 markers spanning 4,271.43 cM were mapped to 44 sex-averaged linkage groups, with an average marker distance of 0.7 cM. An integration analysis linked 5,885 genome scaffolds and 1,504 BAC clones to the linkage map. Based on the high-density linkage map, several QTLs for body weight and body length were detected. This high-density genetic linkage map reveals basic genomic architecture and will be useful for comparative genomics research, genome assembly and genetic improvement of L. vannamei and other penaeid shrimp species. PMID:26503227

Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

PubMed

Thullier, Philippe; Avril, Arnaud; Mathieu, Jacques; Behrens, Christian K; Pellequer, Jean-Luc; Pelat, Thibaut

2013-01-01

The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

PubMed Central

Kilimann, M W; DeGennaro, L J

1985-01-01

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:3933975

How weak values emerge in joint measurements on cloned quantum systems.

PubMed

Hofmann, Holger F

2012-07-13

A statistical analysis of optimal universal cloning shows that it is possible to identify an ideal (but nonpositive) copying process that faithfully maps all properties of the original Hilbert space onto two separate quantum systems, resulting in perfect correlations for all observables. The joint probabilities for noncommuting measurements on separate clones then correspond to the real parts of the complex joint probabilities observed in weak measurements on a single system, where the measurements on the two clones replace the corresponding sequence of weak measurement and postselection. The imaginary parts of weak measurement statics can be obtained by replacing the cloning process with a partial swap operation. A controlled-swap operation combines both processes, making the complete weak measurement statistics accessible as a well-defined contribution to the joint probabilities of fully resolved projective measurements on the two output systems.

Construction of a reference genetic linkage map for carnation (Dianthus caryophyllus L.)

PubMed Central

2013-01-01

Background Genetic linkage maps are important tools for many genetic applications including mapping of quantitative trait loci (QTLs), identifying DNA markers for fingerprinting, and map-based gene cloning. Carnation (Dianthus caryophyllus L.) is an important ornamental flower worldwide. We previously reported a random amplified polymorphic DNA (RAPD)-based genetic linkage map derived from Dianthus capitatus ssp. andrezejowskianus and a simple sequence repeat (SSR)-based genetic linkage map constructed using data from intraspecific F2 populations; however, the number of markers was insufficient, and so the number of linkage groups (LGs) did not coincide with the number of chromosomes (x = 15). Therefore, we aimed to produce a high-density genetic map to improve its usefulness for breeding purposes and genetic research. Results We improved the SSR-based genetic linkage map using SSR markers derived from a genomic library, expression sequence tags, and RNA-seq data. Linkage analysis revealed that 412 SSR loci (including 234 newly developed SSR loci) could be mapped to 17 linkage groups (LGs) covering 969.6 cM. Comparison of five minor LGs covering less than 50 cM with LGs in our previous RAPD-based genetic map suggested that four LGs could be integrated into two LGs by anchoring common SSR loci. Consequently, the number of LGs corresponded to the number of chromosomes (x = 15). We added 192 new SSRs, eight RAPD, and two sequence-tagged site loci to refine the RAPD-based genetic linkage map, which comprised 15 LGs consisting of 348 loci covering 978.3 cM. The two maps had 125 SSR loci in common, and most of the positions of markers were conserved between them. We identified 635 loci in carnation using the two linkage maps. We also mapped QTLs for two traits (bacterial wilt resistance and anthocyanin pigmentation in the flower) and a phenotypic locus for flower-type by analyzing previously reported genotype and phenotype data. Conclusions The improved genetic linkage maps and SSR markers developed in this study will serve as reference genetic linkage maps for members of the genus Dianthus, including carnation, and will be useful for mapping QTLs associated with various traits, and for improving carnation breeding programs. PMID:24160306

Construction of a reference genetic linkage map for carnation (Dianthus caryophyllus L.).

PubMed

Yagi, Masafumi; Yamamoto, Toshiya; Isobe, Sachiko; Hirakawa, Hideki; Tabata, Satoshi; Tanase, Koji; Yamaguchi, Hiroyasu; Onozaki, Takashi

2013-10-26

Genetic linkage maps are important tools for many genetic applications including mapping of quantitative trait loci (QTLs), identifying DNA markers for fingerprinting, and map-based gene cloning. Carnation (Dianthus caryophyllus L.) is an important ornamental flower worldwide. We previously reported a random amplified polymorphic DNA (RAPD)-based genetic linkage map derived from Dianthus capitatus ssp. andrezejowskianus and a simple sequence repeat (SSR)-based genetic linkage map constructed using data from intraspecific F2 populations; however, the number of markers was insufficient, and so the number of linkage groups (LGs) did not coincide with the number of chromosomes (x = 15). Therefore, we aimed to produce a high-density genetic map to improve its usefulness for breeding purposes and genetic research. We improved the SSR-based genetic linkage map using SSR markers derived from a genomic library, expression sequence tags, and RNA-seq data. Linkage analysis revealed that 412 SSR loci (including 234 newly developed SSR loci) could be mapped to 17 linkage groups (LGs) covering 969.6 cM. Comparison of five minor LGs covering less than 50 cM with LGs in our previous RAPD-based genetic map suggested that four LGs could be integrated into two LGs by anchoring common SSR loci. Consequently, the number of LGs corresponded to the number of chromosomes (x = 15). We added 192 new SSRs, eight RAPD, and two sequence-tagged site loci to refine the RAPD-based genetic linkage map, which comprised 15 LGs consisting of 348 loci covering 978.3 cM. The two maps had 125 SSR loci in common, and most of the positions of markers were conserved between them. We identified 635 loci in carnation using the two linkage maps. We also mapped QTLs for two traits (bacterial wilt resistance and anthocyanin pigmentation in the flower) and a phenotypic locus for flower-type by analyzing previously reported genotype and phenotype data. The improved genetic linkage maps and SSR markers developed in this study will serve as reference genetic linkage maps for members of the genus Dianthus, including carnation, and will be useful for mapping QTLs associated with various traits, and for improving carnation breeding programs.

Development of an In Silico Metabolic Simulator and Searchable Metabolism Database for Chemical Risk Assessments

EPA Science Inventory

The US EPA is faced with long lists of chemicals that need to be assessed for hazard, and a gap in evaluating chemical risk is accounting for metabolic activation resulting in increased toxicity. The goals of this project are to develop a capability to predict metabolic maps of x...

Exploiting rice-sorghum synteny for targeted development of EST-SSRs to enrich the sorghum genetic linkage map.

PubMed

Ramu, P; Kassahun, B; Senthilvel, S; Ashok Kumar, C; Jayashree, B; Folkertsma, R T; Reddy, L Ananda; Kuruvinashetti, M S; Haussmann, B I G; Hash, C T

2009-11-01

The sequencing and detailed comparative functional analysis of genomes of a number of select botanical models open new doors into comparative genomics among the angiosperms, with potential benefits for improvement of many orphan crops that feed large populations. In this study, a set of simple sequence repeat (SSR) markers was developed by mining the expressed sequence tag (EST) database of sorghum. Among the SSR-containing sequences, only those sharing considerable homology with rice genomic sequences across the lengths of the 12 rice chromosomes were selected. Thus, 600 SSR-containing sorghum EST sequences (50 homologous sequences on each of the 12 rice chromosomes) were selected, with the intention of providing coverage for corresponding homologous regions of the sorghum genome. Primer pairs were designed and polymorphism detection ability was assessed using parental pairs of two existing sorghum mapping populations. About 28% of these new markers detected polymorphism in this 4-entry panel. A subset of 55 polymorphic EST-derived SSR markers were mapped onto the existing skeleton map of a recombinant inbred population derived from cross N13 x E 36-1, which is segregating for Striga resistance and the stay-green component of terminal drought tolerance. These new EST-derived SSR markers mapped across all 10 sorghum linkage groups, mostly to regions expected based on prior knowledge of rice-sorghum synteny. The ESTs from which these markers were derived were then mapped in silico onto the aligned sorghum genome sequence, and 88% of the best hits corresponded to linkage-based positions. This study demonstrates the utility of comparative genomic information in targeted development of markers to fill gaps in linkage maps of related crop species for which sufficient genomic tools are not available.

In silico approaches reveal the potential for DNA sequence-dependent histone octamer affinity to influence chromatin structure in vivo.

PubMed

Fraser, Ross M; Allan, James; Simmen, Martin W

2006-12-08

Nucleosome positioning signals embedded within the DNA sequence have the potential to influence the detailed structure of the higher-order chromatin fibre. In two previous studies of long stretches of DNA, encompassing the chicken beta-globin and ovine beta-lactoglobulin genes, respectively, we mapped the relative affinity of every site for the core histone octamer. In both cases a periodic arrangement of the in vitro positioning sites suggests that they might influence the folding of a nucleosome chain into higher-order structure; this hypothesis was borne out in the case of the beta-lactoglobulin gene, where the distribution of the in vitro positioning sites is related to the positions nucleosomes actually occupy in sheep liver cells. Here, we have exploited the in vitro nucleosome positioning datasets to simulate nucleosomal organisation using in silico approaches. We use the high-resolution, quantitative positioning maps to define a one-dimensional positioning energy lattice, which can be populated with a defined number of nucleosomes. Monte Carlo techniques are employed to simulate the behaviour of the model at equilibrium to produce a set of configurations, which provide a probability-based occupancy map. Employing a variety of techniques we show that the occupancy maps are a sensitive function of the histone octamer density (nucleosome repeat length) and find that a minimal change in this property can produce dramatic localised changes in structure. Although simulations generally give rise to regular periodic nucleosomal arrangements, they often show octamer density-dependent discontinuities, which tend to co-localise with sequences that adopt distinctive chromatin structure in vivo. Furthermore, the overall organisation of simulated chromatin structures are more closely related to the situation in vivo than is the original in vitro positioning data, particularly at a nucleosome density corresponding to the in vivo state. Although our model is simplified, we argue that it provides a unique insight into the influence that DNA sequence can have in determining chromatin structure and could serve as a useful basis for the incorporation of other parameters.

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

PubMed

Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P

1996-10-01

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.

Comparative mapping in the Fagaceae and beyond with EST-SSRs

PubMed Central

2012-01-01

Background Genetic markers and linkage mapping are basic prerequisites for comparative genetic analyses, QTL detection and map-based cloning. A large number of mapping populations have been developed for oak, but few gene-based markers are available for constructing integrated genetic linkage maps and comparing gene order and QTL location across related species. Results We developed a set of 573 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and located 397 markers (EST-SSRs and genomic SSRs) on the 12 oak chromosomes (2n = 2x = 24) on the basis of Mendelian segregation patterns in 5 full-sib mapping pedigrees of two species: Quercus robur (pedunculate oak) and Quercus petraea (sessile oak). Consensus maps for the two species were constructed and aligned. They showed a high degree of macrosynteny between these two sympatric European oaks. We assessed the transferability of EST-SSRs to other Fagaceae genera and a subset of these markers was mapped in Castanea sativa, the European chestnut. Reasonably high levels of macrosynteny were observed between oak and chestnut. We also obtained diversity statistics for a subset of EST-SSRs, to support further population genetic analyses with gene-based markers. Finally, based on the orthologous relationships between the oak, Arabidopsis, grape, poplar, Medicago, and soybean genomes and the paralogous relationships between the 12 oak chromosomes, we propose an evolutionary scenario of the 12 oak chromosomes from the eudicot ancestral karyotype. Conclusions This study provides map locations for a large set of EST-SSRs in two oak species of recognized biological importance in natural ecosystems. This first step toward the construction of a gene-based linkage map will facilitate the assignment of future genome scaffolds to pseudo-chromosomes. This study also provides an indication of the potential utility of new gene-based markers for population genetics and comparative mapping within and beyond the Fagaceae. PMID:22931513

IN SILICO METHODOLOGIES FOR PREDICTIVE EVALUATION OF TOXICITY BASED ON INTEGRATION OF DATABASES

EPA Science Inventory

In silico methodologies for predictive evaluation of toxicity based on integration of databases

Chihae Yang1 and Ann M. Richard2, 1LeadScope, Inc. 1245 Kinnear Rd. Columbus, OH. 43212 2National Health & Environmental Effects Research Lab, U.S. EPA, Research Triangle Park, ...

Assignment of Alzheimer's presenilin-2 (PS-2) gene to 1q42.1 by fluorescence in situ hybridization.

PubMed

Takano, T; Sahara, N; Yamanouchi, Y; Mori, H

1997-01-17

Presenilin-2 (PS-2) was suggested to be localized on 1q31-42 based on linkage analysis and cDNA cloning. The final identification of PS-2 as the causal gene for early-onset familial Alzheimer's disease in Voga-German pedigrees was concluded based on the point mutation found in the candidate cDNA isolated from this familial AD. We present evidence of its physical genome mapping of PS-2 on chromosome 1q42.1 by fluorescence in situ hybridization method.

Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

PubMed Central

Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

2009-01-01

Background To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. Results An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. Conclusion We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies. PMID:19383142

Gene composer: database software for protein construct design, codon engineering, and gene synthesis.

PubMed

Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

2009-04-21

To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies.

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

PubMed Central

Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

2013-01-01

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

PubMed

Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y; Cheung, Nai-Kong V

2015-05-22

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Construction of BAC Libraries from Flow-Sorted Chromosomes.

PubMed

Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

2016-01-01

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

PubMed

Colbere-Garapin, F; Chousterman, S; Horodniceanu, F; Kourilsky, P; Garapin, A C

1979-08-01

A herpes simplex virus DNA fragment that is produced by digestion with BamHI endonuclease and carries the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been cloned in Escherichia coli. A recombinat plasmid, pFG5, has been analyzed extensively and a detailed restriction map is presented. pFG5 DNA efficiently transforms TK- mouse L cells. The TK coding sequence in the cloned fragment has been localized and a smaller recombinant plasmid, pAG0, also carrying an active TK gene, has been constructed to serve as a more convenient vector for transfer, into TK- cells, of genes previously cloned in E. coli.

Positional cloning of disease genes on chromosome 16

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

Doggett, N.; Bruening, M.; Callen, D.

1996-04-01

The project seeks to elucidate the molecular basis of an important genetic disease (Batten`s disease) by molecular cloning of the affected gene by utilizing an overlapping clone map of chromosome 16. Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressive loss of vision, seizures, and psychomoter disturbances. The Batten disease gene was genetically mapped to the chromosome region 16p 12.1 in close linkage with the genetic markers D16S299 and D16S298. Exon amplification of a cosmid containing D16S298 yielded a candidate gene that was disrupted by a 1 kb genomicmore » deletion in all patients containing the most common haplotype for the disease. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the gene as the Batten disease gene. The disease gene encodes a novel 438 amino acid membrane binding protein of unknown function.« less

Comparative Physical Mapping of the Apospory-Specific Genomic Region in Two Apomictic Grasses: Pennisetum squamulatum and Cenchrus ciliaris

PubMed Central

Goel, Shailendra; Chen, Zhenbang; Akiyama, Yukio; Conner, Joann A.; Basu, Manojit; Gualtieri, Gustavo; Hanna, Wayne W.; Ozias-Akins, Peggy

2006-01-01

In gametophytic apomicts of the aposporous type, each cell of the embryo sac is genetically identical to somatic cells of the ovule because they are products of mitosis, not of meiosis. The egg of the aposporous embryo sac follows parthenogenetic development into an embryo; therefore, uniform progeny result even from heterozygous plants, a trait that would be valuable for many crop species. Attempts to introgress apomixis from wild relatives into major crops through traditional breeding have been hindered by low or no recombination within the chromosomal region governing this trait (the apospory-specific genomic region or ASGR). The lack of recombination also has been a major obstacle to positional cloning of key genes. To further delineate and characterize the nonrecombinant ASGR, we have identified eight new ASGR-linked, AFLP-based molecular markers, only one of which showed recombination with the trait for aposporous embryo sac development. Bacterial artificial chromosome (BAC) clones identified with the ASGR-linked AFLPs or previously mapped markers, when mapped by fluorescence in situ hybridization in Pennisetum squamulatum and Cenchrus ciliaris, showed almost complete macrosynteny between the two apomictic grasses throughout the ASGR, although with an inverted order. A BAC identified with the recombinant AFLP marker mapped most proximal to the centromere of the ASGR-carrier chromosome in P. squamulatum but was not located on the ASGR-carrier chromosome in C. ciliaris. Exceptional regions where synteny was disrupted probably are nonessential for expression of the aposporous trait. The ASGR appears to be maintained as a haplotype even though its position in the genome can be variable. PMID:16547108

QTL analysis of late blight resistance in a diploid potato family of Solanum phureja x S. stenotomum.

PubMed

Costanzo, S; Simko, I; Christ, B J; Haynes, K G

2005-08-01

Field resistance to Phytophthora infestans (Mont.) de Bary, the causal agent of late blight in potatoes, has been characterized in a potato segregating family of 230 full-sib progenies derived from a cross between two hybrid Solanum phureja x S. stenotomum clones. The distribution of area under the disease progress curve values, measured in different years and locations, was consistent with the inheritance of multigenic resistance. Relatively high levels of resistance and transgressive segregations were also observed within this family. A genetic linkage map of this population was constructed with the intent of mapping quantitative trait loci (QTLs) associated with this late blight field resistance. A total of 132 clones from this family were genotyped based on 162 restriction fragment length polymorphism (RFLP) markers. The genome coverage by the map (855.2 cM) is estimated to be at least 70% and includes 112 segregating RFLP markers and two phenotypic markers, with an average distance of 7.7 cM between two markers. Two methods were employed to determine trait-marker association, the non-parametric Kruskal-Wallis test and interval mapping analysis. Three major QTLs were detected on linkage group III, V, and XI, explaining 23, 17, and 10%, respectively, of the total phenotypic variation. The present study revealed the presence of potentially new genetic loci in this diploid potato family contributing to general resistance against late blight. The identification of these QTLs represents the first step toward their introgression into cultivated tetraploid potato cultivars through marker-assisted selection.

Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal Agents

PubMed Central

Richie, Daryl L.; Thompson, Katherine V.; Studer, Christian; Prindle, Vivian C.; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A.; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Jürg; Schuierer, Sven; Hartmann, Nicole; Movva, N. Rao; Tallarico, John A.

2013-01-01

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo. PMID:23478965

Cloning and in-silico analysis of beta-1,3-xylanase from psychrophilic yeast, Glaciozyma antarctica PI12

NASA Astrophysics Data System (ADS)

Nor, Nooraisyah Mohamad; Bakar, Farah Diba Abu; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul

2015-09-01

A beta-1,3-xylanase (EC 3.2.1.32) gene from psychrophilic yeast, Glaciozyma antarctica has been identified via genome data mining. The enzyme was grouped into GH26 family based on Carbohydrate Active Enzyme (CaZY) database. The molecular weight of this protein was predicted to be 42 kDa and is expected to be soluble for expression. The presence of signal peptide suggested that this enzyme may be released extracellularly into the marine environment of the host's habitat. This supports the theory that such enzymatic activity is required for degradation of nutrients of polysaccharide origins into simpler carbohydrates outside the environment before it could be taken up inside the cell. The sequence for this protein showed very little conservation (< 30%) with other beta-1,3-xylanases from available databases. Based on the phylogenetic analysis, this protein also showed distant relationship to other xylanases from eukaryotic origin. The protein may have undergone major substitution in its gene sequence order to adapt to the cold climate. This is the first report of beta-1,3-xylanase gene isolated from a psychrophilic yeast.

Physical and genetic mapping of the dipeptidase gene DPEP1 to 16q24. 3

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

Austruy, E.; Jeanpierre, C.; Junien, C.

1993-03-01

The authors report the subregional physical and genetic mapping on chromosome 16q of a cDNA clone selected as a potential tumor/growth suppressor sequence. By DNA sequencing and RNA expression pattern, this clone was identified as part of the renal dipeptidase gene (DPEP1). Using somatic cell hybrids carrying either different human chromosomes or chromosome 16 segments, they confirm and refine the physical mapping of DPEP1 to the chromosome 16 subregion q24.3. Two RFLPs, a biallelic polymorphism detected by TaqI and a VNTR detected by BamHI, EcoRI, and BglII, are described. Using the VNTR polymorphism, DPEP1 was shown to be linked tomore » D16S7 with a maximum lod score of 5.8 at a recombination fraction of 0.03. 14 refs., 2 figs., 2 tabs.« less

Mapping protein-protein interactions using yeast two-hybrid assays.

PubMed

Mehla, Jitender; Caufield, J Harry; Uetz, Peter

2015-05-01

Yeast two-hybrid (Y2H) screens are an efficient system for mapping protein-protein interactions and whole interactomes. The screens can be performed using random libraries or collections of defined open reading frames (ORFs) called ORFeomes. This protocol describes both library and array-based Y2H screening, with an emphasis on array-based assays. Array-based Y2H is commonly used to test a number of "prey" proteins for interactions with a single "bait" (target) protein or pool of proteins. The advantage of this approach is the direct identification of interacting protein pairs without further downstream experiments: The identity of the preys is known and does not require further confirmation. In contrast, constructing and screening a random prey library requires identification of individual prey clones and systematic retesting. Retesting is typically performed in an array format. © 2015 Cold Spring Harbor Laboratory Press.

High-density linkage mapping revealed suppression of recombination at the sex determination locus in papaya.

PubMed Central

Ma, Hao; Moore, Paul H; Liu, Zhiyong; Kim, Minna S; Yu, Qingyi; Fitch, Maureen M M; Sekioka, Terry; Paterson, Andrew H; Ming, Ray

2004-01-01

A high-density genetic map of papaya (Carica papaya L.) was constructed using 54 F(2) plants derived from cultivars Kapoho and SunUp with 1501 markers, including 1498 amplified fragment length polymorphism (AFLP) markers, the papaya ringspot virus coat protein marker, morphological sex type, and fruit flesh color. These markers were mapped into 12 linkage groups at a LOD score of 5.0 and recombination frequency of 0.25. The 12 major linkage groups covered a total length of 3294.2 cM, with an average distance of 2.2 cM between adjacent markers. This map revealed severe suppression of recombination around the sex determination locus with a total of 225 markers cosegregating with sex types. The cytosine bases were highly methylated in this region on the basis of the distribution of methylation-sensitive and -insensitive markers. This high-density genetic map is essential for cloning of specific genes of interest such as the sex determination gene and for the integration of genetic and physical maps of papaya. PMID:15020433

Predicting dermal penetration for ToxCast chemicals using in silico estimates for diffusion in combination with physiologically based pharmacokinetic (PBPK) modeling.

EPA Science Inventory

Predicting dermal penetration for ToxCast chemicals using in silico estimates for diffusion in combination with physiologically based pharmacokinetic (PBPK) modeling.Evans, M.V., Sawyer, M.E., Isaacs, K.K, and Wambaugh, J.With the development of efficient high-throughput (HT) in ...

In Silico Studies of the Toxcast Chemicals Interacting with Biomolecular targets

EPA Science Inventory

Molecular docking, a structure-based in silico tool for chemical library pre-screening in drug discovery, can be used to explore the potential toxicity of environmental chemicals acting at specific biomelcular targets.

Twenty-seven nonoverlapping zinc finger cDNAs from human T cells map to nine different chromosomes with apparent clustering.

PubMed Central

Huebner, K; Druck, T; Croce, C M; Thiesen, H J

1991-01-01

cDNA clones encoding zinc finger structures were isolated by screening Molt4 and Jurkat cDNA libraries with zinc finger consensus sequences. Candidate clones were partially sequenced to verify the presence of zinc finger-encoding regions; nonoverlapping cDNA clones were chosen on the basis of sequences and genomic hybridization pattern. Zinc finger structure-encoding clones, which were designated by the term "Kox" and a number from 1 to 32 and which were apparently unique (i.e., distinct from each other and distinct from those isolated by other laboratories), were chosen for mapping in the human genome. DNAs from rodent-human somatic cell hybrids retaining defined complements of human chromosomes were analyzed for the presence of each of the Kox genes. Correlation between the presence of specific human chromosome regions and specific Kox genes established the chromosomal locations. Multiple Kox loci were mapped to 7q (Kox 18 and 25 and a locus detected by both Kox 8 cDNA and Kox 27 cDNA), 8q24 5' to the myc locus (Kox 9 and 32), 10cen----q24 (Kox 2, 15, 19, 21, 30, and 31), 12q13-qter (Kox 1 and 20), 17p13 (Kox 11 and 26), and 19q (Kox 5, 6, 10, 22, 24, and 28). Single Kox loci were mapped to 7p22 (Kox 3), 18q12 (Kox 17), 19p (Kox 13), 22q11 between IG lambda and BCR-1 (locus detected by both Kox 8 cDNA and Kox 27 cDNA), and Xp (Kox 14). Several of the Kox loci map to regions in which other zinc finger structure-encoding loci have already been localized, indicating possible zinc finger gene clusters. In addition, Kox genes at 8q24, 17p13, and 22q11--and perhaps other Kox genes--are located near recurrent chromosomal translocation breakpoints. Others, such as those on 7p and 7q, may be near regions specifically active in T cells. Images Figure 4 Figure 5 Figure 2 Figure 3 PMID:2014798

Characterization of Centromeric Histone H3 (CENH3) Variants in Cultivated and Wild Carrots (Daucus sp.)

PubMed Central

Dunemann, Frank; Schrader, Otto; Budahn, Holger; Houben, Andreas

2014-01-01

In eukaryotes, centromeres are the assembly sites for the kinetochore, a multi-protein complex to which spindle microtubules are attached at mitosis and meiosis, thereby ensuring segregation of chromosomes during cell division. They are specified by incorporation of CENH3, a centromere specific histone H3 variant which replaces canonical histone H3 in the nucleosomes of functional centromeres. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, in the presented research we aimed at the identification and cloning of functional CENH3 genes in Daucus carota and three distantly related wild species of genus Daucus varying in basic chromosome numbers. Based on mining the carrot transcriptome followed by a subsequent PCR-based cloning, homologous coding sequences for CENH3s of the four Daucus species were identified. The ORFs of the CENH3 variants were very similar, and an amino acid sequence length of 146 aa was found in three out of the four species. Comparison of Daucus CENH3 amino acid sequences with those of other plant CENH3s as well as their phylogenetic arrangement among other dicot CENH3s suggest that the identified genes are authentic CENH3 homologs. To verify the location of the CENH3 protein in the kinetochore regions of the Daucus chromosomes, a polyclonal antibody based on a peptide corresponding to the N-terminus of DcCENH3 was developed and used for anti-CENH3 immunostaining of mitotic root cells. The chromosomal location of CENH3 proteins in the centromere regions of the chromosomes could be confirmed. For genetic localization of the CENH3 gene in the carrot genome, a previously constructed linkage map for carrot was used for mapping a CENH3-specific simple sequence repeat (SSR) marker, and the CENH3 locus was mapped on the carrot chromosome 9. PMID:24887084

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase.

PubMed

Bansal, Ankita; Kar, Debasish; Murugan, Rajagopal A; Mallick, Sathi; Dutta, Mouparna; Pandey, Satya Deo; Chowdhury, Chiranjit; Ghosh, Anindya S

2015-05-01

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities. © 2015 The Authors.

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

PubMed Central

Klarenbeek, Alex; Mazouari, Khalil El; Desmyter, Aline; Blanchetot, Christophe; Hultberg, Anna; de Jonge, Natalie; Roovers, Rob C; Cambillau, Christian; Spinelli, Sylvia; Del-Favero, Jurgen; Verrips, Theo; de Haard, Hans J; Achour, Ikbel

2015-01-01

Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1β and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform. PMID:26018625

Toward a theory of multilevel evolution: long-term information integration shapes the mutational landscape and enhances evolvability.

PubMed

Hogeweg, Paulien

2012-01-01

Most of evolutionary theory has abstracted away from how information is coded in the genome and how this information is transformed into traits on which selection takes place. While in the earliest stages of biological evolution, in the RNA world, the mapping from the genotype into function was largely predefined by the physical-chemical properties of the evolving entities (RNA replicators, e.g. from sequence to folded structure and catalytic sites), in present-day organisms, the mapping itself is the result of evolution. I will review results of several in silico evolutionary studies which examine the consequences of evolving the genetic coding, and the ways this information is transformed, while adapting to prevailing environments. Such multilevel evolution leads to long-term information integration. Through genome, network, and dynamical structuring, the occurrence and/or effect of random mutations becomes nonrandom, and facilitates rapid adaptation. This is what does happen in the in silico experiments. Is it also what did happen in biological evolution? I will discuss some data that suggest that it did. In any case, these results provide us with novel search images to tackle the wealth of biological data.

STAYGREEN (CsSGR) is a candidate for the anthracnose (Colletotrichum orbiculare) resistance locus cla in Gy14 cucumber.

PubMed

Pan, Junsong; Tan, Junyi; Wang, Yuhui; Zheng, Xiangyang; Owens, Ken; Li, Dawei; Li, Yuhong; Weng, Yiqun

2018-04-21

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants. Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14 × 9930 recombinant inbred lines and 1043 F 2 individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

Towards the isolation of the idiopathic hemochromatosis disease gene

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

Chorney, M.J.; Venditti, C.P.; Harris, J.M.

1994-09-01

Despite the existence of many useful reagents which exist to aid in the positional cloning of the idiopathic hermochromatosis disease gene (HFE), the nature and precise location of this common genetic disease has remained elusive. Our group has pursued an MHC-based positional cloning approach which has centered on the precise physical definition of HLA-A variant chromosomes. Using deletion breakpoint locations in combination with genetic data derived from the Brittany founder population, we have used cDNA selection techniques to isolate new members of distinct multigene families which reside in the HFE critical region (distal to the HLA-A9 breakpoint/proximal to HLA-F). Wemore » have also initiated an independent set of cytogenetic and physical mapping studies to position the marker D6S105 with respect to the telomeric end of the class I subregion. Toward this end, we have performed double labelling FISH experiments which have allowed the localization of D6S105-containing YACs with respect to the HLA-A subregion and to the major G-bands which contain these loci. We have also derived single-copy probes, cosmids and cDNA clones from the region which have been used to create a physical map around D6S105. The combination of the cytogenetic and physical mapping data indicate that D6S105 is at least 2 Mb from HLA-A and that the distal limit of the MHC class I region may extend much further into the the euchromatic region of 6p21.3 than previously expected. A mega-YAC walk is now in progress to link the two loci. Finally, we have identified and characterized a family which is segregating a balanced inversion in phase with HFE. The breakpoint locations of this mutant chromosome may be important in the precise positioning of the HFE gene and attempts to define coding sequences in the proximity of this rearrangement are underway.« less

A transcription map of the regions surrounding the CSF1R locus on human chromosome 5q31: Candidate genes for diastrophic dysplasia

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

Clines, G.; Lovett, M.

1994-09-01

Diastrophic dysplasia (DTD) is an autosomal recessive disorder of unknown pathogenesis that is characterized by abnormal skeletal and cartilage growth. Phenotypic characteristics of the disorder include short stature, scoliosis, and deformation of the first metacarpal. The diastrophic dysplasia gene has been localized to chromosome 5q31-33, within {approximately}60 kb of the colony stimulating factor 1 receptor gene (CSF1R). We have used direct cDNA selection to build a transcription map across {approximately}250 kb surrounding and including the CSF1R locus. cDNA pools from human placenta, activated T cells, cerebellum, Hela cells, fetal brain, chondrocytes, chondrosarcomas and osteosarcomas were multiplexed in these selections. Aftermore » two rounds of selection, an analysis revealed that {approximately}70% of the selected cDNAs were contained within the contig. DNA sequencing and cosmid mapping data from a collection of 310 clones revealed the presence of three new genes in this region that show no appreciable homologies on sequence database searches, as well as cDNA clones from the CSF1R and the PDGFRB loci (another of the known genes in the region). An additional cDNA was found with 100% homology to the gene encoding human ribosomal protein L7 (RPL7). This cDNA comprised {approximately}25% of all selected clones. However, further analysis of the genomic contig revealed the presence of an RPL7 processed pseudogene in very close proximity to the CSF1R and PDGFRB genes. The selection of processed pseudogenes is one previously anticipated artifact of selection metholodolgies, but has not been previously observed. Mutational analysis of the three new genes is underway in diastrophic dysplasia families, as is derivation of full length cDNA clones and the expansion of this detailed transcription map into a larger genomic contig.« less

Family-Based Benchmarking of Copy Number Variation Detection Software.

PubMed

Nutsua, Marcel Elie; Fischer, Annegret; Nebel, Almut; Hofmann, Sylvia; Schreiber, Stefan; Krawczak, Michael; Nothnagel, Michael

2015-01-01

The analysis of structural variants, in particular of copy-number variations (CNVs), has proven valuable in unraveling the genetic basis of human diseases. Hence, a large number of algorithms have been developed for the detection of CNVs in SNP array signal intensity data. Using the European and African HapMap trio data, we undertook a comparative evaluation of six commonly used CNV detection software tools, namely Affymetrix Power Tools (APT), QuantiSNP, PennCNV, GLAD, R-gada and VEGA, and assessed their level of pair-wise prediction concordance. The tool-specific CNV prediction accuracy was assessed in silico by way of intra-familial validation. Software tools differed greatly in terms of the number and length of the CNVs predicted as well as the number of markers included in a CNV. All software tools predicted substantially more deletions than duplications. Intra-familial validation revealed consistently low levels of prediction accuracy as measured by the proportion of validated CNVs (34-60%). Moreover, up to 20% of apparent family-based validations were found to be due to chance alone. Software using Hidden Markov models (HMM) showed a trend to predict fewer CNVs than segmentation-based algorithms albeit with greater validity. PennCNV yielded the highest prediction accuracy (60.9%). Finally, the pairwise concordance of CNV prediction was found to vary widely with the software tools involved. We recommend HMM-based software, in particular PennCNV, rather than segmentation-based algorithms when validity is the primary concern of CNV detection. QuantiSNP may be used as an additional tool to detect sets of CNVs not detectable by the other tools. Our study also reemphasizes the need for laboratory-based validation, such as qPCR, of CNVs predicted in silico.

Varicella zoster virus DNA exists as two isomers.

PubMed Central

Ecker, J R; Hyman, R W

1982-01-01

Fragments of varicella zoster virus DNA produced by EcoRI endonuclease cleavage were cloned in vector pACYC 184 and those produced by HindIII cleavage were cloned in pBR322. Restriction enzyme cleavage maps established by double digestion and blot hybridization showed that varicella zoster virus DNA has a Mr of 80 +/- 3 x 10(6) and exists as a population of two isomers. Images PMID:6275385

Genetic Alterations in Familial Breast Cancer: Mapping and Cloning Genes Other Than BRCAl

DTIC Science & Technology

1997-09-01

predisposition to breast cancer in families. The gene PTEN was successfully cloned by this project, and simultaneously by others (for a different ...with germline translocations’and breast cancer for the identification of tumor suppressor genes. 14. SUBJECT TERMS Breast cancer 17. SECURITY...would limit the statistical power of linkage analysis. Therefore, we decided to integrate linkage analysis with the analysis of germline chromosomal

Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells.

PubMed Central

Cuenda, A; Alonso, G; Morrice, N; Jones, M; Meier, R; Cohen, P; Nebreda, A R

1996-01-01

Two chromatographically distinct stress-activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress- or interleukin-1-stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha-stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N-terminus. SAPKK3 is identical to the recently cloned MKK6. Images PMID:8861944

High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping

PubMed Central

2012-01-01

Background Cucurbita pepo is a member of the Cucurbitaceae family, the second- most important horticultural family in terms of economic importance after Solanaceae. The "summer squash" types, including Zucchini and Scallop, rank among the highest-valued vegetables worldwide. There are few genomic tools available for this species. The first Cucurbita transcriptome, along with a large collection of Single Nucleotide Polymorphisms (SNP), was recently generated using massive sequencing. A set of 384 SNP was selected to generate an Illumina GoldenGate assay in order to construct the first SNP-based genetic map of Cucurbita and map quantitative trait loci (QTL). Results We herein present the construction of the first SNP-based genetic map of Cucurbita pepo using a population derived from the cross of two varieties with contrasting phenotypes, representing the main cultivar groups of the species' two subspecies: Zucchini (subsp. pepo) × Scallop (subsp. ovifera). The mapping population was genotyped with 384 SNP, a set of selected EST-SNP identified in silico after massive sequencing of the transcriptomes of both parents, using the Illumina GoldenGate platform. The global success rate of the assay was higher than 85%. In total, 304 SNP were mapped, along with 11 SSR from a previous map, giving a map density of 5.56 cM/marker. This map was used to infer syntenic relationships between C. pepo and cucumber and to successfully map QTL that control plant, flowering and fruit traits that are of benefit to squash breeding. The QTL effects were validated in backcross populations. Conclusion Our results show that massive sequencing in different genotypes is an excellent tool for SNP discovery, and that the Illumina GoldenGate platform can be successfully applied to constructing genetic maps and performing QTL analysis in Cucurbita. This is the first SNP-based genetic map in the Cucurbita genus and is an invaluable new tool for biological research, especially considering that most of these markers are located in the coding regions of genes involved in different physiological processes. The platform will also be useful for future mapping and diversity studies, and will be essential in order to accelerate the process of breeding new and better-adapted squash varieties. PMID:22356647

Isolation and expression of homeobox genes from the embryonic chicken eye.

PubMed

Dhawan, R R; Schoen, T J; Beebe, D C

1997-06-11

To identify homeobox-containing genes that may play a role in the differentiation of ocular tissues. Total RNA was isolated from microdissected chicken embryo eye tissues at 3.5 days of development (embryonic day 3.5; E3.5). An "anchor-oligo-dT primer" was used for the synthesis of cDNA. Degenerate oligonucleotides designed from highly-conserved sequences in the third helix of the homeobox and the "anchor-primer" were used to amplify cDNAs by polymerase chain reaction (PCR). PCR products were cloned and sequenced. The spatial and temporal expression of selected transcripts was mapped by whole-mount in situ hybridization and northern blot analysis. After sequencing eighteen clones we identified a member of the distal-less family (dlx-3) in cDNA from presumptive neural retina and three chicken homologs of the Xenopus "anterior neural fold" (Xanf-1) in cDNA from anterior eye tissue. Dlx transcripts were mapped by in situ hybridization. Expression began at Hamburger and Hamilton stage 14 (E2.5) and was widely distributed in embryonic mesenchyme on E3 and E4. Expression increased in the retina during early development and persisted until after hatching. The one anf clone selected for further study was not detected by in situ or northern blot analysis. It is feasible to isolate homeobox cDNAs directly from microdissected embryonic tissues. Chicken dlx-3 mRNA has a wider distribution in the embryo than expected, based on the expression of the mouse homolog. Dlx-3 may play a role in establishing or maintaining the differentiation of the retina.

Development of eSSR-Markers in Setaria italica and Their Applicability in Studying Genetic Diversity, Cross-Transferability and Comparative Mapping in Millet and Non-Millet Species

PubMed Central

Misra, Gopal; Gupta, Sarika; Subramanian, Alagesan; Parida, Swarup Kumar; Chattopadhyay, Debasis; Prasad, Manoj

2013-01-01

Foxtail millet ( Setaria italica L.) is a tractable experimental model crop for studying functional genomics of millets and bioenergy grasses. But the limited availability of genomic resources, particularly expressed sequence-based genic markers is significantly impeding its genetic improvement. Considering this, we attempted to develop EST-derived-SSR (eSSR) markers and utilize them in germplasm characterization, cross-genera transferability and in silico comparative mapping. From 66,027 foxtail millet EST sequences 24,828 non-redundant ESTs were deduced, representing ~16 Mb, which revealed 534 (~2%) eSSRs in 495 SSR containing ESTs at a frequency of 1/30 kb. A total of 447 pp were successfully designed, of which 327 were mapped physically onto nine chromosomes. About 106 selected primer pairs representing the foxtail millet genome showed high-level of cross-genera amplification at an average of ~88% in eight millets and four non-millet species. Broad range of genetic diversity (0.02–0.65) obtained in constructed phylogenetic tree using 40 eSSR markers demonstrated its utility in germplasm characterizations and phylogenetics. Comparative mapping of physically mapped eSSR markers showed considerable proportion of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (~68%), maize (~61%) and rice (~42%) chromosomes. Synteny analysis of eSSRs of foxtail millet, rice, maize and sorghum suggested the nested chromosome fusion frequently observed in grass genomes. Thus, for the first time we had generated large-scale eSSR markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species. PMID:23805325

Development of eSSR-Markers in Setaria italica and Their Applicability in Studying Genetic Diversity, Cross-Transferability and Comparative Mapping in Millet and Non-Millet Species.

PubMed

Kumari, Kajal; Muthamilarasan, Mehanathan; Misra, Gopal; Gupta, Sarika; Subramanian, Alagesan; Parida, Swarup Kumar; Chattopadhyay, Debasis; Prasad, Manoj

2013-01-01

Foxtail millet (Setariaitalica L.) is a tractable experimental model crop for studying functional genomics of millets and bioenergy grasses. But the limited availability of genomic resources, particularly expressed sequence-based genic markers is significantly impeding its genetic improvement. Considering this, we attempted to develop EST-derived-SSR (eSSR) markers and utilize them in germplasm characterization, cross-genera transferability and in silico comparative mapping. From 66,027 foxtail millet EST sequences 24,828 non-redundant ESTs were deduced, representing ~16 Mb, which revealed 534 (~2%) eSSRs in 495 SSR containing ESTs at a frequency of 1/30 kb. A total of 447 pp were successfully designed, of which 327 were mapped physically onto nine chromosomes. About 106 selected primer pairs representing the foxtail millet genome showed high-level of cross-genera amplification at an average of ~88% in eight millets and four non-millet species. Broad range of genetic diversity (0.02-0.65) obtained in constructed phylogenetic tree using 40 eSSR markers demonstrated its utility in germplasm characterizations and phylogenetics. Comparative mapping of physically mapped eSSR markers showed considerable proportion of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (~68%), maize (~61%) and rice (~42%) chromosomes. Synteny analysis of eSSRs of foxtail millet, rice, maize and sorghum suggested the nested chromosome fusion frequently observed in grass genomes. Thus, for the first time we had generated large-scale eSSR markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.

Integrative transcriptome, proteome, phosphoproteome and genetic mapping reveals new aspects in a fiberless mutant of cotton

PubMed Central

Ma, Qi-Feng; Wu, Chun-Hui; Wu, Man; Pei, Wen-Feng; Li, Xing-Li; Wang, Wen-Kui; Zhang, Jinfa; Yu, Ji-Wen; Yu, Shu-Xun

2016-01-01

To investigate the molecular mechanisms of fiber initiation in cotton (Gossypium spp.), an integrated approach combining transcriptome, iTRAQ-based proteome and genetic mapping was taken to compare the ovules of the Xuzhou 142 wild type (WT) with its fuzzless-lintless (fl) mutant at −3 and 0 day post-anthesis. A total of 1,953 mRNAs, 187 proteins, and 131 phosphoproteins were differentially expressed (DE) between WT and fl, and the levels of transcripts and their encoded proteins and phosphoproteins were highly congruent. A functional analysis suggested that the abundance of proteins were mainly involved in amino sugar, nucleotide sugar and fatty acid metabolism, one carbon pool for folate metabolism and flavonoid biosynthesis. qRT-PCR, Western blotting, and enzymatic assays were performed to confirm the regulation of these transcripts and proteins. A molecular mapping located the lintless gene li3 in the fl mutant on chromosome 26 for the first time. A further in-silico physical mapping of DE genes with sequence variations between fl and WT identified one and four candidate genes in the li3 and n2 regions, respectively. Taken together, the transcript abundance, phosphorylation status of proteins at the fiber initiation stage and candidate genes have provided insights into regulatory processes underlying cotton fiber initiation. PMID:27075604

Development of a quantitative pachytene chromosome map and its unification with somatic chromosome and linkage maps of rice (Oryza sativa L.).

PubMed

Ohmido, Nobuko; Iwata, Aiko; Kato, Seiji; Wako, Toshiyuki; Fukui, Kiichi

2018-01-01

A quantitative pachytene chromosome map of rice (Oryza sativa L.) was developed using imaging methods. The map depicts not only distribution patterns of chromomeres specific to pachytene chromosomes, but also the higher order information of chromosomal structures, such as heterochromatin (condensed regions), euchromatin (decondensed regions), the primary constrictions (centromeres), and the secondary constriction (nucleolar organizing regions, NOR). These features were image analyzed and quantitatively mapped onto the map by Chromosome Image Analyzing System ver. 4.0 (CHIAS IV). Correlation between H3K9me2, an epigenetic marker and formation and/or maintenance of heterochromatin, thus was, clearly visualized. Then the pachytene chromosome map was unified with the existing somatic chromosome and linkage maps by physically mapping common DNA markers among them, such as a rice A genome specific tandem repeat sequence (TrsA), 5S and 45S ribosomal RNA genes, five bacterial artificial chromosome (BAC) clones, four P1 bacteriophage artificial chromosome (PAC) clones using multicolor fluorescence in situ hybridization (FISH). Detailed comparison between the locations of the DNA probes on the pachytene chromosomes using multicolor FISH, and the linkage map enabled determination of the chromosome number and short/long arms of individual pachytene chromosomes using the chromosome number and arm assignment designated for the linkage map. As a result, the quantitative pachytene chromosome map was unified with two other major rice chromosome maps representing somatic prometaphase chromosomes and genetic linkages. In conclusion, the unification of the three rice maps serves as an indispensable basic information, not only for an in-depth comparison between genetic and chromosomal data, but also for practical breeding programs.

Epigenomics

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Chromosomes

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Transcriptome

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Construction of a high-density genetic map for grape using specific length amplified fragment (SLAF) sequencing

PubMed Central

Guo, Yinshan; Xing, Huiyang; Zhao, Yuhui; Liu, Zhendong; Li, Kun; Guo, Xiuwu

2017-01-01

Genetic maps are important tools in plant genomics and breeding. We report a large-scale discovery of single nucleotide polymorphisms (SNPs) using the specific length amplified fragment sequencing (SLAF-seq) technique for the construction of high-density genetic maps for two elite wine grape cultivars, ‘Chardonnay’ and ‘Beibinghong’, and their 130 F1 plants. A total of 372.53 M paired-end reads were obtained after preprocessing. The average sequencing depth was 33.81 for ‘Chardonnay’ (the female parent), 48.20 for ‘Beibinghong’ (the male parent), and 12.66 for the F1 offspring. We detected 202,349 high-quality SLAFs of which 144,972 were polymorphic; 10,042 SNPs were used to construct a genetic map that spanned 1,969.95 cM, with an average genetic distance of 0.23 cM between adjacent markers. This genetic map contains the largest molecular marker number of the grape maps so far reported. We thus demonstrate that SLAF-seq is a promising strategy for the construction of high-density genetic maps; the map that we report here is a good potential resource for QTL mapping of genes linked to major economic and agronomic traits, map-based cloning, and marker-assisted selection of grape. PMID:28746364

High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus.

PubMed

Muchero, Wellington; Guo, Jianjun; DiFazio, Stephen P; Chen, Jin-Gui; Ranjan, Priya; Slavov, Gancho T; Gunter, Lee E; Jawdy, Sara; Bryan, Anthony C; Sykes, Robert; Ziebell, Angela; Klápště, Jaroslav; Porth, Ilga; Skyba, Oleksandr; Unda, Faride; El-Kassaby, Yousry A; Douglas, Carl J; Mansfield, Shawn D; Martin, Joel; Schackwitz, Wendy; Evans, Luke M; Czarnecki, Olaf; Tuskan, Gerald A

2015-01-23

QTL cloning for the discovery of genes underlying polygenic traits has historically been cumbersome in long-lived perennial plants like Populus. Linkage disequilibrium-based association mapping has been proposed as a cloning tool, and recent advances in high-throughput genotyping and whole-genome resequencing enable marker saturation to levels sufficient for association mapping with no a priori candidate gene selection. Here, multiyear and multienvironment evaluation of cell wall phenotypes was conducted in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree and two partially overlapping populations of unrelated P. trichocarpa genotypes using pyrolysis molecular beam mass spectrometry, saccharification, and/ or traditional wet chemistry. QTL mapping was conducted using a high-density genetic map with 3,568 SNP markers. As a fine-mapping approach, chromosome-wide association mapping targeting a QTL hot-spot on linkage group XIV was performed in the two P. trichocarpa populations. Both populations were genotyped using the 34 K Populus Infinium SNP array and whole-genome resequencing of one of the populations facilitated marker-saturation of candidate intervals for gene identification. Five QTLs ranging in size from 0.6 to 1.8 Mb were mapped on linkage group XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6-carbon sugars using the mapping pedigree. Six candidate loci exhibiting significant associations with phenotypes were identified within QTL intervals. These associations were reproducible across multiple environments, two independent genotyping platforms, and different plant growth stages. cDNA sequencing for allelic variants of three of the six loci identified polymorphisms leading to variable length poly glutamine (PolyQ) stretch in a transcription factor annotated as an ANGUSTIFOLIA C-terminus Binding Protein (CtBP) and premature stop codons in a KANADI transcription factor as well as a protein kinase. Results from protoplast transient expression assays suggested that each of the polymorphisms conferred allelic differences in the activation of cellulose, hemicelluloses, and lignin pathway marker genes. This study illustrates the utility of complementary QTL and association mapping as tools for gene discovery with no a priori candidate gene selection. This proof of concept in a perennial organism opens up opportunities for discovery of novel genetic determinants of economically important but complex traits in plants.

Identification and mapping of ts (tender spines), a gene involved in soft spine development in Cucumis sativus.

PubMed

Guo, Chunli; Yang, Xuqin; Wang, Yunli; Nie, Jingtao; Yang, Yi; Sun, Jingxian; Du, Hui; Zhu, Wenying; Pan, Jian; Chen, Yue; Lv, Duo; He, Huanle; Lian, Hongli; Pan, Junsong; Cai, Run

2018-01-01

Using map-based cloning of ts gene, we identified a new sort of gene involved in the initiation of multicellular tender spine in cucumber. The cucumber (Cucumis sativus L.) fruit contains spines on the surface, which is an extremely valuable quality trait affecting the selection of customers. In this study, we elaborated cucumber line NC072 with wild type (WT) hard fruit spines and its spontaneous mutant NC073, possessing tender and soft spines on fruits. The mutant trait was named as tender spines (ts), which is controlled by a single recessive nuclear gene. We identified the gene ts by map-based cloning with an F 2 segregating population of 721 individuals generated from NC073 and WT line SA419-2. It was located between two markers Indel6239679 and Indel6349344, 109.7 kb physical distance on chromosome 1 containing fifteen putative genes. With sequencing and quantitative reverse transcription-polymerase chain reaction analysis, the Csa1G056960 gene was considered as the most possible candidate gene of ts. In the mutant, Csa1G056960 has a nucleotide change in the 5' splicing site of the second intron, which causes different splicing to delete the second exon, resulting in a N-terminal deletion in the predicted amino acid sequence. The gene encodes a C-type lectin receptor-like tyrosine-protein kinase which would play an important role in the formation of cucumber fruit. This is firstly reported of a receptor kinase gene regulating the development of multicellular spines/trichomes in plants. The ts allele could accelerate the molecular breeding of cucumber soft spines.

Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

DTIC Science & Technology

2012-07-01

Description HRAS Homo sapiens v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), transcript 1 CDC25C Homo sapiens cell division cycle 25...homolog C (CDC25C), transcript variant 1 MYC Homo sapiens v-myc myeloctomatosis viral oncogene homolog (avian) (MYC) MAP3K7 Homo sapiens mitogen...activated protein kinase kinase kinase 7 (MAP3K7) MAP3K8 Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8) SF3B1 Homo sapiens splicing

Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

DTIC Science & Technology

2013-01-01

Description HRAS Homo sapiens v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), transcript 1 CDC25C Homo sapiens cell division cycle 25...homolog C (CDC25C), transcript variant 1 MYC Homo sapiens v-myc myeloctomatosis viral oncogene homolog (avian) (MYC) MAP3K7 Homo sapiens mitogen...activated protein kinase kinase kinase 7 (MAP3K7) MAP3K8 Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8) SF3B1 Homo sapiens

Structural insights into pharmacophore-assisted in silico identification of protein-protein interaction inhibitors for inhibition of human toll-like receptor 4 - myeloid differentiation factor-2 (hTLR4-MD-2) complex.

PubMed

Mishra, Vinita; Pathak, Chandramani

2018-05-29

Toll-like receptor 4 (TLR4) is a member of Toll-Like Receptors (TLRs) family that serves as a receptor for bacterial lipopolysaccharide (LPS). TLR4 alone cannot recognize LPS without aid of co-receptor myeloid differentiation factor-2 (MD-2). Binding of LPS with TLR4 forms a LPS-TLR4-MD-2 complex and directs downstream signaling for activation of immune response, inflammation and NF-κB activation. Activation of TLR4 signaling is associated with various pathophysiological consequences. Therefore, targeting protein-protein interaction (PPI) in TLR4-MD-2 complex formation could be an attractive therapeutic approach for targeting inflammatory disorders. The aim of present study was directed to identify small molecule PPI inhibitors (SMPPIIs) using pharmacophore mapping-based approach of computational drug discovery. Here, we had retrieved the information about the hot spot residues and their pharmacophoric features at both primary (TLR4-MD-2) and dimerization (MD-2-TLR4*) protein-protein interaction interfaces in TLR4-MD-2 homo-dimer complex using in silico methods. Promising candidates were identified after virtual screening, which may restrict TLR4-MD-2 protein-protein interaction. In silico off-target profiling over the virtually screened compounds revealed other possible molecular targets. Two of the virtually screened compounds (C11 and C15) were predicted to have an inhibitory concentration in μM range after HYDE assessment. Molecular dynamics simulation study performed for these two compounds in complex with target protein confirms the stability of the complex. After virtual high throughput screening we found selective hTLR4-MD-2 inhibitors, which may have therapeutic potential to target chronic inflammatory diseases.

In-silico mining, type and frequency analysis of genic microsatellites of finger millet (Eleusine coracana (L.) Gaertn.): a comparative genomic analysis of NBS-LRR regions of finger millet with rice.

PubMed

Kalyana Babu, B; Pandey, Dinesh; Agrawal, P K; Sood, Salej; Kumar, Anil

2014-05-01

In recent years, the increased availability of the DNA sequences has given the possibility to develop and explore the expressed sequence tags (ESTs) derived SSR markers. In the present study, a total of 1956 ESTs of finger millet were used to find the microsatellite type, distribution, frequency and developed a total of 545 primer pairs from the ESTs of finger millet. Thirty-two EST sequences had more than two microsatellites and 1357 sequences did not have any SSR repeats. The most frequent type of repeats was trimeric motif, however the second place was occupied by dimeric motif followed by tetra-, hexa- and penta repeat motifs. The most common dimer repeat motif was GA and in case of trimeric SSRs, it was CGG. The EST sequences of NBS-LRR region of finger millet and rice showed higher synteny and were found on nearly same positions on the rice chromosome map. A total of eight, out of 15 EST based SSR primers were polymorphic among the selected resistant and susceptible finger millet genotypes. The primer FMBLEST5 could able to differentiate them into resistant and susceptible genotypes. The alleles specific to the resistant and susceptible genotypes were sequenced using the ABI 3130XL genetic analyzer and found similarity to NBS-LRR regions of rice and finger millet and contained the characteristic kinase-2 and kinase 3a motifs of plant R-genes belonged to NBS-LRR region. The In-silico and comparative analysis showed that the genes responsible for blast resistance can be identified, mapped and further introgressed through molecular breeding approaches for enhancing the blast resistance in finger millet.

A Saturated Genetic Linkage Map of Autotetraploid Alfalfa (Medicago sativa L.) Developed Using Genotyping-by-Sequencing Is Highly Syntenous with the Medicago truncatula Genome

PubMed Central

Li, Xuehui; Wei, Yanling; Acharya, Ananta; Jiang, Qingzhen; Kang, Junmei; Brummer, E. Charles

2014-01-01

A genetic linkage map is a valuable tool for quantitative trait locus mapping, map-based gene cloning, comparative mapping, and whole-genome assembly. Alfalfa, one of the most important forage crops in the world, is autotetraploid, allogamous, and highly heterozygous, characteristics that have impeded the construction of a high-density linkage map using traditional genetic marker systems. Using genotyping-by-sequencing (GBS), we constructed low-cost, reasonably high-density linkage maps for both maternal and paternal parental genomes of an autotetraploid alfalfa F1 population. The resulting maps contain 3591 single-nucleotide polymorphism markers on 64 linkage groups across both parents, with an average density of one marker per 1.5 and 1.0 cM for the maternal and paternal haplotype maps, respectively. Chromosome assignments were made based on homology of markers to the M. truncatula genome. Four linkage groups representing the four haplotypes of each alfalfa chromosome were assigned to each of the eight Medicago chromosomes in both the maternal and paternal parents. The alfalfa linkage groups were highly syntenous with M. truncatula, and clearly identified the known translocation between Chromosomes 4 and 8. In addition, a small inversion on Chromosome 1 was identified between M. truncatula and M. sativa. GBS enabled us to develop a saturated linkage map for alfalfa that greatly improved genome coverage relative to previous maps and that will facilitate investigation of genome structure. GBS could be used in breeding populations to accelerate molecular breeding in alfalfa. PMID:25147192

A saturated genetic linkage map of autotetraploid alfalfa (Medicago sativa L.) developed using genotyping-by-sequencing is highly syntenous with the Medicago truncatula genome.

PubMed

Li, Xuehui; Wei, Yanling; Acharya, Ananta; Jiang, Qingzhen; Kang, Junmei; Brummer, E Charles

2014-08-21

A genetic linkage map is a valuable tool for quantitative trait locus mapping, map-based gene cloning, comparative mapping, and whole-genome assembly. Alfalfa, one of the most important forage crops in the world, is autotetraploid, allogamous, and highly heterozygous, characteristics that have impeded the construction of a high-density linkage map using traditional genetic marker systems. Using genotyping-by-sequencing (GBS), we constructed low-cost, reasonably high-density linkage maps for both maternal and paternal parental genomes of an autotetraploid alfalfa F1 population. The resulting maps contain 3591 single-nucleotide polymorphism markers on 64 linkage groups across both parents, with an average density of one marker per 1.5 and 1.0 cM for the maternal and paternal haplotype maps, respectively. Chromosome assignments were made based on homology of markers to the M. truncatula genome. Four linkage groups representing the four haplotypes of each alfalfa chromosome were assigned to each of the eight Medicago chromosomes in both the maternal and paternal parents. The alfalfa linkage groups were highly syntenous with M. truncatula, and clearly identified the known translocation between Chromosomes 4 and 8. In addition, a small inversion on Chromosome 1 was identified between M. truncatula and M. sativa. GBS enabled us to develop a saturated linkage map for alfalfa that greatly improved genome coverage relative to previous maps and that will facilitate investigation of genome structure. GBS could be used in breeding populations to accelerate molecular breeding in alfalfa. Copyright © 2014 Li et al.

Biological Pathways

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

PubMed

Siow, Hwee-Leng; Lim, Theam Soon; Gan, Chee-Yuen

2017-01-01

The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04μmol, respectively, whereas CSP6 was about 0.10 and 0.15μmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Faster experimental validation of microRNA targets using cold fusion cloning and a dual firefly-Renilla luciferase reporter assay.

PubMed

Alvarez, M Lucrecia

2014-01-01

Different target prediction algorithms have been developed to provide a list of candidate target genes for a given animal microRNAs (miRNAs). However, these computational approaches provide both false-positive and false-negative predictions. Therefore, the target genes of a specific miRNA identified in silico should be experimentally validated. In this chapter, we describe a step-by-step protocol for the experimental validation of a direct miRNA target using a faster Dual Firefly-Renilla Luciferase Reporter Assay. We describe how to construct reporter plasmids using the simple, fast, and highly efficient cold fusion cloning technology, which does not require ligase, phosphatase, or restriction enzymes. In addition, we provide a protocol for co-transfection of reporter plasmids with either miRNA mimics or miRNA inhibitors in human embryonic kidney 293 (HEK293) cells, as well as a description on how to measure Firefly and Renilla luciferase activity using the Dual-Glo Luciferase Assay kit. As an example of the use of this technology, we will validate glucose-6-phosphate dehydrogenase (G6PD) as a direct target of miR-1207-5p.

Discovery and molecular characterization of a new cryptovirus dsRNA genome from Japanese persimmon through conventional cloning and high-throughput sequencing.

PubMed

Morelli, M; Chiumenti, M; De Stradis, A; La Notte, P; Minafra, A

2015-02-01

Through the application of next generation sequencing, in synergy with conventional cloning of DOP-PCR fragments, two double-stranded RNA (dsRNA) molecules of about 1.5 kbp in size were isolated from leaf tissue of a Japanese persimmon (accession SSPI) from Apulia (southern Italy) showing veinlets necrosis. High-throughput sequencing allowed whole genome sequence assembly, yielding a 1,577 and a 1,491 bp contigs identified as dsRNA-1 and dsRNA-2 of a previously undescribed virus, provisionally named as Persimmon cryptic virus (PeCV). In silico analysis showed that both dsRNA fragments were monocistronic and comprised the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP) genes, respectively. Phylogenetic reconstruction revealed a close relationship of these dsRNAs with those of cryptoviruses described in woody and herbaceous hosts, recently gathered in genus Deltapartitivirus. Virus-specific primers for RT-PCR, designed in the CP cistron, detected viral RNAs also in symptomless persimmon trees sampled from the same geographical area of SSPI, thus proving that PeCV infection may be fairly common and presumably latent.

Chromosomal localization of seven PAX genes and cloning of a novel family member, PAX-9.

PubMed

Stapleton, P; Weith, A; Urbánek, P; Kozmik, Z; Busslinger, M

1993-04-01

In the human paired box-containing (PAX) gene family, only two members, PAX-3 and PAX-6, which are associated with Waardenburg's syndrome and aniridia, respectively have been mapped to human chromosomes. We have now isolated cosmids for six additional human PAX genes (PAX-1,-2,-5,-7,-8,-9) and a polymerase chain reaction fragment for PAX-4. PAX-9 is a novel family member which is closely related in its paired domain to PAX-1. The chromosomal location of all cloned PAX genes was determined by analysis of somatic cell hybrids and (except PAX-4) by fluorescence in situ hybridization to metaphase chromosomes. PAX-1 and PAX-7 map to chromosomal regions containing previously assigned disease loci.

A consensus linkage map of lentil based on DArT markers from three RIL mapping populations.

PubMed

Ates, Duygu; Aldemir, Secil; Alsaleh, Ahmad; Erdogmus, Semih; Nemli, Seda; Kahriman, Abdullah; Ozkan, Hakan; Vandenberg, Albert; Tanyolac, Bahattin

2018-01-01

Lentil (Lens culinaris ssp. culinaris Medikus) is a diploid (2n = 2x = 14), self-pollinating grain legume with a haploid genome size of about 4 Gbp and is grown throughout the world with current annual production of 4.9 million tonnes. A consensus map of lentil (Lens culinaris ssp. culinaris Medikus) was constructed using three different lentils recombinant inbred line (RIL) populations, including "CDC Redberry" x "ILL7502" (LR8), "ILL8006" x "CDC Milestone" (LR11) and "PI320937" x "Eston" (LR39). The lentil consensus map was composed of 9,793 DArT markers, covered a total of 977.47 cM with an average distance of 0.10 cM between adjacent markers and constructed 7 linkage groups representing 7 chromosomes of the lentil genome. The consensus map had no gap larger than 12.67 cM and only 5 gaps were found to be between 12.67 cM and 6.0 cM (on LG3 and LG4). The localization of the SNP markers on the lentil consensus map were in general consistent with their localization on the three individual genetic linkage maps and the lentil consensus map has longer map length, higher marker density and shorter average distance between the adjacent markers compared to the component linkage maps. This high-density consensus map could provide insight into the lentil genome. The consensus map could also help to construct a physical map using a Bacterial Artificial Chromosome library and map based cloning studies. Sequence information of DArT may help localization of orientation scaffolds from Next Generation Sequencing data.

A consensus linkage map of lentil based on DArT markers from three RIL mapping populations

PubMed Central

Ates, Duygu; Aldemir, Secil; Alsaleh, Ahmad; Erdogmus, Semih; Nemli, Seda; Kahriman, Abdullah; Ozkan, Hakan; Vandenberg, Albert

2018-01-01

Background Lentil (Lens culinaris ssp. culinaris Medikus) is a diploid (2n = 2x = 14), self-pollinating grain legume with a haploid genome size of about 4 Gbp and is grown throughout the world with current annual production of 4.9 million tonnes. Materials and methods A consensus map of lentil (Lens culinaris ssp. culinaris Medikus) was constructed using three different lentils recombinant inbred line (RIL) populations, including “CDC Redberry” x “ILL7502” (LR8), “ILL8006” x “CDC Milestone” (LR11) and “PI320937” x “Eston” (LR39). Results The lentil consensus map was composed of 9,793 DArT markers, covered a total of 977.47 cM with an average distance of 0.10 cM between adjacent markers and constructed 7 linkage groups representing 7 chromosomes of the lentil genome. The consensus map had no gap larger than 12.67 cM and only 5 gaps were found to be between 12.67 cM and 6.0 cM (on LG3 and LG4). The localization of the SNP markers on the lentil consensus map were in general consistent with their localization on the three individual genetic linkage maps and the lentil consensus map has longer map length, higher marker density and shorter average distance between the adjacent markers compared to the component linkage maps. Conclusion This high-density consensus map could provide insight into the lentil genome. The consensus map could also help to construct a physical map using a Bacterial Artificial Chromosome library and map based cloning studies. Sequence information of DArT may help localization of orientation scaffolds from Next Generation Sequencing data. PMID:29351563

InSilico DB genomic datasets hub: an efficient starting point for analyzing genome-wide studies in GenePattern, Integrative Genomics Viewer, and R/Bioconductor.

PubMed

Coletta, Alain; Molter, Colin; Duqué, Robin; Steenhoff, David; Taminau, Jonatan; de Schaetzen, Virginie; Meganck, Stijn; Lazar, Cosmin; Venet, David; Detours, Vincent; Nowé, Ann; Bersini, Hugues; Weiss Solís, David Y

2012-11-18

Genomics datasets are increasingly useful for gaining biomedical insights, with adoption in the clinic underway. However, multiple hurdles related to data management stand in the way of their efficient large-scale utilization. The solution proposed is a web-based data storage hub. Having clear focus, flexibility and adaptability, InSilico DB seamlessly connects genomics dataset repositories to state-of-the-art and free GUI and command-line data analysis tools. The InSilico DB platform is a powerful collaborative environment, with advanced capabilities for biocuration, dataset sharing, and dataset subsetting and combination. InSilico DB is available from https://insilicodb.org.

Identification of alsterpaullone as a novel small molecule inhibitor to target group 3 medulloblastoma.

PubMed

Faria, Claudia C; Agnihotri, Sameer; Mack, Stephen C; Golbourn, Brian J; Diaz, Roberto J; Olsen, Samantha; Bryant, Melissa; Bebenek, Matthew; Wang, Xin; Bertrand, Kelsey C; Kushida, Michelle; Head, Renee; Clark, Ian; Dirks, Peter; Smith, Christian A; Taylor, Michael D; Rutka, James T

2015-08-28

Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma.

Porcine MAP3K5 analysis: molecular cloning, characterization, tissue expression pattern, and copy number variations associated with residual feed intake.

PubMed

Pu, L; Zhang, L C; Zhang, J S; Song, X; Wang, L G; Liang, J; Zhang, Y B; Liu, X; Yan, H; Zhang, T; Yue, J W; Li, N; Wu, Q Q; Wang, L X

2016-08-12

Mitogen-activated protein kinase kinase kinase 5 (MAP3K5) is essential for apoptosis, proliferation, differentiation, and immune responses, and is a candidate marker for residual feed intake (RFI) in pig. We cloned the full-length cDNA sequence of porcine MAP3K5 by rapid-amplification of cDNA ends. The 5451-bp gene contains a 5'-untranslated region (UTR) (718 bp), a coding region (3738 bp), and a 3'-UTR (995 bp), and encodes a peptide of 1245 amino acids, which shares 97, 99, 97, 93, 91, and 84% sequence identity with cattle, sheep, human, mouse, chicken, and zebrafish MAP3K5, respectively. The deduced MAP3K5 protein sequence contains two conserved domains: a DUF4071 domain and a protein kinase domain. Phylogenetic analysis showed that porcine MAP3K5 forms a separate branch to vicugna and camel MAP3K5. Tissue expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR) revealed that MAP3K5 was expressed in the heart, liver, spleen, lung, kidney, muscle, fat, pancrea, ileum, and stomach tissues. Copy number variation was detected for porcine MAP3K5 and validated by qRT-PCR. Furthermore, a significant increase in average copy number was detected in the low RFI group when compared to the high RFI group in a Duroc pig population. These results provide useful information regarding the influence of MAP3K5 on RFI in pigs.

Discovery of agents that eradicate leukemia stem cells using an in silico screen of public gene expression data

PubMed Central

Hassane, Duane C.; Guzman, Monica L.; Corbett, Cheryl; Li, Xiaojie; Abboud, Ramzi; Young, Fay; Liesveld, Jane L.; Carroll, Martin

2008-01-01

Increasing evidence indicates that malignant stem cells are important for the pathogenesis of acute myelogenous leukemia (AML) and represent a reservoir of cells that drive the development of AML and relapse. Therefore, new treatment regimens are necessary to prevent relapse and improve therapeutic outcomes. Previous studies have shown that the sesquiterpene lactone, parthenolide (PTL), ablates bulk, progenitor, and stem AML cells while causing no appreciable toxicity to normal hematopoietic cells. Thus, PTL must evoke cellular responses capable of mediating AML selective cell death. Given recent advances in chemical genomics such as gene expression-based high-throughput screening (GE-HTS) and the Connectivity Map, we hypothesized that the gene expression signature resulting from treatment of primary AML with PTL could be used to search for similar signatures in publicly available gene expression profiles deposited into the Gene Expression Omnibus (GEO). We therefore devised a broad in silico screen of the GEO database using the PTL gene expression signature as a template and discovered 2 new agents, celastrol and 4-hydroxy-2-nonenal, that effectively eradicate AML at the bulk, progenitor, and stem cell level. These findings suggest the use of multicenter collections of high-throughput data to facilitate discovery of leukemia drugs and drug targets. PMID:18305216

Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32.

PubMed

Kumar, Vikram; Damodharan, S; Pandaranayaka, Eswari P J; Madathiparambil, Madanan G; Tennyson, Jebasingh

2016-01-01

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6.

Integrating In Silico Resources to Map a Signaling Network

PubMed Central

Liu, Hanqing; Beck, Tim N.; Golemis, Erica A.; Serebriiskii, Ilya G.

2013-01-01

The abundance of publicly available life science databases offer a wealth of information that can support interpretation of experimentally derived data and greatly enhance hypothesis generation. Protein interaction and functional networks are not simply new renditions of existing data: they provide the opportunity to gain insights into the specific physical and functional role a protein plays as part of the biological system. In this chapter, we describe different in silico tools that can quickly and conveniently retrieve data from existing data repositories and discuss how the available tools are best utilized for different purposes. While emphasizing protein-protein interaction databases (e.g., BioGrid and IntAct), we also introduce metasearch platforms such as STRING and GeneMANIA, pathway databases (e.g., BioCarta and Pathway Commons), text mining approaches (e.g., PubMed and Chilibot), and resources for drug-protein interactions, genetic information for model organisms and gene expression information based on microarray data mining. Furthermore, we provide a simple step-by-step protocol to building customized protein-protein interaction networks in Cytoscape, a powerful network assembly and visualization program, integrating data retrieved from these various databases. As we illustrate, generation of composite interaction networks enables investigators to extract significantly more information about a given biological system than utilization of a single database or sole reliance on primary literature. PMID:24233784

In Silico, Experimental, Mechanistic Model for Extended-Release Felodipine Disposition Exhibiting Complex Absorption and a Highly Variable Food Interaction

PubMed Central

Kim, Sean H. J.; Jackson, Andre J.; Hunt, C. Anthony

2014-01-01

The objective of this study was to develop and explore new, in silico experimental methods for deciphering complex, highly variable absorption and food interaction pharmacokinetics observed for a modified-release drug product. Toward that aim, we constructed an executable software analog of study participants to whom product was administered orally. The analog is an object- and agent-oriented, discrete event system, which consists of grid spaces and event mechanisms that map abstractly to different physiological features and processes. Analog mechanisms were made sufficiently complicated to achieve prespecified similarity criteria. An equation-based gastrointestinal transit model with nonlinear mixed effects analysis provided a standard for comparison. Subject-specific parameterizations enabled each executed analog’s plasma profile to mimic features of the corresponding six individual pairs of subject plasma profiles. All achieved prespecified, quantitative similarity criteria, and outperformed the gastrointestinal transit model estimations. We observed important subject-specific interactions within the simulation and mechanistic differences between the two models. We hypothesize that mechanisms, events, and their causes occurring during simulations had counterparts within the food interaction study: they are working, evolvable, concrete theories of dynamic interactions occurring within individual subjects. The approach presented provides new, experimental strategies for unraveling the mechanistic basis of complex pharmacological interactions and observed variability. PMID:25268237

Potential Value of Major Antigenic Protein 2 for Serological Diagnosis of Heartwater and Related Ehrlichial Infections

PubMed Central

Bowie, Michael V.; Reddy, G. Roman; Semu, Shalt M.; Mahan, Suman M.; Barbet, Anthony F.

1999-01-01

Cowdria ruminantium is the etiologic agent of heartwater, a disease causing major economic loss in ruminants in sub-Saharan Africa and the Caribbean. Development of a serodiagnostic test is essential for determining the carrier status of animals from regions where heartwater is endemic, but most available tests give false-positive reactions with sera against related Erhlichia species. Current approaches rely on molecular methods to define proteins and epitopes that may allow specific diagnosis. Two major antigenic proteins (MAPs), MAP1 and MAP2, have been examined for their use as antigens in the serodiagnosis of heartwater. The objectives of this study were (i) to determine if MAP2 is conserved among five geographically divergent strains of C. ruminantium and (ii) to determine if MAP2 homologs are present in Ehrlichia canis, the causative agent of canine ehrlichiosis, and Ehrlichia chaffeensis, the organism responsible for human monocytic ehrlichiosis. These two agents are closely related to C. ruminantium. The map2 gene from four strains of C. ruminantium was cloned, sequenced, and compared with the previously reported map2 gene from the Crystal Springs strain. Only 10 nucleic acid differences between the strains were identified, and they translate to only 3 amino acid changes, indicating that MAP2 is highly conserved. Genes encoding MAP2 homologs from E. canis and E. chaffeensis also were cloned and sequenced. Amino acid analysis of MAP2 homologs of E. chaffeensis and E. canis with MAP2 of C. ruminantium revealed 83.4 and 84.4% identities, respectively. Further analysis of MAP2 and its homologs revealed that the whole protein lacks specificity for heartwater diagnosis. The development of epitope-specific assays using this sequence information may produce diagnostic tests suitable for C. ruminantium and also other related rickettsiae. PMID:10066656

Construction of a high-density genetic map and lint percentage and cottonseed nutrient trait QTL identification in upland cotton (Gossypium hirsutum L.).

PubMed

Liu, Dexin; Liu, Fang; Shan, Xiaoru; Zhang, Jian; Tang, Shiyi; Fang, Xiaomei; Liu, Xueying; Wang, Wenwen; Tan, Zhaoyun; Teng, Zhonghua; Zhang, Zhengsheng; Liu, Dajun

2015-10-01

Upland cotton plays a critical role not only in the textile industry, but also in the production of important secondary metabolites, such as oil and proteins. Construction of a high-density linkage map and identifying yield and seed trait quantitative trail loci (QTL) are prerequisites for molecular marker-assisted selective breeding projects. Here, we update a high-density upland cotton genetic map from recombinant inbred lines. A total of 25,313 SSR primer pairs were screened for polymorphism between Yumian 1 and T586, and 1712 SSR primer pairs were used to genotype the mapping population and construct a map. An additional 1166 loci have been added to our previously published map with 509 SSR markers. The updated genetic map spans a total recombinant length of 3338.2 cM and contains 1675 SSR loci and nine morphological markers, with an average interval of 1.98 cM between adjacent markers. Green lint (Lg) mapped on chromosome 15 in a previous report is mapped in an interval of 2.6 cM on chromosome 21. Based on the map and phenotypic data from multiple environments, 79 lint percentage and seed nutrient trait QTL are detected. These include 8 lint percentage, 13 crude protein, 15 crude oil, 8 linoleic, 10 oleic, 13 palmitic, and 12 stearic acid content QTL. They explain 3.5-62.7 % of the phenotypic variation observed. Four morphological markers identified have a major impact on lint percentage and cottonseed nutrients traits. In this study, our genetic map provides new sights into the tetraploid cotton genome. Furthermore, the stable QTL and morphological markers could be used for fine-mapping and map-based cloning.

Large-scale mapping of mutations affecting zebrafish development.

PubMed

Geisler, Robert; Rauch, Gerd-Jörg; Geiger-Rudolph, Silke; Albrecht, Andrea; van Bebber, Frauke; Berger, Andrea; Busch-Nentwich, Elisabeth; Dahm, Ralf; Dekens, Marcus P S; Dooley, Christopher; Elli, Alexandra F; Gehring, Ines; Geiger, Horst; Geisler, Maria; Glaser, Stefanie; Holley, Scott; Huber, Matthias; Kerr, Andy; Kirn, Anette; Knirsch, Martina; Konantz, Martina; Küchler, Axel M; Maderspacher, Florian; Neuhauss, Stephan C; Nicolson, Teresa; Ober, Elke A; Praeg, Elke; Ray, Russell; Rentzsch, Brit; Rick, Jens M; Rief, Eva; Schauerte, Heike E; Schepp, Carsten P; Schönberger, Ulrike; Schonthaler, Helia B; Seiler, Christoph; Sidi, Samuel; Söllner, Christian; Wehner, Anja; Weiler, Christian; Nüsslein-Volhard, Christiane

2007-01-09

Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.

In silico mining of putative microsatellite markers from whole genome sequence of water buffalo (Bubalus bubalis) and development of first BuffSatDB

PubMed Central

2013-01-01

Background Though India has sequenced water buffalo genome but its draft assembly is based on cattle genome BTau 4.0, thus de novo chromosome wise assembly is a major pending issue for global community. The existing radiation hybrid of buffalo and these reported STR can be used further in final gap plugging and “finishing” expected in de novo genome assembly. QTL and gene mapping needs mining of putative STR from buffalo genome at equal interval on each and every chromosome. Such markers have potential role in improvement of desirable characteristics, such as high milk yields, resistance to diseases, high growth rate. The STR mining from whole genome and development of user friendly database is yet to be done to reap the benefit of whole genome sequence. Description By in silico microsatellite mining of whole genome, we have developed first STR database of water buffalo, BuffSatDb (Buffalo MicroSatellite Database (http://cabindb.iasri.res.in/buffsatdb/) which is a web based relational database of 910529 microsatellite markers, developed using PHP and MySQL database. Microsatellite markers have been generated using MIcroSAtellite tool. It is simple and systematic web based search for customised retrieval of chromosome wise and genome-wide microsatellites. Search has been enabled based on chromosomes, motif type (mono-hexa), repeat motif and repeat kind (simple and composite). The search may be customised by limiting location of STR on chromosome as well as number of markers in that range. This is a novel approach and not been implemented in any of the existing marker database. This database has been further appended with Primer3 for primer designing of the selected markers enabling researcher to select markers of choice at desired interval over the chromosome. The unique add-on of degenerate bases further helps in resolving presence of degenerate bases in current buffalo assembly. Conclusion Being first buffalo STR database in the world , this would not only pave the way in resolving current assembly problem but shall be of immense use for global community in QTL/gene mapping critically required to increase knowledge in the endeavour to increase buffalo productivity, especially for third world country where rural economy is significantly dependent on buffalo productivity. PMID:23336431

In silico mining of putative microsatellite markers from whole genome sequence of water buffalo (Bubalus bubalis) and development of first BuffSatDB.

PubMed

Sarika; Arora, Vasu; Iquebal, Mir Asif; Rai, Anil; Kumar, Dinesh

2013-01-19

Though India has sequenced water buffalo genome but its draft assembly is based on cattle genome BTau 4.0, thus de novo chromosome wise assembly is a major pending issue for global community. The existing radiation hybrid of buffalo and these reported STR can be used further in final gap plugging and "finishing" expected in de novo genome assembly. QTL and gene mapping needs mining of putative STR from buffalo genome at equal interval on each and every chromosome. Such markers have potential role in improvement of desirable characteristics, such as high milk yields, resistance to diseases, high growth rate. The STR mining from whole genome and development of user friendly database is yet to be done to reap the benefit of whole genome sequence. By in silico microsatellite mining of whole genome, we have developed first STR database of water buffalo, BuffSatDb (Buffalo MicroSatellite Database (http://cabindb.iasri.res.in/buffsatdb/) which is a web based relational database of 910529 microsatellite markers, developed using PHP and MySQL database. Microsatellite markers have been generated using MIcroSAtellite tool. It is simple and systematic web based search for customised retrieval of chromosome wise and genome-wide microsatellites. Search has been enabled based on chromosomes, motif type (mono-hexa), repeat motif and repeat kind (simple and composite). The search may be customised by limiting location of STR on chromosome as well as number of markers in that range. This is a novel approach and not been implemented in any of the existing marker database. This database has been further appended with Primer3 for primer designing of the selected markers enabling researcher to select markers of choice at desired interval over the chromosome. The unique add-on of degenerate bases further helps in resolving presence of degenerate bases in current buffalo assembly. Being first buffalo STR database in the world , this would not only pave the way in resolving current assembly problem but shall be of immense use for global community in QTL/gene mapping critically required to increase knowledge in the endeavour to increase buffalo productivity, especially for third world country where rural economy is significantly dependent on buffalo productivity.

[Prediction of ETA oligopeptides antagonists from Glycine max based on in silico proteolysis].

PubMed

Qiao, Lian-Sheng; Jiang, Lu-di; Luo, Gang-Gang; Lu, Fang; Chen, Yan-Kun; Wang, Ling-Zhi; Li, Gong-Yu; Zhang, Yan-Ling

2017-02-01

Oligopeptides are one of the the key pharmaceutical effective constituents of traditional Chinese medicine(TCM). Systematic study on composition and efficacy of TCM oligopeptides is essential for the analysis of material basis and mechanism of TCM. In this study, the potential anti-hypertensive oligopeptides from Glycine max and their endothelin receptor A (ETA) antagonistic activity were discovered and predicted based on in silico technologies.Main protein sequences of G. max were collected and oligopeptides were obtained using in silico gastrointestinal tract proteolysis. Then, the pharmacophore of ETA antagonistic peptides was constructed and included one hydrophobic feature, one ionizable negative feature, one ring aromatic feature and five excluded volumes. Meanwhile, three-dimensional structure of ETA was developed by homology modeling methods for further docking studies. According to docking analysis and consensus score, the key amino acid of GLN165 was identified for ETA antagonistic activity. And 27 oligopeptides from G. max were predicted as the potential ETA antagonists by pharmacophore and docking studies.In silico proteolysis could be used to analyze the protein sequences from TCM. According to combination of in silico proteolysis and molecular simulation, the biological activities of oligopeptides could be predicted rapidly based on the known TCM protein sequence. It might provide the methodology basis for rapidly and efficiently implementing the mechanism analysis of TCM oligopeptides. Copyright© by the Chinese Pharmaceutical Association.

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

PubMed

Lu, L; Komada, M; Kitamura, N

1998-06-15

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

Contiguous deletion of the NDP, MAOA, MAOB, and EFHC2 genes in a patient with Norrie disease, severe psychomotor retardation and myoclonic epilepsy.

PubMed

Rodriguez-Revenga, L; Madrigal, I; Alkhalidi, L S; Armengol, L; González, E; Badenas, C; Estivill, X; Milà, M

2007-05-01

Norrie disease (ND) is an X-linked disorder, inherited as a recessive trait that, therefore, mostly affects males. The gene responsible for ND, called NDP, maps to the short arm of chromosome X (Xp11.4-p11.3). We report here an atypical case of ND, consisting of a patient harboring a large submicroscopic deletion affecting not only the NDP gene but also the MAOA, MAOB, and EFHC2 genes. Microarray comparative genomic hybridization (CGH) analysis showed that 11 consecutive bacterial artificial chromosome (BAC) clones, mapping around the NDP gene, were deleted. These clones span a region of about 1 Mb on Xp11.3. The deletion was ascertained by fluorescent in situ hybridization (FISH) analysis with different BAC clones located within the region. Clinical features of the proband include bilateral retinal detachment, microcephaly, severe psychomotor retardation without verbal language skills acquired, and epilepsy. The identification and molecular characterization of this case reinforces the idea of a new contiguous gene syndrome that would explain the complex phenotype shared by atypical ND patients.

Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana.

PubMed

Wittenberg, Alexander H J; van der Lee, Theo; Cayla, Cyril; Kilian, Andrzej; Visser, Richard G F; Schouten, Henk J

2005-08-01

Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. This paper demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana. Restriction fragments from a genomic representation of the ecotype Landsberg erecta (Ler) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypes Columbia (Col) and Ler and of individuals from an F(2) population obtained from a Col x Ler cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the Ler markers and alignment with the available Col genome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms.

Mutations in CsPID encoding a Ser/Thr protein kinase are responsible for round leaf shape in cucumber (Cucumis sativus L.).

PubMed

Zhang, Chaowen; Chen, Feifan; Zhao, Ziyao; Hu, Liangliang; Liu, Hanqiang; Cheng, Zhihui; Weng, Yiqun; Chen, Peng; Li, Yuhong

2018-06-01

Two round-leaf mutants, rl-1 and rl-2, were identified from EMS-induced mutagenesis. High throughput sequencing and map-based cloning suggested CsPID encoding a Ser/Thr protein kinase as the most possible candidate for rl-1. Rl-2 was allelic to Rl-1. Leaf shape is an important plant architecture trait that is affected by plant hormones, especially auxin. In Arabidopsis, PINOID (PID), a regulator for the auxin polar transporter PIN (PIN-FORMED) affects leaf shape formation, but this function of PID in crop plants has not been well studied. From an EMS mutagenesis population, we identified two round-leaf (rl) mutants, C356 and C949. Segregation analysis suggested that both mutations were controlled by single recessive genes, rl-1 and rl-2, respectively. With map-based cloning, we show that CsPID as the candidate gene of rl-1; a non-synonymous SNP in the second exon of CsPID resulted in an amino acid substitution and the round leaf phenotype. As compared in the wild type plant, CsPID had significantly lower expression in the root, leaf and female flowers in C356, which may result in the less developed roots, round leaves and abnormal female flowers, respectively in the rl-1 mutant. Among the three copies of PID genes, CsPID, CsPID2 and CSPID2L (CsPID2-like) in the cucumber genome, CsPID was the only one with significantly differential expression in adult leaves between WT and C356 suggesting CsPID plays a main role in leaf shape formation. The rl-2 mutation in C949 was also cloned, which was due to another SNP in a nearby location of rl-1 in the same CsPID gene. The two round leaf mutants and the work presented herein provide a good foundation for understanding the molecular mechanisms of CsPID in cucumber leaf development.

In Silico Identification Software (ISIS): A Machine Learning Approach to Tandem Mass Spectral Identification of Lipids

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

Kangas, Lars J.; Metz, Thomas O.; Isaac, Georgis

2012-05-15

Liquid chromatography-mass spectrometry-based metabolomics has gained importance in the life sciences, yet it is not supported by software tools for high throughput identification of metabolites based on their fragmentation spectra. An algorithm (ISIS: in silico identification software) and its implementation are presented and show great promise in generating in silico spectra of lipids for the purpose of structural identification. Instead of using chemical reaction rate equations or rules-based fragmentation libraries, the algorithm uses machine learning to find accurate bond cleavage rates in a mass spectrometer employing collision-induced dissocia-tion tandem mass spectrometry. A preliminary test of the algorithm with 45 lipidsmore » from a subset of lipid classes shows both high sensitivity and specificity.« less

Cloning and sequence analysis of the human brain beta-adrenergic receptor. Evolutionary relationship to rodent and avian beta-receptors and porcine muscarinic receptors.

PubMed

Chung, F Z; Lentes, K U; Gocayne, J; Fitzgerald, M; Robinson, D; Kerlavage, A R; Fraser, C M; Venter, J C

1987-01-26

Two cDNA clones, lambda-CLFV-108 and lambda-CLFV-119, encoding for the beta-adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV-517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain beta-receptor consists of 413 amino acids with a calculated Mr of 46480. The gene contains three potential glucocorticoid receptor-binding sites. The beta-receptor expressed in human brain was homology with rodent (88%) and avian (52%) beta-receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci. USA 81, 272 276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.

Testing chromosomal phylogenies and inversion breakpoint reuse in Drosophila. The martensis cluster revisited.

PubMed

Prada, Carlos F; Delprat, Alejandra; Ruiz, Alfredo

2011-02-01

The chromosomal relationships of the four martensis cluster species are among the most complex and intricate within the entire Drosophila repleta group, due to the so-called sharing of inversions. Here, we have revised these relationships using comparative mapping of bacterial artificial chromosome (BAC) clones on the salivary gland chromosomes. A physical map of chromosome 2 of Drosophila uniseta (one of the cluster members) was generated by in situ hybridization of 82 BAC clones from the physical map of the Drosophila buzzatii genome (an outgroup that represents the ancestral arrangement). By comparing the marker positions, we determined the number, order, and orientation of conserved chromosomal segments between chromosome 2 of D. buzzatii and D. uniseta. GRIMM software was used to infer that a minimum of five chromosomal inversions are necessary to transform the chromosome 2 of D. buzzatii into that of D. uniseta. Two of these inversions have been overlooked in previous cytological analyses. The five fixed inversions entail two breakpoint reuses because only nine syntenic segments and eight interruptions were observed. We tested for the presence of the five inversions fixed in D. uniseta in the other three species of the martensis cluster by in situ hybridization of eight breakpoint-bearing BAC clones. The results shed light on the chromosomal phylogeny of the martensis cluster, yet leave a number of questions open.

A RAD-Based Genetic Map for Anchoring Scaffold Sequences and Identifying QTLs in Bitter Gourd (Momordica charantia)

PubMed Central

Cui, Junjie; Luo, Shaobo; Niu, Yu; Huang, Rukui; Wen, Qingfang; Su, Jianwen; Miao, Nansheng; He, Weiming; Dong, Zhensheng; Cheng, Jiaowen; Hu, Kailin

2018-01-01

Genetic mapping is a basic tool necessary for anchoring assembled scaffold sequences and for identifying QTLs controlling important traits. Though bitter gourd (Momordica charantia) is both consumed and used as a medicinal, research on its genomics and genetic mapping is severely limited. Here, we report the construction of a restriction site associated DNA (RAD)-based genetic map for bitter gourd using an F2 mapping population comprising 423 individuals derived from two cultivated inbred lines, the gynoecious line ‘K44’ and the monoecious line ‘Dali-11.’ This map comprised 1,009 SNP markers and spanned a total genetic distance of 2,203.95 cM across the 11 linkage groups. It anchored a total of 113 assembled scaffolds that covered about 251.32 Mb (85.48%) of the 294.01 Mb assembled genome. In addition, three horticulturally important traits including sex expression, fruit epidermal structure, and immature fruit color were evaluated using a combination of qualitative and quantitative data. As a result, we identified three QTL/gene loci responsible for these traits in three environments. The QTL/gene gy/fffn/ffn, controlling sex expression involved in gynoecy, first female flower node, and female flower number was detected in the reported region. Particularly, two QTLs/genes, Fwa/Wr and w, were found to be responsible for fruit epidermal structure and white immature fruit color, respectively. This RAD-based genetic map promotes the assembly of the bitter gourd genome and the identified genetic loci will accelerate the cloning of relevant genes in the future. PMID:29706980

Genome Mapping and Molecular Breeding of Tomato

PubMed Central

Foolad, Majid R.

2007-01-01

The cultivated tomato, Lycopersicon esculentum, is the second most consumed vegetable worldwide and a well-studied crop species in terms of genetics, genomics, and breeding. It is one of the earliest crop plants for which a genetic linkage map was constructed, and currently there are several molecular maps based on crosses between the cultivated and various wild species of tomato. The high-density molecular map, developed based on an L. esculentum × L. pennellii cross, includes more than 2200 markers with an average marker distance of less than 1 cM and an average of 750 kbp per cM. Different types of molecular markers such as RFLPs, AFLPs, SSRs, CAPS, RGAs, ESTs, and COSs have been developed and mapped onto the 12 tomato chromosomes. Markers have been used extensively for identification and mapping of genes and QTLs for many biologically and agriculturally important traits and occasionally for germplasm screening, fingerprinting, and marker-assisted breeding. The utility of MAS in tomato breeding has been restricted largely due to limited marker polymorphism within the cultivated species and economical reasons. Also, when used, MAS has been employed mainly for improving simply-inherited traits and not much for improving complex traits. The latter has been due to unavailability of reliable PCR-based markers and problems with linkage drag. Efforts are being made to develop high-throughput markers with greater resolution, including SNPs. The expanding tomato EST database, which currently includes ∼214 000 sequences, the new microarray DNA chips, and the ongoing sequencing project are expected to aid development of more practical markers. Several BAC libraries have been developed that facilitate map-based cloning of genes and QTLs. Sequencing of the euchromatic portions of the tomato genome is paving the way for comparative and functional analysis of important genes and QTLs. PMID:18364989

A reference linkage map for Eucalyptus

PubMed Central

2012-01-01

Background Genetic linkage maps are invaluable resources in plant research. They provide a key tool for many genetic applications including: mapping quantitative trait loci (QTL); comparative mapping; identifying unlinked (i.e. independent) DNA markers for fingerprinting, population genetics and phylogenetics; assisting genome sequence assembly; relating physical and recombination distances along the genome and map-based cloning of genes. Eucalypts are the dominant tree species in most Australian ecosystems and of economic importance globally as plantation trees. The genome sequence of E. grandis has recently been released providing unprecedented opportunities for genetic and genomic research in the genus. A robust reference linkage map containing sequence-based molecular markers is needed to capitalise on this resource. Several high density linkage maps have recently been constructed for the main commercial forestry species in the genus (E. grandis, E. urophylla and E. globulus) using sequenced Diversity Arrays Technology (DArT) and microsatellite markers. To provide a single reference linkage map for eucalypts a composite map was produced through the integration of data from seven independent mapping experiments (1950 individuals) using a marker-merging method. Results The composite map totalled 1107 cM and contained 4101 markers; comprising 3880 DArT, 213 microsatellite and eight candidate genes. Eighty-one DArT markers were mapped to two or more linkage groups, resulting in the 4101 markers being mapped to 4191 map positions. Approximately 13% of DArT markers mapped to identical map positions, thus the composite map contained 3634 unique loci at an average interval of 0.31 cM. Conclusion The composite map represents the most saturated linkage map yet produced in Eucalyptus. As the majority of DArT markers contained on the map have been sequenced, the map provides a direct link to the E. grandis genome sequence and will serve as an important reference for progressing eucalypt research. PMID:22702473

High resolution physical mapping of single gene fragments on pachytene chromosome 4 and 7 of Rosa.

PubMed

Kirov, Ilya V; Van Laere, Katrijn; Khrustaleva, Ludmila I

2015-07-02

Rosaceae is a family containing many economically important fruit and ornamental species. Although fluorescence in situ hybridization (FISH)-based physical mapping of plant genomes is a valuable tool for map-based cloning, comparative genomics and evolutionary studies, no studies using high resolution physical mapping have been performed in this family. Previously we proved that physical mapping of single-copy genes as small as 1.1 kb is possible on mitotic metaphase chromosomes of Rosa wichurana using Tyramide-FISH. In this study we aimed to further improve the physical map of Rosa wichurana by applying high resolution FISH to pachytene chromosomes. Using high resolution Tyramide-FISH and multicolor Tyramide-FISH, 7 genes (1.7-3 kb) were successfully mapped on pachytene chromosomes 4 and 7 of Rosa wichurana. Additionally, by using multicolor Tyramide-FISH three closely located genes were simultaneously visualized on chromosome 7. A detailed map of heterochromatine/euchromatine patterns of chromosome 4 and 7 was developed with indication of the physical position of these 7 genes. Comparison of the gene order between Rosa wichurana and Fragaria vesca revealed a poor collinearity for chromosome 7, but a perfect collinearity for chromosome 4. High resolution physical mapping of short probes on pachytene chromosomes of Rosa wichurana was successfully performed for the first time. Application of Tyramide-FISH on pachytene chromosomes allowed the mapping resolution to be increased up to 20 times compared to mitotic metaphase chromosomes. High resolution Tyramide-FISH and multicolor Tyramide-FISH might become useful tools for further physical mapping of single-copy genes and for the integration of physical and genetic maps of Rosa wichurana and other members of the Rosaceae.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo) and chicken (Gallus gallus) genomes

PubMed Central

2011-01-01

Background A robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. Results The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. Conclusion The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species. PMID:21906286

Genetic mapping of a food preference gene in the silkworm, Bombyx mori, using restriction fragment length polymorphisms (RFLPs).

PubMed

Mase, Keisuke; Iizuka, Tetsuya; Yamamoto, Toshio; Okada, Eiji; Hara, Wajirou

2007-06-01

The domesticated silkworm, Bombyx mori, has strict food preferences and grows by feeding on mulberry leaves. However, "Sawa-J", an abnormal feeding habit strain selected from the genetic stock, feeds on an artificial diet without mulberry leaf powder. In this study, the food preference gene in Sawa-J was genetically identified using restriction fragment length polymorphisms (RFLPs) of a cDNA clone on each linkage group. Taking advantage of a lack of genetic recombination in females, reciprocal backcrossed F1 (BC1) progenies were independently prepared using a non-feeding strain, C108, as a mating partner of Sawa-J. Our results of linkage analysis and mapping proved that the feeding behavior is primarily controlled by a major recessive gene mapped at 20.2 cM on RFLP linkage group 9 (RFLG9), and clone e73 at a distance of 4.2 cM was found as the first linked molecular marker.

Recombination and Transfection Mapping of Cistron 5 of Bacteriophage Sp82g

PubMed Central

Green, D. MacDonald; Urban, Margeret I.

1972-01-01

Recombination between transfecting SP82G DNA molecules has been studied in Bacillus subtilis. Recombinant progeny issuing from transfected cells show many of the features that characterize progeny production in multiplicity reactivated bacteriophage, such as: majority recombinant clones, non-reciprocity of recombinant clones and the frequent absence of input alleles. While transfection substantially lowers the linkage observed between markers in normal phage crosses, linkage is observed at small map distances in transfection either by plating transfected bacteria or the progeny phage. Maps constructed from transfection crosses are identical to those of normal phage crosses, except in magnitude.—Examination of the concentration response of two marker biparental crosses, and three marker triparental crosses using transfecting DNA leads to the conclusion that at all concentrations, transfective centers are saturated with respect to the number of molecules that can be taken up. Thus, the frequency of recombinant infective centers, or recombinant progeny is independent of concentration effects. PMID:17248556

A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations.

PubMed

Liu, Xia; Li, Tuoping; Hart, Darren J; Gao, Song; Wang, Hongling; Gao, Herui; Xu, Shumin; Zhang, Yifeng; Liu, Yifei; An, Yingfeng

2018-03-18

Currently, the most widely used strategies for molecular cloning are sticky-end ligation-based cloning, TA cloning, blunt-end ligation-based cloning and ligase-independent cloning. In this study we have developed a novel mini-vector pANY1 which can simultaneously meet the requirements of all these cloning strategies. In addition, the selection of appropriate restriction digestion sites is difficult in some cases because of the presence of internal sites. In this study, an annealing of PCR products (APP)-based sticky-end cloning strategy was introduced to avoid this issue. Additionally, false positives occur during molecular cloning, which increases the workload of isolating positive clones. The plasmid pANY1 contains a ccdB cassette between multiple cloning sites, which efficiently avoids these false positives. Therefore, this mini-vector should serve as a useful tool with wide applications in biosciences, agriculture, food technologies, etc. Copyright © 2018 Elsevier Inc. All rights reserved.

The Virtual Anemia Trial: An Assessment of Model‐Based In Silico Clinical Trials of Anemia Treatment Algorithms in Patients With Hemodialysis

PubMed Central

Topping, Alice; Kappel, Franz; Thijssen, Stephan; Kotanko, Peter

2018-01-01

In silico approaches have been proposed as a novel strategy to increase the repertoire of clinical trial designs. Realistic simulations of clinical trials can provide valuable information regarding safety and limitations of treatment protocols and have been shown to assist in the cost‐effective planning of clinical studies. In this report, we present a blueprint for the stepwise integration of internal, external, and ecological validity considerations in virtual clinical trials (VCTs). We exemplify this approach in the context of a model‐based in silico clinical trial aimed at anemia treatment in patients undergoing hemodialysis (HD). Hemoglobin levels and subsequent anemia treatment were simulated on a per patient level over the course of a year and compared to real‐life clinical data of 79,426 patients undergoing HD. The novel strategies presented here, aimed to improve external and ecological validity of a VCT, significantly increased the predictive power of the discussed in silico trial. PMID:29368434

Versatile P(acman) BAC Libraries for Transgenesis Studies in Drosophila melanogaster

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

Venken, Koen J.T.; Carlson, Joseph W.; Schulze, Karen L.

2009-04-21

We constructed Drosophila melanogaster BAC libraries with 21-kb and 83-kb inserts in the P(acman) system. Clones representing 12-fold coverage and encompassing more than 95percent of annotated genes were mapped onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using Phi C31 integrase to rescue mutations. They can be modified through recombineering, for example to incorporate protein tags and assess expression patterns.

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5.

PubMed

Kapanadze, B; Kashuba, V; Baranova, A; Rasool, O; van Everdink, W; Liu, Y; Syomov, A; Corcoran, M; Poltaraus, A; Brodyansky, V; Syomova, N; Kazakov, A; Ibbotson, R; van den Berg, A; Gizatullin, R; Fedorova, L; Sulimova, G; Zelenin, A; Deaven, L; Lehrach, H; Grander, D; Buys, C; Oscier, D; Zabarovsky, E R; Einhorn, S; Yankovsky, N

1998-04-17

B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc-finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B-CLL rearrangements.

Development of a sensitive Luminex xMAP-based microsphere immunoassay for specific detection of Iris yellow spot virus.

PubMed

Yu, Cui; Yang, Cuiyun; Song, Shaoyi; Yu, Zixiang; Zhou, Xueping; Wu, Jianxiang

2018-04-04

Iris yellow spot virus (IYSV) is an Orthotospovirus that infects most Allium species. Very few approaches for specific detection of IYSV from infected plants are available to date. We report the development of a high-sensitive Luminex xMAP-based microsphere immunoassay (MIA) for specific detection of IYSV. The nucleocapsid (N) gene of IYSV was cloned and expressed in Escherichia coli to produce the His-tagged recombinant N protein. A panel of monoclonal antibodies (MAbs) against IYSV was generated by immunizing the mice with recombinant N protein. Five specific MAbs (16D9, 11C6, 7F4, 12C10, and 14H12) were identified and used for developing the Luminex xMAP-based MIA systems along with a polyclonal antibody against IYSV. Comparative analyses of their sensitivity and specificity in detecting IYSV from infected tobacco leaves identified 7F4 as the best-performed MAb in MIA. We then optimized the working conditions of Luminex xMAP-based MIA in specific detection of IYSV from infected tobacco leaves by using appropriate blocking buffer and proper concentration of biotin-labeled antibodies as well as the suitable ratio between the antibodies and the streptavidin R-phycoerythrin (SA-RPE). Under the optimized conditions the Luminex xMAP-based MIA was able to specifically detect IYSV with much higher sensitivity than conventional enzyme-linked immunosorbent assay (ELISA). Importantly, the Luminex xMAP-based MIA is time-saving and the whole procedure could be completed within 2.5 h. We generated five specific MAbs against IYSV and developed the Luminex xMAP-based MIA method for specific detection of IYSV in plants. This assay provides a sensitive, high-specific, easy to perform and likely cost-effective approach for IYSV detection from infected plants, implicating potential broad usefulness of MIA in plant virus diagnosis.

In Silico Constraint-Based Strain Optimization Methods: the Quest for Optimal Cell Factories

PubMed Central

Maia, Paulo; Rocha, Miguel

2015-01-01

SUMMARY Shifting from chemical to biotechnological processes is one of the cornerstones of 21st century industry. The production of a great range of chemicals via biotechnological means is a key challenge on the way toward a bio-based economy. However, this shift is occurring at a pace slower than initially expected. The development of efficient cell factories that allow for competitive production yields is of paramount importance for this leap to happen. Constraint-based models of metabolism, together with in silico strain design algorithms, promise to reveal insights into the best genetic design strategies, a step further toward achieving that goal. In this work, a thorough analysis of the main in silico constraint-based strain design strategies and algorithms is presented, their application in real-world case studies is analyzed, and a path for the future is discussed. PMID:26609052

Identification of BRCA1 and 2 Other Tumor Suppressor Genes on Chromosome 17 Through Positional Cloning

DTIC Science & Technology

2000-04-01

Genes, LOH Mapping, Chromosome 17, Physical Mapping, Genetic Mapping, CDNA Screening, Humans, Anatomical 81 Samples, Mutation Detection, Breast Cancer...According to the established model for LOH involving tumor suppressor genes, the allele remaining in the tumor sample would harbor the deleterious mutation ...sequencing on an AB1373A sequencer (Applied Biosystems, Foster City, CA). As none of the samples we have sequenced have revealed any mutations , we have

EST-derived SSR markers used as anchor loci for the construction of a consensus linkage map in ryegrass (Lolium spp.)

PubMed Central

2010-01-01

Background Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm. Results A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2. Conclusions The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species. PMID:20712870

Genome-wide Association Mapping of Qualitatively Inherited Traits in a Germplasm Collection.

PubMed

Bandillo, Nonoy B; Lorenz, Aaron J; Graef, George L; Jarquin, Diego; Hyten, David L; Nelson, Randall L; Specht, James E

2017-07-01

Genome-wide association (GWA) has been used as a tool for dissecting the genetic architecture of quantitatively inherited traits. We demonstrate here that GWA can also be highly useful for detecting many major genes governing categorically defined phenotype variants that exist for qualitatively inherited traits in a germplasm collection. Genome-wide association mapping was applied to categorical phenotypic data available for 10 descriptive traits in a collection of ∼13,000 soybean [ (L.) Merr.] accessions that had been genotyped with a 50,000 single nucleotide polymorphism (SNP) chip. A GWA on a panel of accessions of this magnitude can offer substantial statistical power and mapping resolution, and we found that GWA mapping resulted in the identification of strong SNP signals for 24 classical genes as well as several heretofore unknown genes controlling the phenotypic variants in those traits. Because some of these genes had been cloned, we were able to show that the narrow GWA mapping SNP signal regions that we detected for the phenotypic variants had chromosomal bp spans that, with just one exception, overlapped the bp region of the cloned genes, despite local variation in SNP number and nonuniform SNP distribution in the chip set. Copyright © 2017 Crop Science Society of America.

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H/sub 2/ metabolism in Escherichia coli

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

Sankar, P.; Lee, J.H.; Shanmugam, K.T.

1985-04-01

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2more » (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.« less

In silico characterization of a novel pathogenic deletion mutation identified in XPA gene in a Pakistani family with severe xeroderma pigmentosum.

PubMed

Nasir, Muhammad; Ahmad, Nafees; Sieber, Christian M K; Latif, Amir; Malik, Salman Akbar; Hameed, Abdul

2013-09-24

Xeroderma Pigmentosum (XP) is a rare skin disorder characterized by skin hypersensitivity to sunlight and abnormal pigmentation. The aim of this study was to investigate the genetic cause of a severe XP phenotype in a consanguineous Pakistani family and in silico characterization of any identified disease-associated mutation. The XP complementation group was assigned by genotyping of family for known XP loci. Genotyping data mapped the family to complementation group A locus, involving XPA gene. Mutation analysis of the candidate XP gene by DNA sequencing revealed a novel deletion mutation (c.654del A) in exon 5 of XPA gene. The c.654del A, causes frameshift, which pre-maturely terminates protein and result into a truncated product of 222 amino acid (aa) residues instead of 273 (p.Lys218AsnfsX5). In silico tools were applied to study the likelihood of changes in structural motifs and thus interaction of mutated protein with binding partners. In silico analysis of mutant protein sequence, predicted to affect the aa residue which attains coiled coil structure. The coiled coil structure has an important role in key cellular interactions, especially with DNA damage-binding protein 2 (DDB2), which has important role in DDB-mediated nucleotide excision repair (NER) system. Our findings support the fact of genetic and clinical heterogeneity in XP. The study also predicts the critical role of DDB2 binding region of XPA protein in NER pathway and opens an avenue for further research to study the functional role of the mutated protein domain.

Linkage analysis by genotyping of sibling populations: a genetic map for the potato cyst nematode constructed using a "pseudo-F2" mapping strategy.

PubMed

Rouppe van der Voort, J N; van Eck, H J; van Zandvoort, P M; Overmars, H; Helder, J; Bakker, J

1999-07-01

A mapping strategy is described for the construction of a linkage map of a non-inbred species in which individual offspring genotypes are not amenable to marker analysis. After one extra generation of random mating, the segregating progeny was propagated, and bulked populations of offspring were analyzed. Although the resulting population structure is different from that of commonly used mapping populations, we show that the maximum likelihood formula for a normal F2 is applicable for the estimation of recombination. This "pseudo-F2" mapping strategy, in combination with the development of an AFLP assay for single cysts, facilitated the construction of a linkage map for the potato cyst nematode Globodera rostochiensis. Using 12 pre-selected AFLP primer combinations, a total of 66 segregating markers were identified, 62 of which were mapped to nine linkage groups. These 62 AFLP markers are randomly distributed and cover about 65% of the genome. An estimate of the physical size of the Globodera genome was obtained from comparisons of the number of AFLP fragments obtained with the values for Caenorhabditis elegans. The methodology presented here resulted in the first genomic map for a cyst nematode. The low value of the kilobase/centimorgan (kb/cM) ratio for the Globodera genome will facilitate map-based cloning of genes that mediate the interaction between the nematode and its host plant.

Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

PubMed

Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

2016-12-01

Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes.

PubMed

Haiminen, Niina; Feltus, F Alex; Parida, Laxmi

2011-04-15

We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies.

Unravelling and managing fusiform rust disease: a model approach for coevolved forest tree pathosystems.

Treesearch

CD Nelson; TL Kubisiak; HV Amerson

2010-01-01

Fusiform rust disease remains the most destructive disease in pine plantations in the southern United States. Our ongoing research is designed to identify, map, and clone the interacting genes of the host and pathogen. Several resistance (R) genes have been identified and genetically mapped using informative pine families and single-spore isolate inoculations. In...

Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

PubMed

Chao, Mei; Lin, Chia-Chi; Lin, Feng-Ming; Li, Hsin-Pai; Iang, Shan-Bei

2015-12-01

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

Brief Guide to Genomics: DNA, Genes and Genomes

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Sequence Ready Characterization of the Pericentromeric Region of 19p12

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

Evan E. Eichler

2006-08-31

Current mapping and sequencing strategies have been inadequate within the proximal portion of 19p12 due, in part, to the presence of a recently expanded ZNF (zinc-finger) gene family and the presence of large (25-50 kb) inverted beta-satellite repeat structures which bracket this tandemly duplicated gene family. The virtual of absence of classically defined “unique” sequence within the region has hampered efforts to identify and characterize a suitable minimal tiling path of clones which can be used as templates required for finished sequencing of the region. The goal of this proposal is to develop and implement a novel sequence-anchor strategy tomore » generate a contiguous BAC map of the most proximal portion of chromosome 19p12 for the purpose of complete sequence characterization. The target region will be an estimated 4.5 Mb of DNA extending from STS marker D19S450 (the beginning of the ZNF gene cluster) to the centromeric (alpha-satellite) junction of 19p11. The approach will entail 1) pre-selection of 19p12 BAC and cosmid clones (NIH approved library) utilizing both 19p12 -unique and 19p12-SPECIFIC repeat probes (Eichler et al., 1998); 2) the generation of a BAC/cosmid end-sequence map across the region with a density of one marker every 8kb; 3) the development of a second-generation of STS (sequence tagged sites) which will be used to identify and verify clonal overlap at the level of the sequence; 4) incorporation of these sequence-anchored overlapping clones into existing cosmid/BAC restriction maps developed at Livermore National Laboratory; and 5) validation of the organization of this region utilizing high-resolution FISH techniques (extended chromatin analysis) on monochromosomal 19 somatic cell hybrids and parental cell lines of source material. The data generated will be used in the selection of the most parsimonious tiling path of BAC clones to be sequenced as part of the JGI effort on chromosome 19 and should serve as a model for the sequence characterization of other difficult regions of the human genome« less

Molecular mechanisms underlying variations in lung function: a systems genetics analysis

PubMed Central

Obeidat, Ma’en; Hao, Ke; Bossé, Yohan; Nickle, David C; Nie, Yunlong; Postma, Dirkje S; Laviolette, Michel; Sandford, Andrew J; Daley, Denise D; Hogg, James C; Elliott, W Mark; Fishbane, Nick; Timens, Wim; Hysi, Pirro G; Kaprio, Jaakko; Wilson, James F; Hui, Jennie; Rawal, Rajesh; Schulz, Holger; Stubbe, Beate; Hayward, Caroline; Polasek, Ozren; Järvelin, Marjo-Riitta; Zhao, Jing Hua; Jarvis, Deborah; Kähönen, Mika; Franceschini, Nora; North, Kari E; Loth, Daan W; Brusselle, Guy G; Smith, Albert Vernon; Gudnason, Vilmundur; Bartz, Traci M; Wilk, Jemma B; O’Connor, George T; Cassano, Patricia A; Tang, Wenbo; Wain, Louise V; Artigas, María Soler; Gharib, Sina A; Strachan, David P; Sin, Don D; Tobin, Martin D; London, Stephanie J; Hall, Ian P; Paré, Peter D

2016-01-01

Summary Background Lung function measures reflect the physiological state of the lung, and are essential to the diagnosis of chronic obstructive pulmonary disease (COPD). The SpiroMeta-CHARGE consortium undertook the largest genome-wide association study (GWAS) so far (n=48 201) for forced expiratory volume in 1 s (FEV1) and the ratio of FEV1 to forced vital capacity (FEV1/FVC) in the general population. The lung expression quantitative trait loci (eQTLs) study mapped the genetic architecture of gene expression in lung tissue from 1111 individuals. We used a systems genetics approach to identify single nucleotide polymorphisms (SNPs) associated with lung function that act as eQTLs and change the level of expression of their target genes in lung tissue; termed eSNPs. Methods The SpiroMeta-CHARGE GWAS results were integrated with lung eQTLs to map eSNPs and the genes and pathways underlying the associations in lung tissue. For comparison, a similar analysis was done in peripheral blood. The lung mRNA expression levels of the eSNP-regulated genes were tested for associations with lung function measures in 727 individuals. Additional analyses identified the pleiotropic effects of eSNPs from the published GWAS catalogue, and mapped enrichment in regulatory regions from the ENCODE project. Finally, the Connectivity Map database was used to identify potential therapeutics in silico that could reverse the COPD lung tissue gene signature. Findings SNPs associated with lung function measures were more likely to be eQTLs and vice versa. The integration mapped the specific genes underlying the GWAS signals in lung tissue. The eSNP-regulated genes were enriched for developmental and inflammatory pathways; by comparison, SNPs associated with lung function that were eQTLs in blood, but not in lung, were only involved in inflammatory pathways. Lung function eSNPs were enriched for regulatory elements and were over-represented among genes showing differential expression during fetal lung development. An mRNA gene expression signature for COPD was identified in lung tissue and compared with the Connectivity Map. This in-silico drug repurposing approach suggested several compounds that reverse the COPD gene expression signature, including a nicotine receptor antagonist. These findings represent novel therapeutic pathways for COPD. Interpretation The system genetics approach identified lung tissue genes driving the variation in lung function and susceptibility to COPD. The identification of these genes and the pathways in which they are enriched is essential to understand the pathophysiology of airway obstruction and to identify novel therapeutic targets and biomarkers for COPD, including drugs that reverse the COPD gene signature in silico. Funding The research reported in this article was not specifically funded by any agency. See Acknowledgments for a full list of funders of the lung eQTL study and the Spiro-Meta CHARGE GWAS. PMID:26404118

A High Density Consensus Genetic Map of Tetraploid Cotton That Integrates Multiple Component Maps through Molecular Marker Redundancy Check

PubMed Central

Blenda, Anna; Fang, David D.; Rami, Jean-François; Garsmeur, Olivier; Luo, Feng; Lacape, Jean-Marc

2012-01-01

A consensus genetic map of tetraploid cotton was constructed using six high-density maps and after the integration of a sequence-based marker redundancy check. Public cotton SSR libraries (17,343 markers) were curated for sequence redundancy using 90% as a similarity cutoff. As a result, 20% of the markers (3,410) could be considered as redundant with some other markers. The marker redundancy information had been a crucial part of the map integration process, in which the six most informative interspecific Gossypium hirsutum×G. barbadense genetic maps were used for assembling a high density consensus (HDC) map for tetraploid cotton. With redundant markers being removed, the HDC map could be constructed thanks to the sufficient number of collinear non-redundant markers in common between the component maps. The HDC map consists of 8,254 loci, originating from 6,669 markers, and spans 4,070 cM, with an average of 2 loci per cM. The HDC map presents a high rate of locus duplications, as 1,292 markers among the 6,669 were mapped in more than one locus. Two thirds of the duplications are bridging homoeologous AT and DT chromosomes constitutive of allopolyploid cotton genome, with an average of 64 duplications per AT/DT chromosome pair. Sequences of 4,744 mapped markers were used for a mutual blast alignment (BBMH) with the 13 major scaffolds of the recently released Gossypium raimondii genome indicating high level of homology between the diploid D genome and the tetraploid cotton genetic map, with only a few minor possible structural rearrangements. Overall, the HDC map will serve as a valuable resource for trait QTL comparative mapping, map-based cloning of important genes, and better understanding of the genome structure and evolution of tetraploid cotton. PMID:23029214

Mutagenesis of the lac promoter region in M13 mp10 phage DNA by 4'-hydroxymethyl-4,5',8-trimethylpsoralen

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

Piette, J.; Decuyper-Debergh, D.; Gamper, H.

Double-stranded M13 phage DNA (M13 mp10 replicative form) was photoreacted with 4'-hydroxymethyl-4,5',8-trimethylpsoralen, using light of wavelength greater than 320 nm or greater than 390 nm to generate predominantly crosslinks or monoadducts, respectively. The damaged DNAs were scored for inactivation and mutagenesis after transfection into Escherichia coli. The appearance of light-blue or colorless plaques on indicator medium showed that mutation had occurred in the lac insert of the viral DNA. A comparison of the consequences of the two phototreatments with psoralen supports the idea that crosslinks are both more lethal and more mutagenic than monoadducts. Numerous mutant clones partially or totallymore » deficient in beta-galactosidase were plaque-purified and amplified. The viral DNA of each clone was sequenced by the dideoxy chain-terminating procedure. All of the observed base-pair changes were mapped to the lac promoter region and consisted of 3 transition, 14 transversion, and 6 single base-pair frame-shift mutations. The predominant mutation was a T.A----G.C transversion.« less

Development of 5123 Intron-Length Polymorphic Markers for Large-Scale Genotyping Applications in Foxtail Millet

PubMed Central

Muthamilarasan, Mehanathan; Venkata Suresh, B.; Pandey, Garima; Kumari, Kajal; Parida, Swarup Kumar; Prasad, Manoj

2014-01-01

Generating genomic resources in terms of molecular markers is imperative in molecular breeding for crop improvement. Though development and application of microsatellite markers in large-scale was reported in the model crop foxtail millet, no such large-scale study was conducted for intron-length polymorphic (ILP) markers. Considering this, we developed 5123 ILP markers, of which 4049 were physically mapped onto 9 chromosomes of foxtail millet. BLAST analysis of 5123 expressed sequence tags (ESTs) suggested the function for ∼71.5% ESTs and grouped them into 5 different functional categories. About 440 selected primer pairs representing the foxtail millet genome and the different functional groups showed high-level of cross-genera amplification at an average of ∼85% in eight millets and five non-millet species. The efficacy of the ILP markers for distinguishing the foxtail millet is demonstrated by observed heterozygosity (0.20) and Nei's average gene diversity (0.22). In silico comparative mapping of physically mapped ILP markers demonstrated substantial percentage of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (∼50%), maize (∼46%), rice (∼21%) and Brachypodium (∼21%) chromosomes. Hence, for the first time, we developed large-scale ILP markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species. PMID:24086082

VIRTUAL LIVER: AN IN SILICO FRAMEWORK FOR ANALYZING CHEMICAL-INDUCED HEPATOTOXICITY

EPA Science Inventory

The US EPA Virtual Liver (v-LiverTM) is an in silico framework for the dose-dependent perturbation of normal hepatic functions by chemicals using in vitro data. The framework consists of a computable knowledge-base (KB) to infer putative pathways in hepatotoxicity and a cellular...

Development of a Computational (in silico) Model of Ocular Teratogenesis

EPA Science Inventory

EPA’s ToxCast™ project is profiling the in vitro bioactivity of chemical compounds to assess pathway-level and cell-based signatures that are highly correlated with observed in vivo toxicity. In silico models provide a framework for interpreting the in vitro results and for simul...

Advances in In Vitro and In Silico Tools for Toxicokinetic Dose Modeling and Predictive Toxicology (WC10)

EPA Science Inventory

Recent advances in vitro assays, in silico tools, and systems biology approaches provide opportunities for refined mechanistic understanding for chemical safety assessment that will ultimately lead to reduced reliance on animal-based methods. With the U.S. commercial chemical lan...

A new in silico classification model for ready biodegradability, based on molecular fragments.

PubMed

Lombardo, Anna; Pizzo, Fabiola; Benfenati, Emilio; Manganaro, Alberto; Ferrari, Thomas; Gini, Giuseppina

2014-08-01

Regulations such as the European REACH (Registration, Evaluation, Authorization and restriction of Chemicals) often require chemicals to be evaluated for ready biodegradability, to assess the potential risk for environmental and human health. Because not all chemicals can be tested, there is an increasing demand for tools for quick and inexpensive biodegradability screening, such as computer-based (in silico) theoretical models. We developed an in silico model starting from a dataset of 728 chemicals with ready biodegradability data (MITI-test Ministry of International Trade and Industry). We used the novel software SARpy to automatically extract, through a structural fragmentation process, a set of substructures statistically related to ready biodegradability. Then, we analysed these substructures in order to build some general rules. The model consists of a rule-set made up of the combination of the statistically relevant fragments and of the expert-based rules. The model gives good statistical performance with 92%, 82% and 76% accuracy on the training, test and external set respectively. These results are comparable with other in silico models like BIOWIN developed by the United States Environmental Protection Agency (EPA); moreover this new model includes an easily understandable explanation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Error Checking and Graphical Representation of Multiple–Complete–Digest (MCD) Restriction-Fragment Maps

PubMed Central

Thayer, Edward C.; Olson, Maynard V.; Karp, Richard M.

1999-01-01

Genetic and physical maps display the relative positions of objects or markers occurring within a target DNA molecule. In constructing maps, the primary objective is to determine the ordering of these objects. A further objective is to assign a coordinate to each object, indicating its distance from a reference end of the target molecule. This paper describes a computational method and a body of software for assigning coordinates to map objects, given a solution or partial solution to the ordering problem. We describe our method in the context of multiple–complete–digest (MCD) mapping, but it should be applicable to a variety of other mapping problems. Because of errors in the data or insufficient clone coverage to uniquely identify the true ordering of the map objects, a partial ordering is typically the best one can hope for. Once a partial ordering has been established, one often seeks to overlay a metric along the map to assess the distances between the map objects. This problem often proves intractable because of data errors such as erroneous local length measurements (e.g., large clone lengths on low-resolution physical maps). We present a solution to the coordinate assignment problem for MCD restriction-fragment mapping, in which a coordinated set of single-enzyme restriction maps are simultaneously constructed. We show that the coordinate assignment problem can be expressed as the solution of a system of linear constraints. If the linear system is free of inconsistencies, it can be solved using the standard Bellman–Ford algorithm. In the more typical case where the system is inconsistent, our program perturbs it to find a new consistent system of linear constraints, close to those of the given inconsistent system, using a modified Bellman–Ford algorithm. Examples are provided of simple map inconsistencies and the methods by which our program detects candidate data errors and directs the user to potential suspect regions of the map. PMID:9927487

Cloning and characterization of XiR1, a locus responsible for dagger nematode resistance in grape.

PubMed

Hwang, Chin-Feng; Xu, Kenong; Hu, Rong; Zhou, Rita; Riaz, Summaira; Walker, M Andrew

2010-08-01

The dagger nematode, Xiphinema index, feeds aggressively on grape roots and in the process, vectors grapevine fanleaf virus (GFLV) leading to the severe viral disease known as fanleaf degeneration. Resistance to X. index and GFLV has been the key objective of grape rootstock breeding programs. A previous study found that resistance to X. index derived from Vitis arizonica was largely controlled by a major quantitative trait locus, XiR1 (X. index Resistance 1), located on chromosome 19. The study presented here develops high-resolution genetic and physical maps in an effort to identify the XiR1 gene(s). The mapping was carried out with 1,375 genotypes in three populations derived from D8909-15, a resistant selection from a cross of V. rupestris A. de Serres (susceptible) x V. arizonica b42-26 (resistant). Resistance to X. index was evaluated on 99 informative recombinants that were identified by screening the three populations with two markers flanking the XiR1 locus. The high-resolution genetic map of XiR1 was primarily constructed with seven DNA markers developed in this study. Physical mapping of XiR1 was accomplished by screening three bacterial artificial chromosome (BAC) libraries constructed from D8909-15, V. vinifera Cabernet Sauvignon and V. arizonica b42-26. A total of 32 BAC clones were identified and the XiR1 locus was delineated within a 115 kb region. Sequence analysis of three BAC clones identified putative nucleotide binding/leucine-rich repeat (NB-LRR) genes. This is the first report of a closely linked major gene locus responsible for ectoparasitic nematode resistance. The markers developed from this study are being used to expedite the breeding of resistant grape rootstocks.

Cloning and characterization of XiR1, a locus responsible for dagger nematode resistance in grape

PubMed Central

Hwang, Chin-Feng; Xu, Kenong; Hu, Rong; Zhou, Rita; Riaz, Summaira

2010-01-01

The dagger nematode, Xiphinemaindex, feeds aggressively on grape roots and in the process, vectors grapevine fanleaf virus (GFLV) leading to the severe viral disease known as fanleaf degeneration. Resistance to X. index and GFLV has been the key objective of grape rootstock breeding programs. A previous study found that resistance to X. index derived from Vitis arizonica was largely controlled by a major quantitative trait locus, XiR1 (X. index Resistance 1), located on chromosome 19. The study presented here develops high-resolution genetic and physical maps in an effort to identify the XiR1 gene(s). The mapping was carried out with 1,375 genotypes in three populations derived from D8909-15, a resistant selection from a cross of V. rupestris A. de Serres (susceptible) × V. arizonica b42-26 (resistant). Resistance to X. index was evaluated on 99 informative recombinants that were identified by screening the three populations with two markers flanking the XiR1 locus. The high-resolution genetic map of XiR1 was primarily constructed with seven DNA markers developed in this study. Physical mapping of XiR1 was accomplished by screening three bacterial artificial chromosome (BAC) libraries constructed from D8909-15, V. vinifera Cabernet Sauvignon and V. arizonica b42-26. A total of 32 BAC clones were identified and the XiR1 locus was delineated within a 115 kb region. Sequence analysis of three BAC clones identified putative nucleotide binding/leucine-rich repeat (NB-LRR) genes. This is the first report of a closely linked major gene locus responsible for ectoparasitic nematode resistance. The markers developed from this study are being used to expedite the breeding of resistant grape rootstocks. PMID:20490447

Purely in silico BCS classification: science based quality standards for the world's drugs.

PubMed

Dahan, Arik; Wolk, Omri; Kim, Young Hoon; Ramachandran, Chandrasekharan; Crippen, Gordon M; Takagi, Toshihide; Bermejo, Marival; Amidon, Gordon L

2013-11-04

BCS classification is a vital tool in the development of both generic and innovative drug products. The purpose of this work was to provisionally classify the world's top selling oral drugs according to the BCS, using in silico methods. Three different in silico methods were examined: the well-established group contribution (CLogP) and atom contribution (ALogP) methods, and a new method based solely on the molecular formula and element contribution (KLogP). Metoprolol was used as the benchmark for the low/high permeability class boundary. Solubility was estimated in silico using a thermodynamic equation that relies on the partition coefficient and melting point. The validity of each method was affirmed by comparison to reference data and literature. We then used each method to provisionally classify the orally administered, IR drug products found in the WHO Model list of Essential Medicines, and the top-selling oral drug products in the United States (US), Great Britain (GB), Spain (ES), Israel (IL), Japan (JP), and South Korea (KR). A combined list of 363 drugs was compiled from the various lists, and 257 drugs were classified using the different in silico permeability methods and literature solubility data, as well as BDDCS classification. Lastly, we calculated the solubility values for 185 drugs from the combined set using in silico approach. Permeability classification with the different in silico methods was correct for 69-72.4% of the 29 reference drugs with known human jejunal permeability, and for 84.6-92.9% of the 14 FDA reference drugs in the set. The correlations (r(2)) between experimental log P values of 154 drugs and their CLogP, ALogP and KLogP were 0.97, 0.82 and 0.71, respectively. The different in silico permeability methods produced comparable results: 30-34% of the US, GB, ES and IL top selling drugs were class 1, 27-36.4% were class 2, 22-25.5% were class 3, and 5.46-14% were class 4 drugs, while ∼8% could not be classified. The WHO list included significantly less class 1 and more class 3 drugs in comparison to the countries' lists, probably due to differences in commonly used drugs in developing vs industrial countries. BDDCS classified more drugs as class 1 compared to in silico BCS, likely due to the more lax benchmark for metabolism (70%), in comparison to the strict permeability benchmark (metoprolol). For 185 out of the 363 drugs, in silico solubility values were calculated, and successfully matched the literature solubility data. In conclusion, relatively simple in silico methods can be used to estimate both permeability and solubility. While CLogP produced the best correlation to experimental values, even KLogP, the most simplified in silico method that is based on molecular formula with no knowledge of molecular structure, produced comparable BCS classification to the sophisticated methods. This KLogP, when combined with a mean melting point and estimated dose, can be used to provisionally classify potential drugs from just molecular formula, even before synthesis. 49-59% of the world's top-selling drugs are highly soluble (class 1 and class 3), and are therefore candidates for waivers of in vivo bioequivalence studies. For these drugs, the replacement of expensive human studies with affordable in vitro dissolution tests would ensure their bioequivalence, and encourage the development and availability of generic drug products in both industrial and developing countries.

A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis.

PubMed

Bansal, Ankita; Kar, Debasish; Pandey, Satya Deo; Matcha, Ashok; Kumar, N Ganesh; Nathan, Soshina; Ghosh, Anindya S

2017-06-01

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.

Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein.

PubMed

Kaushik, Himani; Deshmukh, Sachin; Mathur, Deepika Dayal; Tiwari, Archana; Garg, Lalit C

2013-01-01

Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM1 ganglioside receptor of LTB. The rLTB.Etx40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens. aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli.

Genotyping by Sequencing in Almond: SNP Discovery, Linkage Mapping, and Marker Design

PubMed Central

Goonetilleke, Shashi N.; March, Timothy J.; Wirthensohn, Michelle G.; Arús, Pere; Walker, Amanda R.; Mather, Diane E.

2017-01-01

In crop plant genetics, linkage maps provide the basis for the mapping of loci that affect important traits and for the selection of markers to be applied in crop improvement. In outcrossing species such as almond (Prunus dulcis Mill. D. A. Webb), application of a double pseudotestcross mapping approach to the F1 progeny of a biparental cross leads to the construction of a linkage map for each parent. Here, we report on the application of genotyping by sequencing to discover and map single nucleotide polymorphisms in the almond cultivars “Nonpareil” and “Lauranne.” Allele-specific marker assays were developed for 309 tag pairs. Application of these assays to 231 Nonpareil × Lauranne F1 progeny provided robust linkage maps for each parent. Analysis of phenotypic data for shell hardness demonstrated the utility of these maps for quantitative trait locus mapping. Comparison of these maps to the peach genome assembly confirmed high synteny and collinearity between the peach and almond genomes. The marker assays were applied to progeny from several other Nonpareil crosses, providing the basis for a composite linkage map of Nonpareil. Applications of the assays to a panel of almond clones and a panel of rootstocks used for almond production demonstrated the broad applicability of the markers and provide subsets of markers that could be used to discriminate among accessions. The sequence-based linkage maps and single nucleotide polymorphism assays presented here could be useful resources for the genetic analysis and genetic improvement of almond. PMID:29141988

Construction of a high-density, high-resolution genetic map and its integration with BAC-based physical map in channel catfish

PubMed Central

Li, Yun; Liu, Shikai; Qin, Zhenkui; Waldbieser, Geoff; Wang, Ruijia; Sun, Luyang; Bao, Lisui; Danzmann, Roy G.; Dunham, Rex; Liu, Zhanjiang

2015-01-01

Construction of genetic linkage map is essential for genetic and genomic studies. Recent advances in sequencing and genotyping technologies made it possible to generate high-density and high-resolution genetic linkage maps, especially for the organisms lacking extensive genomic resources. In the present work, we constructed a high-density and high-resolution genetic map for channel catfish with three large resource families genotyped using the catfish 250K single-nucleotide polymorphism (SNP) array. A total of 54,342 SNPs were placed on the linkage map, which to our knowledge had the highest marker density among aquaculture species. The estimated genetic size was 3,505.4 cM with a resolution of 0.22 cM for sex-averaged genetic map. The sex-specific linkage maps spanned a total of 4,495.1 cM in females and 2,593.7 cM in males, presenting a ratio of 1.7 : 1 between female and male in recombination fraction. After integration with the previously established physical map, over 87% of physical map contigs were anchored to the linkage groups that covered a physical length of 867 Mb, accounting for ∼90% of the catfish genome. The integrated map provides a valuable tool for validating and improving the catfish whole-genome assembly and facilitates fine-scale QTL mapping and positional cloning of genes responsible for economically important traits. PMID:25428894

Single strand conformation polymorphism based SNP and Indel markers for genetic mapping and synteny analysis of common bean (Phaseolus vulgaris L.)

PubMed Central

2009-01-01

Background Expressed sequence tags (ESTs) are an important source of gene-based markers such as those based on insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). Several gel based methods have been reported for the detection of sequence variants, however they have not been widely exploited in common bean, an important legume crop of the developing world. The objectives of this project were to develop and map EST based markers using analysis of single strand conformation polymorphisms (SSCPs), to create a transcript map for common bean and to compare synteny of the common bean map with sequenced chromosomes of other legumes. Results A set of 418 EST based amplicons were evaluated for parental polymorphisms using the SSCP technique and 26% of these presented a clear conformational or size polymorphism between Andean and Mesoamerican genotypes. The amplicon based markers were then used for genetic mapping with segregation analysis performed in the DOR364 × G19833 recombinant inbred line (RIL) population. A total of 118 new marker loci were placed into an integrated molecular map for common bean consisting of 288 markers. Of these, 218 were used for synteny analysis and 186 presented homology with segments of the soybean genome with an e-value lower than 7 × 10-12. The synteny analysis with soybean showed a mosaic pattern of syntenic blocks with most segments of any one common bean linkage group associated with two soybean chromosomes. The analysis with Medicago truncatula and Lotus japonicus presented fewer syntenic regions consistent with the more distant phylogenetic relationship between the galegoid and phaseoloid legumes. Conclusion The SSCP technique is a useful and inexpensive alternative to other SNP or Indel detection techniques for saturating the common bean genetic map with functional markers that may be useful in marker assisted selection. In addition, the genetic markers based on ESTs allowed the construction of a transcript map and given their high conservation between species allowed synteny comparisons to be made to sequenced genomes. This synteny analysis may support positional cloning of target genes in common bean through the use of genomic information from these other legumes. PMID:20030833

Mapping of a major QTL for salt tolerance of mature field-grown maize plants based on SNP markers.

PubMed

Luo, Meijie; Zhao, Yanxin; Zhang, Ruyang; Xing, Jinfeng; Duan, Minxiao; Li, Jingna; Wang, Naishun; Wang, Wenguang; Zhang, Shasha; Chen, Zhihui; Zhang, Huasheng; Shi, Zi; Song, Wei; Zhao, Jiuran

2017-08-15

Salt stress significantly restricts plant growth and production. Maize is an important food and economic crop but is also a salt sensitive crop. Identification of the genetic architecture controlling salt tolerance facilitates breeders to select salt tolerant lines. However, the critical quantitative trait loci (QTLs) responsible for the salt tolerance of field-grown maize plants are still unknown. To map the main genetic factors contributing to salt tolerance in mature maize, a double haploid population (240 individuals) and 1317 single nucleotide polymorphism (SNP) markers were employed to produce a genetic linkage map covering 1462.05 cM. Plant height of mature maize cultivated in the saline field (SPH) and plant height-based salt tolerance index (ratio of plant height between saline and control fields, PHI) were used to evaluate salt tolerance of mature maize plants. A major QTL for SPH was detected on Chromosome 1 with the LOD score of 22.4, which explained 31.2% of the phenotypic variation. In addition, the major QTL conditioning PHI was also mapped at the same position on Chromosome 1, and two candidate genes involving in ion homeostasis were identified within the confidence interval of this QTL. The detection of the major QTL in adult maize plant establishes the basis for the map-based cloning of genes associated with salt tolerance and provides a potential target for marker assisted selection in developing maize varieties with salt tolerance.

The gene for pancreatic polypeptide (PPY) and the anonymous marker D17S78 are within 45 kb of each other on chromosome 17q21

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

Chandrasekharappa, S.C.; King, S.E.; Lee, Y.H.

1994-05-15

A gene for early-onset breast and ovarian cancer (BRCA1) has been localized to a small region of chromosome 17q21. A combination of genetic linkage studies, radiation-reduced hybrid analysis, and physical mapping by FISH has identified several genes/markers that lie in this interval. Among these are the gene encoding pancreatic polypeptide (PPY) and a polymorphic marker at locus D17S78. Efforts to construct a physical map of this region by isolating a large number of yeast artificial chromosome (YAC) and cosmid clones demonstrate that PPY and D17S78 are present within the same cosmid clone, and therefore no farther than 45 kb apart.more » This observation takes on particular significance since it excludes a recently described BRCA1 candidate gene from the interval defined by meiotic mapping. Although PPY and D17S78 were found to be no farther than 45 kb apart, identification of a smaller fragment that hybridizes to both probes would indicate that these two are much closer. The probe p131 and the gene PPY were previously mapped to 17q21-q23 and to the proximal long arm of chromosome 17, respectively. The demonstration of the close proximity of these markers should allow them to be treated as a single locus in terms of long-range genomic mapping of this region, and the genomic clones isolated should serve as useful resources for the identification of the BRCA1 gene. Analysis of a large number of a familial and spordic breast and ovarian cancers has identified frequent loss of heterozygosity near the BRCA1 locus. A recent report has suggested the responsible interval lies just telomeric to PPY, and a suggested candidate gene (MCD) for BRCA1 was found to be somatically rearranged in two of several hundred sporadic breast tumors.« less

Application of Genome Wide Association and Genomic Prediction for Improvement of Cacao Productivity and Resistance to Black and Frosty Pod Diseases

PubMed Central

Romero Navarro, J. Alberto; Phillips-Mora, Wilbert; Arciniegas-Leal, Adriana; Mata-Quirós, Allan; Haiminen, Niina; Mustiga, Guiliana; Livingstone III, Donald; van Bakel, Harm; Kuhn, David N.; Parida, Laxmi; Kasarskis, Andrew; Motamayor, Juan C.

2017-01-01

Chocolate is a highly valued and palatable confectionery product. Chocolate is primarily made from the processed seeds of the tree species Theobroma cacao. Cacao cultivation is highly relevant for small-holder farmers throughout the tropics, yet its productivity remains limited by low yields and widespread pathogens. A panel of 148 improved cacao clones was assembled based on productivity and disease resistance, and phenotypic single-tree replicated clonal evaluation was performed for 8 years. Using high-density markers, the diversity of clones was expressed relative to 10 known ancestral cacao populations, and significant effects of ancestry were observed in productivity and disease resistance. Genome-wide association (GWA) was performed, and six markers were significantly associated with frosty pod disease resistance. In addition, genomic selection was performed, and consistent with the observed extensive linkage disequilibrium, high predictive ability was observed at low marker densities for all traits. Finally, quantitative trait locus mapping and differential expression analysis of two cultivars with contrasting disease phenotypes were performed to identify genes underlying frosty pod disease resistance, identifying a significant quantitative trait locus and 35 differentially expressed genes using two independent differential expression analyses. These results indicate that in breeding populations of heterozygous and recently admixed individuals, mapping approaches can be used for low complexity traits like pod color cacao, or in other species single gene disease resistance, however genomic selection for quantitative traits remains highly effective relative to mapping. Our results can help guide the breeding process for sustainable improved cacao productivity. PMID:29184558

Effect of Different Sampling Schedules on Results of Bioavailability and Bioequivalence Studies: Evaluation by Means of Monte Carlo Simulations.

PubMed

Kano, Eunice Kazue; Chiann, Chang; Fukuda, Kazuo; Porta, Valentina

2017-08-01

Bioavailability and bioequivalence study is one of the most frequently performed investigations in clinical trials. Bioequivalence testing is based on the assumption that 2 drug products will be therapeutically equivalent when they are equivalent in the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed and becomes available at the site of drug action. In recent years there has been a significant growth in published papers that use in silico studies based on mathematical simulations to analyze pharmacokinetic and pharmacodynamic properties of drugs, including bioavailability and bioequivalence aspects. The goal of this study is to evaluate the usefulness of in silico studies as a tool in the planning of bioequivalence, bioavailability and other pharmacokinetic assays, e.g., to determine an appropriate sampling schedule. Monte Carlo simulations were used to define adequate blood sampling schedules for a bioequivalence assay comparing 2 different formulations of cefadroxil oral suspensions. In silico bioequivalence studies comparing different formulation of cefadroxil oral suspensions using various sampling schedules were performed using models. An in vivo study was conducted to confirm in silico results. The results of in silico and in vivo bioequivalence studies demonstrated that schedules with fewer sampling times are as efficient as schedules with larger numbers of sampling times in the assessment of bioequivalence, but only if T max is included as a sampling time. It was also concluded that in silico studies are useful tools in the planning of bioequivalence, bioavailability and other pharmacokinetic in vivo assays. © Georg Thieme Verlag KG Stuttgart · New York.

Lgn1, a gene that determines susceptibility to Legionella pneumophila, maps to mouse chromosome 13

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

Dietrich, W.F.; Damron, D.M.; Lander, E.S.

1995-04-10

The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene. 22 refs., 2 figs., 2 tabs.

Genome-wide identification and characterization of NB-ARC resistant genes in wheat (Triticum aestivum L.) and their expression during leaf rust infection.

PubMed

Chandra, Saket; Kazmi, Andaleeb Z; Ahmed, Zainab; Roychowdhury, Gargi; Kumari, Veena; Kumar, Manish; Mukhopadhyay, Kunal

2017-07-01

NB-ARC domain-containing resistance genes from the wheat genome were identified, characterized and localized on chromosome arms that displayed differential yet positive response during incompatible and compatible leaf rust interactions. Wheat (Triticum aestivum L.) is an important cereal crop; however, its production is affected severely by numerous diseases including rusts. An efficient, cost-effective and ecologically viable approach to control pathogens is through host resistance. In wheat, high numbers of resistance loci are present but only few have been identified and cloned. A comprehensive analysis of the NB-ARC-containing genes in complete wheat genome was accomplished in this study. Complete NB-ARC encoding genes were mined from the Ensembl Plants database to predict 604 NB-ARC containing sequences using the HMM approach. Genome-wide analysis of orthologous clusters in the NB-ARC-containing sequences of wheat and other members of the Poaceae family revealed maximum homology with Oryza sativa indica and Brachypodium distachyon. The identification of overlap between orthologous clusters enabled the elucidation of the function and evolution of resistance proteins. The distributions of the NB-ARC domain-containing sequences were found to be balanced among the three wheat sub-genomes. Wheat chromosome arms 4AL and 7BL had the most NB-ARC domain-containing contigs. The spatio-temporal expression profiling studies exemplified the positive role of these genes in resistant and susceptible wheat plants during incompatible and compatible interaction in response to the leaf rust pathogen Puccinia triticina. Two NB-ARC domain-containing sequences were modelled in silico, cloned and sequenced to analyze their fine structures. The data obtained in this study will augment isolation, characterization and application NB-ARC resistance genes in marker-assisted selection based breeding programs for improving rust resistance in wheat.

Molecular cloning and functional characterization of an antifungal PR-5 protein from Ocimum basilicum.

PubMed

Rather, Irshad Ahmad; Awasthi, Praveen; Mahajan, Vidushi; Bedi, Yashbir S; Vishwakarma, Ram A; Gandhi, Sumit G

2015-03-01

Pathogenesis-related (PR) proteins are involved in biotic and abiotic stress responses of plants and are grouped into 17 families (PR-1 to PR-17). PR-5 family includes proteins related to thaumatin and osmotin, with several members possessing antimicrobial properties. In this study, a PR-5 gene showing a high degree of homology with osmotin-like protein was isolated from sweet basil (Ocimum basilicum L.). A complete open reading frame consisting of 675 nucleotides, coding for a precursor protein, was obtained by PCR amplification. Based on sequence comparisons with tobacco osmotin and other osmotin-like proteins (OLPs), this protein was named ObOLP. The predicted mature protein is 225 amino acids in length and contains 16 cysteine residues that may potentially form eight disulfide bonds, a signature common to most PR-5 proteins. Among the various abiotic stress treatments tested, including high salt, mechanical wounding and exogenous phytohormone/elicitor treatments; methyl jasmonate (MeJA) and mechanical wounding significantly induced the expression of ObOLP gene. The coding sequence of ObOLP was cloned and expressed in a bacterial host resulting in a 25kDa recombinant-HIS tagged protein, displaying antifungal activity. The ObOLP protein sequence appears to contain an N-terminal signal peptide with signatures of secretory pathway. Further, our experimental data shows that ObOLP expression is regulated transcriptionally and in silico analysis suggests that it may be post-transcriptionally and post-translationally regulated through microRNAs and post-translational protein modifications, respectively. This study appears to be the first report of isolation and characterization of osmotin-like protein gene from O. basilicum. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of male fecundity, interindividual distance and anisotropic pollen dispersal on mating success in a Scots pine (Pinus sylvestris) seed orchard

PubMed Central

Torimaru, T; Wennström, U; Lindgren, D; Wang, X-R

2012-01-01

Quantifying the effect of pollen dispersal and flowering traits on mating success is essential for understanding evolutionary responses to changing environments and establishing strategies for forest tree breeding. This study examined, quantitatively, the effects of male fecundity, interindividual distance and anisotropic pollen dispersal on the mating success of Scots pine (Pinus sylvestris), utilizing a well-mapped Scots pine seed orchard. Paternity analysis of 1021 seeds sampled from 87 trees representing 28 clones showed that 53% of the seeds had at least one potential pollen parent within the orchard. Pronounced variation in paternal contribution was observed among clones. Variations in pollen production explained up to 78% of the variation in mating success, which was 11.2 times greater for clones producing the largest amount of pollen than for clones producing the least pollen. Mating success also varied with intertree distance and direction, which explained up to 28% of the variance. Fertilization between neighboring trees 2.3 m apart was 2.4 times more frequent than between trees 4.6 m apart, and up to 12.4 times higher for trees downwind of the presumed prevailing wind direction than for upwind trees. The effective number of pollen donors recorded in the seed orchard (12.2) was smaller than the theoretical expectation (19.7). Based on the empirical observations, a mating model that best describes the gene dispersal pattern in clonal seed orchards was constructed. PMID:21897440

Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

PubMed Central

Liu, Changqing; Bai, Chunyu; Guo, Yu; Liu, Dan; Lu, Taofeng; Li, Xiangchen; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

2014-01-01

Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger. PMID:24608928

Mapping of the 3q27 region involved in Dup(3q) syndrome

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

Rizzu, P.; Baldini, A.; Overhauser, J.

1994-09-01

The duplication 3q syndrome is characterized by partial trisomy of a segment of the long arm of chromosome 3. We have previously found that 3q26.3-3q27 is the minimal region of trisomy overlap. This critical region (CR) is delimited by two patient chromosome breakpoints, approximately 10 cM apart. In order to identify the gene(s) responsible for the Dup(3q) phenotype, we are generating a physical map of the region and identifying expressed sequences. First, we have generated a cytological map using two- and three-color fluorescence in situ hybridization on metaphase and interphase chromosomes. Results allowed us to determine the centromere-telomere orientation, ordermore » and relative distances of six cosmid clones mapped to the CR. Because some of the markers used are part of the consensus chromosome 3 map, our data were easily integrated with existing mapping information. Subsequently, we have included in the map YAC clones positive for polymorphic PCR markers identified by CEPH-Genethon, as well as newly isolated YACs. We have assigned them to the critical region 7 of the Genethon polymorphic markers and linked them to three YAC contigs. Currently our map includes two of the five genes known to map in this region. Interestingly, we found that these two functionally related genes (kininogen and histidin-rich glycoprotein) map to the same 1 Mb genomic fragment. As the physical map is being constructed we are searching for expressed sequences. Positive cDNAs have been found and their characterization is in progress. In conclusion, we will present an integrated map of 3q27 that includes genetic, physical and cytological information as well as gene annotation. As Dup(3q) syndrome is likely to be a contiguous gene syndrome, such a map will be necessary for our understanding of this multiple congenital anomaly.« less

X-linked hypophosphatemia. A phenotype in search of a cause.

PubMed

Tenenhouse, H S; Scriver, C R

1992-05-01

XLH is an important disease, it is the subject of several classic articles in the medical sciences (Scriver et al., 1991), and it has been an important stimulus to study renal hypophosphatemias and how they are involved in rickets and osteomalacia (Scriver, 1974; Scriver and Tenenhouse, 1991). Renal transport is the major determinant of phosphate homeostasis in mammals and it is unlikely that this important biochemical parameter would have been left by evolution to a single renal transport system. Together physiologists and geneticists found that the mammalian kidney has several gene products dedicated to phosphate transport. That has implications for biochemists in search of a membrane protein to clone and explain XLH, for example. Let us suppose the transporter affected in XLH is cloned. Will it be the product of the XLH (or Hyp or Gy) locus? One will not know until the transporter gene is mapped. There is no question of the X-chromosome locus product being protein kinase C for example, since it maps to autosomes. But where does one start in the search for the X-chromosome locus? With the elusive putative diffusible factor or with the transporter, or perhaps with an enzyme in vitamin D hormone metabolism? Which goes to say that it is necessary to know the phenotype to arrive at the right locus. Or is it? Sufficient physical mapping of region Xp22.31-p21.3 will eventually lead to positional cloning of the Hyp gene. What will it be?(ABSTRACT TRUNCATED AT 250 WORDS)

DIANA-TarBase v7.0: indexing more than half a million experimentally supported miRNA:mRNA interactions.

PubMed

Vlachos, Ioannis S; Paraskevopoulou, Maria D; Karagkouni, Dimitra; Georgakilas, Georgios; Vergoulis, Thanasis; Kanellos, Ilias; Anastasopoulos, Ioannis-Laertis; Maniou, Sofia; Karathanou, Konstantina; Kalfakakou, Despina; Fevgas, Athanasios; Dalamagas, Theodore; Hatzigeorgiou, Artemis G

2015-01-01

microRNAs (miRNAs) are short non-coding RNA species, which act as potent gene expression regulators. Accurate identification of miRNA targets is crucial to understanding their function. Currently, hundreds of thousands of miRNA:gene interactions have been experimentally identified. However, this wealth of information is fragmented and hidden in thousands of manuscripts and raw next-generation sequencing data sets. DIANA-TarBase was initially released in 2006 and it was the first database aiming to catalog published experimentally validated miRNA:gene interactions. DIANA-TarBase v7.0 (http://www.microrna.gr/tarbase) aims to provide for the first time hundreds of thousands of high-quality manually curated experimentally validated miRNA:gene interactions, enhanced with detailed meta-data. DIANA-TarBase v7.0 enables users to easily identify positive or negative experimental results, the utilized experimental methodology, experimental conditions including cell/tissue type and treatment. The new interface provides also advanced information ranging from the binding site location, as identified experimentally as well as in silico, to the primer sequences used for cloning experiments. More than half a million miRNA:gene interactions have been curated from published experiments on 356 different cell types from 24 species, corresponding to 9- to 250-fold more entries than any other relevant database. DIANA-TarBase v7.0 is freely available. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea.

PubMed

Martin, Stanton L; Blackmon, Barbara P; Rajagopalan, Ravi; Houfek, Thomas D; Sceeles, Robert G; Denn, Sheila O; Mitchell, Thomas K; Brown, Douglas E; Wing, Rod A; Dean, Ralph A

2002-01-01

We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.

In Silico Genome Mismatch Scanning to Map Breast Cancer Genes in Extended Pedigrees

DTIC Science & Technology

2009-07-01

contiguous window of loci. The current graphical model is applied to both the paternal and maternal haplotypes for each observed individual. The values at... paternal and maternal haplotypes of each individual in the sample in parallel. These affect the observed genotypes shown as white squares. The states...individuals, but also, by listing the parents as zero, allows for samples of unrelated individuals as required by these methods. IntervalLDwill treat

An ultra-high density linkage map and QTL mapping for sex and growth-related traits of common carp (Cyprinus carpio)

PubMed Central

Peng, Wenzhu; Xu, Jian; Zhang, Yan; Feng, Jianxin; Dong, Chuanju; Jiang, Likun; Feng, Jingyan; Chen, Baohua; Gong, Yiwen; Chen, Lin; Xu, Peng

2016-01-01

High density genetic linkage maps are essential for QTL fine mapping, comparative genomics and high quality genome sequence assembly. In this study, we constructed a high-density and high-resolution genetic linkage map with 28,194 SNP markers on 14,146 distinct loci for common carp based on high-throughput genotyping with the carp 250 K single nucleotide polymorphism (SNP) array in a mapping family. The genetic length of the consensus map was 10,595.94 cM with an average locus interval of 0.75 cM and an average marker interval of 0.38 cM. Comparative genomic analysis revealed high level of conserved syntenies between common carp and the closely related model species zebrafish and medaka. The genome scaffolds were anchored to the high-density linkage map, spanning 1,357 Mb of common carp reference genome. QTL mapping and association analysis identified 22 QTLs for growth-related traits and 7 QTLs for sex dimorphism. Candidate genes underlying growth-related traits were identified, including important regulators such as KISS2, IGF1, SMTLB, NPFFR1 and CPE. Candidate genes associated with sex dimorphism were also identified including 3KSR and DMRT2b. The high-density and high-resolution genetic linkage map provides an important tool for QTL fine mapping and positional cloning of economically important traits, and improving common carp genome assembly. PMID:27225429

Cloning, analysis and functional annotation of expressed sequence tags from the Earthworm Eisenia fetida

PubMed Central

Pirooznia, Mehdi; Gong, Ping; Guan, Xin; Inouye, Laura S; Yang, Kuan; Perkins, Edward J; Deng, Youping

2007-01-01

Background Eisenia fetida, commonly known as red wiggler or compost worm, belongs to the Lumbricidae family of the Annelida phylum. Little is known about its genome sequence although it has been extensively used as a test organism in terrestrial ecotoxicology. In order to understand its gene expression response to environmental contaminants, we cloned 4032 cDNAs or expressed sequence tags (ESTs) from two E. fetida libraries enriched with genes responsive to ten ordnance related compounds using suppressive subtractive hybridization-PCR. Results A total of 3144 good quality ESTs (GenBank dbEST accession number EH669363–EH672369 and EL515444–EL515580) were obtained from the raw clone sequences after cleaning. Clustering analysis yielded 2231 unique sequences including 448 contigs (from 1361 ESTs) and 1783 singletons. Comparative genomic analysis showed that 743 or 33% of the unique sequences shared high similarity with existing genes in the GenBank nr database. Provisional function annotation assigned 830 Gene Ontology terms to 517 unique sequences based on their homology with the annotated genomes of four model organisms Drosophila melanogaster, Mus musculus, Saccharomyces cerevisiae, and Caenorhabditis elegans. Seven percent of the unique sequences were further mapped to 99 Kyoto Encyclopedia of Genes and Genomes pathways based on their matching Enzyme Commission numbers. All the information is stored and retrievable at a highly performed, web-based and user-friendly relational database called EST model database or ESTMD version 2. Conclusion The ESTMD containing the sequence and annotation information of 4032 E. fetida ESTs is publicly accessible at . PMID:18047730

In silico design and screening of hypothetical MOF-74 analogs and their experimental synthesis

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

Witman, Matthew; Ling, Sanliang; Anderson, Samantha

2016-01-01

We present thein silico designof MOFs exhibiting 1-dimensional rod topologies by enumerating MOF-74-type analogs based on the PubChem Compounds database. We simulate the adsorption behavior of CO 2in the generated analogs and experimentally validate a novel MOF-74 analog, Mg 2(olsalazine).

Isolation of mini- and microsatellite loci from chromosome 19 library

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

Prosnyak, M.I.; Belajeva, O.V.; Polukarova, L.G.

Mini- and microsatellite sequences are abundant in the human genome and are very useful as genetic markers. We report the isolation of a panel of clones containing marker sequences from chromosome 19. We screened 10,000 clones from the chromosome 19 cosmid library for the presence of di-(CA)n, tri-(TCC)n, (CAC)n microsatellites and M13-like minisatellite sequences. For this we have used synthetic oligonucleotides and polynucleotides, including micro- (CA, TCC, CAC) and minisatellite (M13 core) sequences. Preliminary results indicated that the chromosome 19 cosmid library contained both human and hamster clones. In order to identify human sequences from this library we have developedmore » the technique of colony and blot hybridization with Alu-PCR, L1-PCR and B1-PCR probes. Dozens of clones have been selected, some of which were analyzed by conventional Southern blot analysis and non-radioactive in situ hybridization of chromosomes. Highly informative markers derived from these clones will be used for physical and genetic mapping of chromosome 19.« less

The Chromosome Microdissection and Microcloning Technique.

PubMed

Zhang, Ying-Xin; Deng, Chuan-Liang; Hu, Zan-Min

2016-01-01

Chromosome microdissection followed by microcloning is an efficient tool combining cytogenetics and molecular genetics that can be used for the construction of the high density molecular marker linkage map and fine physical map, the generation of probes for chromosome painting, and the localization and cloning of important genes. Here, we describe a modified technique to microdissect a single chromosome, paint individual chromosomes, and construct single-chromosome DNA libraries.

The Virtual Anemia Trial: An Assessment of Model-Based In Silico Clinical Trials of Anemia Treatment Algorithms in Patients With Hemodialysis.

PubMed

Fuertinger, Doris H; Topping, Alice; Kappel, Franz; Thijssen, Stephan; Kotanko, Peter

2018-04-01

In silico approaches have been proposed as a novel strategy to increase the repertoire of clinical trial designs. Realistic simulations of clinical trials can provide valuable information regarding safety and limitations of treatment protocols and have been shown to assist in the cost-effective planning of clinical studies. In this report, we present a blueprint for the stepwise integration of internal, external, and ecological validity considerations in virtual clinical trials (VCTs). We exemplify this approach in the context of a model-based in silico clinical trial aimed at anemia treatment in patients undergoing hemodialysis (HD). Hemoglobin levels and subsequent anemia treatment were simulated on a per patient level over the course of a year and compared to real-life clinical data of 79,426 patients undergoing HD. The novel strategies presented here, aimed to improve external and ecological validity of a VCT, significantly increased the predictive power of the discussed in silico trial. © 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

An in silico method to identify computer-based protocols worthy of clinical study: An insulin infusion protocol use case

PubMed Central

Wong, Anthony F; Pielmeier, Ulrike; Haug, Peter J; Andreassen, Steen

2016-01-01

Objective Develop an efficient non-clinical method for identifying promising computer-based protocols for clinical study. An in silico comparison can provide information that informs the decision to proceed to a clinical trial. The authors compared two existing computer-based insulin infusion protocols: eProtocol-insulin from Utah, USA, and Glucosafe from Denmark. Materials and Methods The authors used eProtocol-insulin to manage intensive care unit (ICU) hyperglycemia with intravenous (IV) insulin from 2004 to 2010. Recommendations accepted by the bedside clinicians directly link the subsequent blood glucose values to eProtocol-insulin recommendations and provide a unique clinical database. The authors retrospectively compared in silico 18 984 eProtocol-insulin continuous IV insulin infusion rate recommendations from 408 ICU patients with those of Glucosafe, the candidate computer-based protocol. The subsequent blood glucose measurement value (low, on target, high) was used to identify if the insulin recommendation was too high, on target, or too low. Results Glucosafe consistently provided more favorable continuous IV insulin infusion rate recommendations than eProtocol-insulin for on target (64% of comparisons), low (80% of comparisons), or high (70% of comparisons) blood glucose. Aggregated eProtocol-insulin and Glucosafe continuous IV insulin infusion rates were clinically similar though statistically significantly different (Wilcoxon signed rank test P = .01). In contrast, when stratified by low, on target, or high subsequent blood glucose measurement, insulin infusion rates from eProtocol-insulin and Glucosafe were statistically significantly different (Wilcoxon signed rank test, P < .001), and clinically different. Discussion This in silico comparison appears to be an efficient nonclinical method for identifying promising computer-based protocols. Conclusion Preclinical in silico comparison analytical framework allows rapid and inexpensive identification of computer-based protocol care strategies that justify expensive and burdensome clinical trials. PMID:26228765

Proposal of an in silico profiler for categorisation of repeat dose toxicity data of hair dyes.

PubMed

Nelms, M D; Ates, G; Madden, J C; Vinken, M; Cronin, M T D; Rogiers, V; Enoch, S J

2015-05-01

This study outlines the analysis of 94 chemicals with repeat dose toxicity data taken from Scientific Committee on Consumer Safety opinions for commonly used hair dyes in the European Union. Structural similarity was applied to group these chemicals into categories. Subsequent mechanistic analysis suggested that toxicity to mitochondria is potentially a key driver of repeat dose toxicity for chemicals within each of the categories. The mechanistic hypothesis allowed for an in silico profiler consisting of four mechanism-based structural alerts to be proposed. These structural alerts related to a number of important chemical classes such as quinones, anthraquinones, substituted nitrobenzenes and aromatic azos. This in silico profiler is intended for grouping chemicals into mechanism-based categories within the adverse outcome pathway paradigm.

Physical map of the Brucella melitensis 16 M chromosome.

PubMed Central

Allardet-Servent, A; Carles-Nurit, M J; Bourg, G; Michaux, S; Ramuz, M

1991-01-01

We present the first restriction map of the Brucella melitensis 16 M chromosome obtained by Southern blot hybridization of SpeI, XhoI, and XbaI fragments separated by pulsed-field gel electrophoresis. All restriction fragments (a total of 113) were mapped into an open circle. The main difficulty in mapping involved the exceedingly high number of restriction fragments, as was expected considering the 59% G + C content of the Brucella genome. Several cloned genes were placed on this map, especially rRNA operons which are repeated three times. The size of the B. melitensis chromosome, estimated as 2,600 kb long in a previous study, appeared longer (3,130 kb) by restriction mapping. This restriction map is an initial approach to achieve a genetic map of the Brucella chromosome. Images PMID:2007548

Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs

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

Singh, Deepti; Rawat, Surender; Waseem, Mohd

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, K{sub m} values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative propertymore » against BSA and Cu{sup 2+}/H{sub 2}O{sub 2} model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies. - Highlights: • Laccase from Yersinia enterocolitica strain 7 was expressed in Escherichia coli BL21 (DE3). • Recombinant laccase was found to be thermostable and alkali tolerant. • The in silico and experimental studied proves the biotransformation of NSAIDs. • Laccase binds to ligands differentially under different protonation state. • Laccase also possesses free radical scavenging property.« less

Integrated In Silico-In Vitro Identification and Characterization of the SH3-Mediated Interaction between Human PTTG and its Cognate Partners in Medulloblastoma.

PubMed

Liu, Jiangang; Wang, Dapeng; Li, Yanyan; Yao, Hui; Zhang, Nan; Zhang, Xuewen; Zhong, Fangping; Huang, Yulun

2018-06-01

The human pituitary tumor-transforming gene is an oncogenic protein which serves as a central hub in the cellular signaling network of medulloblastoma. The protein contains two vicinal PxxP motifs at its C terminus that are potential binding sites of peptide-recognition SH3 domains. Here, a synthetic protocol that integrated in silico analysis and in vitro assay was described to identify the SH3-binding partners of pituitary tumor-transforming gene in the gene expression profile of medulloblastoma. In the procedure, a variety of structurally diverse, non-redundant SH3 domains with high gene expression in medulloblastoma were compiled, and their three-dimensional structures were either manually retrieved from the protein data bank database or computationally modeled through bioinformatics technique. The binding capability of these domains towards the two PxxP-containing peptides m1p: 161 LGPPSPVK 168 and m2p: 168 KMPSPPWE 175 of pituitary tumor-transforming gene were ranked by structure-based scoring and fluorescence-based assay. Consequently, a number of SH3 domains, including MAP3K and PI3K, were found to have moderate or high affinity for m1p and/or m2p. Interestingly, the two overlapping peptides exhibits a distinct binding profile to these identified domain partners, suggesting that the binding selectivity of m1p and m2p is optimized across the medulloblastoma expression spectrum by competing for domain candidates. In addition, two redesigned versions of m1p peptide ware obtained via a structure-based rational mutation approach, which exhibited an increased affinity for the domain as compared to native peptide.

In silico identification of essential proteins in Corynebacterium pseudotuberculosis based on protein-protein interaction networks.

PubMed

Folador, Edson Luiz; de Carvalho, Paulo Vinícius Sanches Daltro; Silva, Wanderson Marques; Ferreira, Rafaela Salgado; Silva, Artur; Gromiha, Michael; Ghosh, Preetam; Barh, Debmalya; Azevedo, Vasco; Röttger, Richard

2016-11-04

Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis. Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous. The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing according wet-lab studies. The non-host homologous essential proteins are attractive targets for therapeutic and diagnostic proposes. They allow for searching of small molecule inhibitors of binding interactions enabling modern drug discovery. Overall, the predicted Cp PPI networks form a valuable and versatile tool for researchers interested in Corynebacterium pseudotuberculosis.

LipidFrag: Improving reliability of in silico fragmentation of lipids and application to the Caenorhabditis elegans lipidome

PubMed Central

Neumann, Steffen; Schmitt-Kopplin, Philippe

2017-01-01

Lipid identification is a major bottleneck in high-throughput lipidomics studies. However, tools for the analysis of lipid tandem MS spectra are rather limited. While the comparison against spectra in reference libraries is one of the preferred methods, these libraries are far from being complete. In order to improve identification rates, the in silico fragmentation tool MetFrag was combined with Lipid Maps and lipid-class specific classifiers which calculate probabilities for lipid class assignments. The resulting LipidFrag workflow was trained and evaluated on different commercially available lipid standard materials, measured with data dependent UPLC-Q-ToF-MS/MS acquisition. The automatic analysis was compared against manual MS/MS spectra interpretation. With the lipid class specific models, identification of the true positives was improved especially for cases where candidate lipids from different lipid classes had similar MetFrag scores by removing up to 56% of false positive results. This LipidFrag approach was then applied to MS/MS spectra of lipid extracts of the nematode Caenorhabditis elegans. Fragments explained by LipidFrag match known fragmentation pathways, e.g., neutral losses of lipid headgroups and fatty acid side chain fragments. Based on prediction models trained on standard lipid materials, high probabilities for correct annotations were achieved, which makes LipidFrag a good choice for automated lipid data analysis and reliability testing of lipid identifications. PMID:28278196

Genetic dissection and fine mapping of a novel dt gene associated with determinate growth habit in sesame.

PubMed

Zhang, Yanxin; Wang, Linhai; Gao, Yuan; Li, Donghua; Yu, Jingyin; Zhou, Rong; Zhang, Xiurong

2018-06-14

As an important oil crop, growth habit of sesame (Sesamum indicum L.) is naturally indeterminate, which brings about asynchronous maturity of capsules and causes loss of yield. The genetic basis of determinate growth habit in sesame was investigated by classical genetic analysis through multiple populations, results revealed that it was controlled by an unique recessive gene. The genotyping by sequencing (GBS) approach was employed for high-throughput SNP identification and genotyping in the F 2 population, then a high density bin map was constructed, the map was 1086.403 cM in length, which consisted of 1184 bins (13,679 SNPs), with an average of 0.918 cM between adjacent bins. Based on bin mapping in conjunction with SSR markers analysis in targeted region, the novel sesame determinacy gene was mapped on LG09 in a genome region of 41 kb. This study dissected genetic basis of determinate growth habit in sesame, constructed a new high-density bin map and mapped a novel determinacy gene. Results of this study demonstrate that we employed an optimized approach to get fine-accuracy, high-resolution and high-efficiency mapping result in sesame. The findings provided important foundation for sesame determinacy gene cloning and were expected to be applied in breeding for cultivars suited to mechanized production.

Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

PubMed Central

Throop, Andrea L.; LaBaer, Joshua

2015-01-01

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

Cloning and Characterization of the Scalloped Region of Drosophila Melanogaster

PubMed Central

Campbell, S. D.; Duttaroy, A.; Katzen, A. L.; Chovnick, A.

1991-01-01

Viable mutants of the scalloped gene (sd) of Drosophila melanogaster exhibit defects that can include gapping of the wing margin and ectopic bristle formation on the wing. Lethal sd alleles characterized in the present study now implicate this gene in a genetic function essential for normal development. In order to further characterize the developmental role of this gene, we have undertaken to clone and characterize the region where sd maps. A P[ry(+)] transposon insertion at 13F associated with sd([ry+2216]) served as the starting point for a 42-kb chromosomal walk. Molecular lesions associated with viable and lethal sd alleles were characterized by genomic hybridization analysis as a means of defining the extent of the gene. DNA rearrangements associated with 11 viable sd alleles map to a 2-kb interval which appears to be a ``hot spot'' for P element activity. Four of five recessive lethal sd mutations were mapped by denaturing gradient gel electrophoresis to a region 12-14 kb away from the region of viable lesions. In a sd(+) genotype, at least two structurally related and developmentally regulated transcripts hybridize to the genomic region where several sd lethal alleles have been localized. A viable mutation, sd(58), used for comparison in the transcript analysis, makes at least two slightly smaller transcripts that also hybridize to this region. Preliminary analysis of cDNA clones has identified three structurally related transcripts that hybridize to this genomic region. The 5' end of these transcripts extends into the 2-kb genomic region wherein DNA rearrangements were seen in the P element rearrangements. We favor the view that the transcripts represented by these cDNA clones are products of the sd gene. If this is true, the sd gene would include genomic sequences extending over at least 14 kb of the described chromosomal walk, and would appear to be subject to alternative splicing. PMID:1706292

Innate immunity and the new forward genetics.

PubMed

Beutler, Bruce

2016-12-01

As it is a hard-wired system for responses to microbes, innate immunity is particularly susceptible to classical genetic analysis. Mutations led the way to the discovery of many of the molecular elements of innate immune sensing and signaling pathways. In turn, the need for a faster way to find the molecular causes of mutation-induced phenotypes triggered a huge transformation in forward genetics. During the 1980s and 1990s, many heritable phenotypes were ascribed to mutations through positional cloning. In mice, this required three steps. First, a genetic mapping step was used to show that a given phenotype emanated from a circumscribed region of the genome. Second, a physical mapping step was undertaken, in which all of the region was cloned and its gene content determined. Finally, a concerted search for the mutation was performed. Such projects usually lasted for several years, but could produce breakthroughs in our understanding of biological processes. Publication of the annotated mouse genome sequence in 2002 made physical mapping unnecessary. More recently we devised a new technology for automated genetic mapping, which eliminated both genetic mapping and the search for mutations among candidate genes. The cause of phenotype can now be determined instantaneously. We have created more than 100,000 coding/splicing mutations. And by screening for defects of innate and adaptive immunity we have discovered many "new" proteins needed for innate immune function. Copyright © 2016 Elsevier Ltd. All rights reserved.

A candidate region for Nevoid Basal Cell Carcinoma Syndrome defined by genetic and physical mapping

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

Wainwright, B.; Negus, K.; Berkman, J.

1994-09-01

Nevoid Basal Cell Carcinoma Syndrome (NBCCS, or Gorlin`s syndrome) is a cancer predisposition syndrome charcterized by multiple basal cell carcinomas (BCCs) and diverse developmental defects. The gene responsible for NBCCS, which is most likely to be a tumor suppressor gene, has previously been mapped to 9q22.3-q31 in a 12 cM interval between the microsatellite marker loci D9S12 and D9S109. Combined multipoint and haplotype analyses of Australian pedigrees has further refined the localization to a 2 cM interval between markers D9S196 and D9S180. Our loss of heterozygosity (LOH) studies from sporadic (n= 58) and familial (n=41) BCCs indicate that 50% havemore » deletions within the NBCCS candidate region. All LOH is consistent with the genetic mapping of the NBCCS locus. Additionally, one sporadic tumor indicates that the smallest region of overlap in the deletions is within the interval D9S287 (proximal) and D9S180 (distal). A series of YAC clones from within this region has been mapped by FISH to examine chimerism. These clones, which have been mapped with respect to one another, form a contig which encompasses the candidate region from D9S196 to D9S180.« less

Innate immunity and the new forward genetics

PubMed Central

Beutler, Bruce

2016-01-01

As it is a hard-wired system for responses to microbes, innate immunity is particularly susceptible to classical genetic analysis. Mutations led the way to the discovery of many of the molecular elements of innate immune sensing and signaling pathways. In turn, the need for a faster way to find the molecular causes of mutation-induced phenotypes triggered a huge transformation in forward genetics. During the 1980s and 1990s, many heritable phenotypes were ascribed to mutations through positional cloning. In mice, this required three steps. First, a genetic mapping step was used to show that a given phenotype emanated from a circumscribed region of the genome. Second, a physical mapping step was undertaken, in which all of the region was cloned and its gene content determined. Finally, a concerted search for the mutation was performed. Such projects usually lasted for several years, but could produce breakthroughs in our understanding of biological processes. Publication of the annotated mouse genome sequence in 2002 made physical mapping unnecessary. More recently we devised a new technology for automated genetic mapping, which eliminated both genetic mapping and the search for mutations among candidate genes. The cause of phenotype can now be determined instantaneously. We have created more than 100,000 coding/splicing mutations. And by screening for defects of innate and adaptive immunity we have discovered many “new” proteins needed for innate immune function. PMID:27890263

A complete YAC contig of the Prader-Willi/Angelman chromosome region (15q11-q13) and refined localization of the SNRPN gene

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

Mutirangura, A.; Jayakumar, A.; Sutcliffe, J.S.

1993-12-01

Since a previous report of a partial YAC contig of the Prader-Willi/Angelman chromosome region (15q11-q13), a complete contig spanning approximately 3.5 Mb has been developed. YACs were isolated from two human genomic libraries by PCR and hybridization screening methods. Twenty-three sequence-tagged sites (STSs) were mapped within the contig, a density of [approximately]1 per 200 kb. Overlaps between YAC clones were identified by Alu-PCR dot-blot analysis and confirmed by STS mapping or hybridization with ends of YAC inserts. The gene encoding small nuclear ribonucleoprotein-associated peptide N (SNRPN), recently identified as a candidate gene for Prader-Willi syndrome, was localized within this contigmore » between markers PW71 and TD3-21. Loci mapped within and immediately flanking the Prader-Willi/Angelman chromosome region contig are ordered as follows: cen-IR39-ML34-IR4-3R-TD189-1-PW71-SNRPN-TD3-21-LS6-1-GABRB3,D15S97-GABRA5-IR10-1-CMW1-tel. This YAC contig will be a useful resource for more detailed physical mapping of the region, for generation of new DNA markers, and for mapping or cloning candidate genes for the Prader-Willi and Angelman syndromes. 36 refs., 2 figs., 2 tabs.« less

Mapping and characterization of vicriviroc resistance mutations from HIV-1 isolated from treatment-experienced subjects enrolled in a phase II study (VICTOR-E1).

PubMed

McNicholas, Paul M; Mann, Paul A; Wojcik, Lisa; Qiu, Ping; Lee, Erin; McCarthy, Michael; Shen, Junwu; Black, Todd A; Strizki, Julie M

2011-03-01

In the phase 2 VICTOR-E1 study, treatment-experienced subjects receiving 20 mg or 30 mg of the CCR5 antagonist vicriviroc (VCV), with a boosted protease containing optimized background regimen, experienced significantly greater reductions in HIV-1 viral load compared with control subjects. Among the 79 VCV-treated subjects, 15 experienced virologic failure, and of these 5 had VCV-resistant virus. This study investigated the molecular basis for the changes in susceptibility to VCV in these subjects. Sequence analysis and phenotypic susceptibility testing was performed on envelope clones from VCV-resistant virus. For select clones, an exchange of mutations in the V3 loop was performed between phenotypically resistant clones and the corresponding susceptible clones. Phenotypic resistance was manifest by reductions in the maximum percent inhibition. Clonal analysis of envelopes from the 5 subjects identified multiple amino acid changes in gp160 that were exclusive to the resistant clones, however, none of the changes were conserved between subjects. Introduction of V3 loop substitutions from the resistant clones into the matched susceptible clones was not sufficient to reproduce the resistant phenotype. Likewise, changing the substitutions in the V3 loops from resistant clones to match susceptible clones only restored susceptibility in 1 clone. There were no clearly conserved patterns of mutations in gp160 associated with phenotypic resistance to VCV and mutations both within and outside of the V3 loop contributed to the resistance phenotype. These data suggest that genotypic tests for VCV susceptibility may require larger training sets and additional information beyond V3 sequences.

A High-Density Consensus Map of Common Wheat Integrating Four Mapping Populations Scanned by the 90K SNP Array

PubMed Central

Wen, Weie; He, Zhonghu; Gao, Fengmei; Liu, Jindong; Jin, Hui; Zhai, Shengnan; Qu, Yanying; Xia, Xianchun

2017-01-01

A high-density consensus map is a powerful tool for gene mapping, cloning and molecular marker-assisted selection in wheat breeding. The objective of this study was to construct a high-density, single nucleotide polymorphism (SNP)-based consensus map of common wheat (Triticum aestivum L.) by integrating genetic maps from four recombinant inbred line populations. The populations were each genotyped using the wheat 90K Infinium iSelect SNP assay. A total of 29,692 SNP markers were mapped on 21 linkage groups corresponding to 21 hexaploid wheat chromosomes, covering 2,906.86 cM, with an overall marker density of 10.21 markers/cM. Compared with the previous maps based on the wheat 90K SNP chip detected 22,736 (76.6%) of the SNPs with consistent chromosomal locations, whereas 1,974 (6.7%) showed different chromosomal locations, and 4,982 (16.8%) were newly mapped. Alignment of the present consensus map and the wheat expressed sequence tags (ESTs) Chromosome Bin Map enabled assignment of 1,221 SNP markers to specific chromosome bins and 819 ESTs were integrated into the consensus map. The marker orders of the consensus map were validated based on physical positions on the wheat genome with Spearman rank correlation coefficients ranging from 0.69 (4D) to 0.97 (1A, 4B, 5B, and 6A), and were also confirmed by comparison with genetic position on the previously 40K SNP consensus map with Spearman rank correlation coefficients ranging from 0.84 (6D) to 0.99 (6A). Chromosomal rearrangements reported previously were confirmed in the present consensus map and new putative rearrangements were identified. In addition, an integrated consensus map was developed through the combination of five published maps with ours, containing 52,607 molecular markers. The consensus map described here provided a high-density SNP marker map and a reliable order of SNPs, representing a step forward in mapping and validation of chromosomal locations of SNPs on the wheat 90K array. Moreover, it can be used as a reference for quantitative trait loci (QTL) mapping to facilitate exploitation of genes and QTL in wheat breeding. PMID:28848588

In silico pharmacology for drug discovery: applications to targets and beyond

PubMed Central

Ekins, S; Mestres, J; Testa, B

2007-01-01

Computational (in silico) methods have been developed and widely applied to pharmacology hypothesis development and testing. These in silico methods include databases, quantitative structure-activity relationships, similarity searching, pharmacophores, homology models and other molecular modeling, machine learning, data mining, network analysis tools and data analysis tools that use a computer. Such methods have seen frequent use in the discovery and optimization of novel molecules with affinity to a target, the clarification of absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization. The first part of this review discussed the methods that have been used for virtual ligand and target-based screening and profiling to predict biological activity. The aim of this second part of the review is to illustrate some of the varied applications of in silico methods for pharmacology in terms of the targets addressed. We will also discuss some of the advantages and disadvantages of in silico methods with respect to in vitro and in vivo methods for pharmacology research. Our conclusion is that the in silico pharmacology paradigm is ongoing and presents a rich array of opportunities that will assist in expediating the discovery of new targets, and ultimately lead to compounds with predicted biological activity for these novel targets. PMID:17549046

In silico characterization of a novel pathogenic deletion mutation identified in XPA gene in a Pakistani family with severe xeroderma pigmentosum

PubMed Central

2013-01-01

Background Xeroderma Pigmentosum (XP) is a rare skin disorder characterized by skin hypersensitivity to sunlight and abnormal pigmentation. The aim of this study was to investigate the genetic cause of a severe XP phenotype in a consanguineous Pakistani family and in silico characterization of any identified disease-associated mutation. Results The XP complementation group was assigned by genotyping of family for known XP loci. Genotyping data mapped the family to complementation group A locus, involving XPA gene. Mutation analysis of the candidate XP gene by DNA sequencing revealed a novel deletion mutation (c.654del A) in exon 5 of XPA gene. The c.654del A, causes frameshift, which pre-maturely terminates protein and result into a truncated product of 222 amino acid (aa) residues instead of 273 (p.Lys218AsnfsX5). In silico tools were applied to study the likelihood of changes in structural motifs and thus interaction of mutated protein with binding partners. In silico analysis of mutant protein sequence, predicted to affect the aa residue which attains coiled coil structure. The coiled coil structure has an important role in key cellular interactions, especially with DNA damage-binding protein 2 (DDB2), which has important role in DDB-mediated nucleotide excision repair (NER) system. Conclusions Our findings support the fact of genetic and clinical heterogeneity in XP. The study also predicts the critical role of DDB2 binding region of XPA protein in NER pathway and opens an avenue for further research to study the functional role of the mutated protein domain. PMID:24063568

Transcriptional profiles of Rel/NF-κB, inhibitor of NF-κB (IκB), and lipopolysaccharide-induced TNF-α factor (LITAF) in the lipopolysaccharide (LPS) and two Vibrio sp.-exposed intertidal copepod, Tigriopus japonicus.

PubMed

Kim, Bo-Mi; Jeong, Chang-Bum; Rhee, Jae-Sung; Lee, Jae-Seong

2014-02-01

The immune system and the role of immunity-related genes have rarely been studied in copepods, even though copepods have a primitive immune response system and also have a potential in pathogen transport higher trophic levels. In this study, we firstly cloned and characterized three core immune genes such as nuclear factor κB (NF-κB), inhibitor of NF-κB (IκB), and lipopolysaccharide-induced TNF-α factor (LITAF) genes in the intertidal copepod Tigriopus japonicus. Several in silico analyses based on conserved domains, motifs, and phylogenetic relationships were supporting their annotations. To investigate the immune-related role of three genes, we exposed lipopolysaccharide (LPS) and two Vibrio sp. to T. japonicus. After exposure of different concentrations of LPS and two Vibrio sp., transcripts of TJ-IκB and TJ-LITAF genes were significantly elevated during the time course in a dose-dependent manner, while TJ-NF-κB transcripts were not significantly changed during exposure. These findings demonstrated that the copepod T. japonicus has a conserved immunity against infection. Copyright © 2013 Elsevier Ltd. All rights reserved.

A high density genetic map and QTL for agronomic and yield traits in Foxtail millet [Setaria italica (L.) P. Beauv].

PubMed

Fang, Xiaomei; Dong, Kongjun; Wang, Xiaoqin; Liu, Tianpeng; He, Jihong; Ren, Ruiyu; Zhang, Lei; Liu, Rui; Liu, Xueying; Li, Man; Huang, Mengzhu; Zhang, Zhengsheng; Yang, Tianyu

2016-05-04

Foxtail millet [Setaria italica (L.) P. Beauv.], a crop of historical importance in China, has been adopted as a model crop for studying C-4 photosynthesis, stress biology and biofuel traits. Construction of a high density genetic map and identification of stable quantitative trait loci (QTL) lay the foundation for marker-assisted selection for agronomic traits and yield improvement. A total of 10598 SSR markers were developed according to the reference genome sequence of foxtail millet cultivar 'Yugu1'. A total of 1013 SSR markers showing polymorphism between Yugu1 and Longgu7 were used to genotype 167 individuals from a Yugu1 × Longgu7 F2 population, and a high density genetic map was constructed. The genetic map contained 1035 loci and spanned 1318.8 cM with an average distance of 1.27 cM between adjacent markers. Based on agronomic and yield traits identified in 2 years, 29 QTL were identified for 11 traits with combined analysis and single environment analysis. These QTL explained from 7.0 to 14.3 % of phenotypic variation. Favorable QTL alleles for peduncle length originated from Longgu7 whereas favorable alleles for the other traits originated from Yugu1 except for qLMS6.1. New SSR markers, a high density genetic map and QTL identified for agronomic and yield traits lay the ground work for functional gene mapping, map-based cloning and marker-assisted selection in foxtail millet.

Small RNA analysis in Petunia hybrida identifies unusual tissue-specific expression patterns of conserved miRNAs and of a 24mer RNA

PubMed Central

Tedder, Philip; Zubko, Elena; Westhead, David R.; Meyer, Peter

2009-01-01

Two pools of small RNAs were cloned from inflorescences of Petunia hybrida using a 5′-ligation dependent and a 5′-ligation independent approach. The two libraries were integrated into a public website that allows the screening of individual sequences against 359,769 unique clones. The library contains 15 clones with 100% identity and 53 clones with one mismatch to miRNAs described for other plant species. For two conserved miRNAs, miR159 and miR390, we find clear differences in tissue-specific distribution, compared with other species. This shows that evolutionary conservation of miRNA sequences does not necessarily include a conservation of the miRNA expression profile. Almost 60% of all clones in the database are 24-nucleotide clones. In accordance with the role of 24mers in marking repetitive regions, we find them distributed across retroviral and transposable element sequences but other 24mers map to promoter regions and to different transcript regions. For one target region we observe tissue-specific variation of matching 24mers, which demonstrates that, as for 21mers, 24mer concentrations are not necessarily identical in different tissues. Asymmetric distribution of a putative novel miRNA in the two libraries suggests that the cloning method can be selective for the representation of certain small RNAs in a collection. PMID:19369427

Advances in synthetic peptides reagent discovery

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Stratis-Cullum, Dimitra N.

2013-05-01

Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the rapid discovery and development of peptides with interaction targeted to materials ranging from biological hazards through inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial display technology is relatively simple and rapid to make laboratory automation possible. This included extensive study of the protective antigen system of Bacillus anthracis, including development of discovery, characterization, and computational biology capabilities for in-silico optimization. Although the benefits towards CBD goals are evident, the impact is far-reaching due to our ability to understand and harness peptide interactions that are ultimately extendable to the hybrid biomaterials of the future. In this paper, we describe advances in peptide discovery including, new target systems (e.g. non-biological materials), advanced library development and clone analysis including integrated reporting.

Chromosome mapping, molecular cloning and expression analysis of a novel gene response for leaf width in rice.

PubMed

Wu, Yahui; Luo, Lixin; Chen, Likai; Tao, Xingxing; Huang, Ming; Wang, Hui; Chen, Zhiqiang; Xiao, Wuming

2016-11-18

Genetic analysis revealed that narrow leaf, small panicle, thin and slender stems as well as low fertility rate of an Indica rice variety were recessive traits and controlled by a single gene. Applying map-based cloning strategy, a novel narrow leaf gene, which was named nal11 was delimited to an interval of 58.3 kb between the InDel markers N10 and InD5016. There are 9 genes in the mapping interval, and only a heat shock DNAJ protein encode gene (Os07g09450) has a specific G to T SNP, which was occurred at the last base of the second exon of Os07g09450 in ZYX. 5' and 3' RACE result shown that there were two transcripts in NAL11, and the SNP in nal11 leads to a variable shear of mRNA. In addition, this type of mRNA alternative splicing together with a stop codon closely followed the SNP which caused termination of translation destroyed the DNAJ domain of nal11's product. These results suggested that the heat shock DNAJ gene was most likely to be the candidate gene of nal11. The results of RT-PCR and real-time PCR further verified that the SNP in the ZYX-nal11 gene affects mRNA splicing pattern. Phenotype of ZYX may be caused by a statistically significant reduction in the total number of small veins in leaf, size and number of small vascular bundles and cells in stems, similar to several previous reported mutations. The basic molecular information we provide here will be useful for further investigations of the physiological function of the heat shock DNAJ gene, which will be helpful in better understanding the role of the DNAJ family in regulation of plant type traits such as leaf width of rice. Copyright © 2016 Elsevier Inc. All rights reserved.

An Integrated Genomic Approach for Rapid Delineation of Candidate Genes Regulating Agro-Morphological Traits in Chickpea

PubMed Central

Saxena, Maneesha S.; Bajaj, Deepak; Das, Shouvik; Kujur, Alice; Kumar, Vinod; Singh, Mohar; Bansal, Kailash C.; Tyagi, Akhilesh K.; Parida, Swarup K.

2014-01-01

The identification and fine mapping of robust quantitative trait loci (QTLs)/genes governing important agro-morphological traits in chickpea still lacks systematic efforts at a genome-wide scale involving wild Cicer accessions. In this context, an 834 simple sequence repeat and single-nucleotide polymorphism marker-based high-density genetic linkage map between cultivated and wild parental accessions (Cicer arietinum desi cv. ICC 4958 and Cicer reticulatum wild cv. ICC 17160) was constructed. This inter-specific genetic map comprising eight linkage groups spanned a map length of 949.4 cM with an average inter-marker distance of 1.14 cM. Eleven novel major genomic regions harbouring 15 robust QTLs (15.6–39.8% R2 at 4.2–15.7 logarithm of odds) associated with four agro-morphological traits (100-seed weight, pod and branch number/plant and plant hairiness) were identified and mapped on chickpea chromosomes. Most of these QTLs showed positive additive gene effects with effective allelic contribution from ICC 4958, particularly for increasing seed weight (SW) and pod and branch number. One robust SW-influencing major QTL region (qSW4.2) has been narrowed down by combining QTL mapping with high-resolution QTL region-specific association analysis, differential expression profiling and gene haplotype-based association/LD mapping. This enabled to delineate a strong SW-regulating ABI3VP1 transcription factor (TF) gene at trait-specific QTL interval and consequently identified favourable natural allelic variants and superior high seed weight-specific haplotypes in the upstream regulatory region of this gene showing increased transcript expression during seed development. The genes (TFs) harbouring diverse trait-regulating QTLs, once validated and fine-mapped by our developed rapid integrated genomic approach and through gene/QTL map-based cloning, can be utilized as potential candidates for marker-assisted genetic enhancement of chickpea. PMID:25335477

A Genetic Linkage Map for Cattle

PubMed Central

Bishop, M. D.; Kappes, S. M.; Keele, J. W.; Stone, R. T.; Sunden, SLF.; Hawkins, G. A.; Toldo, S. S.; Fries, R.; Grosz, M. D.; Yoo, J.; Beattie, C. W.

1994-01-01

We report the most extensive physically anchored linkage map for cattle produced to date. Three-hundred thirteen genetic markers ordered in 30 linkage groups, anchored to 24 autosomal chromosomes (n = 29), the X and Y chromosomes, four unanchored syntenic groups and two unassigned linkage groups spanning 2464 cM of the bovine genome are summarized. The map also assigns 19 type I loci to specific chromosomes and/or syntenic groups and four cosmid clones containing informative microsatellites to chromosomes 13, 25 and 29 anchoring syntenic groups U11, U7 and U8, respectively. This map provides the skeletal framework prerequisite to development of a comprehensive genetic map for cattle and analysis of economic trait loci (ETL). PMID:7908653

Surpassing the no-cloning limit with a heralded hybrid linear amplifier for coherent states

PubMed Central

Haw, Jing Yan; Zhao, Jie; Dias, Josephine; Assad, Syed M.; Bradshaw, Mark; Blandino, Rémi; Symul, Thomas; Ralph, Timothy C.; Lam, Ping Koy

2016-01-01

The no-cloning theorem states that an unknown quantum state cannot be cloned exactly and deterministically due to the linearity of quantum mechanics. Associated with this theorem is the quantitative no-cloning limit that sets an upper bound to the quality of the generated clones. However, this limit can be circumvented by abandoning determinism and using probabilistic methods. Here, we report an experimental demonstration of probabilistic cloning of arbitrary coherent states that clearly surpasses the no-cloning limit. Our scheme is based on a hybrid linear amplifier that combines an ideal deterministic linear amplifier with a heralded measurement-based noiseless amplifier. We demonstrate the production of up to five clones with the fidelity of each clone clearly exceeding the corresponding no-cloning limit. Moreover, since successful cloning events are heralded, our scheme has the potential to be adopted in quantum repeater, teleportation and computing applications. PMID:27782135

A novel tandem repeat sequence located on human chromosome 4p: isolation and characterization.

PubMed

Kogi, M; Fukushige, S; Lefevre, C; Hadano, S; Ikeda, J E

1997-06-01

In an effort to analyze the genomic region of the distal half of human chromosome 4p, to where Huntington disease and other diseases have been mapped, we have isolated the cosmid clone (CRS447) that was likely to contain a region with specific repeat sequences. Clone CRS447 was subjected to detailed analysis, including chromosome mapping, restriction mapping, and DNA sequencing. Chromosome mapping by both a human-CHO hybrid cell panel and FISH revealed that CRS447 was predominantly located in the 4p15.1-15.3 region. CRS447 was shown to consist of tandem repeats of 4.7-kb units present on chromosome 4p. A single EcoRI unit was subcloned (pRS447), and the complete sequence was determined as 4752 nucleotides. When pRS447 was used as a probe, the number of copies of this repeat per haploid genome was estimated to be 50-70. Sequence analysis revealed that it contained two internal CA repeats and one putative ORF. Database search established that this sequence was unreported. However, two homologous STS markers were found in the database. We concluded that CRS447/pRS447 is a novel tandem repeat sequence that is mainly specific to human chromosome 4p.

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

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

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

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

Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats.

PubMed

Gibbs, M; Dawson, D A; McCamley, C; Wardle, A F; Armour, J A; Burke, T

1997-12-01

The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0.1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.

Recent progress on the genetics and molecular breeding of brown planthopper resistance in rice.

PubMed

Hu, Jie; Xiao, Cong; He, Yuqing

2016-12-01

Brown planthopper (BPH) is the most devastating pest of rice. Host-plant resistance is the most desirable and economic strategy in the management of BPH. To date, 29 major BPH resistance genes have been identified from indica cultivars and wild rice species, and more than ten genes have been fine mapped to chromosome regions of less than 200 kb. Four genes (Bph14, Bph26, Bph17 and bph29) have been cloned. The increasing number of fine-mapped and cloned genes provide a solid foundation for development of functional markers for use in breeding. Several BPH resistant introgression lines (ILs), near-isogenic lines (NILs) and pyramided lines (PLs) carrying single or multiple resistance genes were developed by marker assisted backcross breeding (MABC). Here we review recent progress on the genetics and molecular breeding of BPH resistance in rice. Prospect for developing cultivars with durable, broad-spectrum BPH resistance are discussed.

Genomic stability in the archaeae Haloferax volcanii and Haloferax mediterranei.

PubMed Central

López-García, P; St Jean, A; Amils, R; Charlebois, R L

1995-01-01

Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10(7) years of these species' divergent evolution. PMID:7868620

Anhidrotic ectodermal dysplasia gene region cloned in yeast artificial chromosomes

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

Kere, J.; Grzeschik, K.H.; Limon, J.

1993-05-01

Anhidrotic ectodermal dysplasia (EDA), an X-chromosomal recessive disorder, is expressed in a few females with chromosomal translocations involving bands Xq12-q13. Using available DNA markers from the region and somatic cell hybrids the authors mapped the X-chromosomal breakpoints in two such translocations. The breakpoints were further mapped within a yeast artificial chromosome contig constructed by chromosome walking techniques. Genomic DNA markers that map between the two translocation breakpoints were recovered representing putative portions of the EDA gene. 32 refs., 3 figs., 1 tab.

Forgotten forests--issues and prospects in biome mapping using Seasonally Dry Tropical Forests as a case study.

PubMed

Särkinen, Tiina; Iganci, João R V; Linares-Palomino, Reynaldo; Simon, Marcelo F; Prado, Darién E

2011-11-24

South America is one of the most species diverse continents in the world. Within South America diversity is not distributed evenly at both local and continental scales and this has led to the recognition of various areas with unique species assemblages. Several schemes currently exist which divide the continental-level diversity into large species assemblages referred to as biomes. Here we review five currently available biome maps for South America, including the WWF Ecoregions, the Americas basemap, the Land Cover Map of South America, Morrone's Biogeographic regions of Latin America, and the Ecological Systems Map. The comparison is performed through a case study on the Seasonally Dry Tropical Forest (SDTF) biome using herbarium data of habitat specialist species. Current biome maps of South America perform poorly in depicting SDTF distribution. The poor performance of the maps can be attributed to two main factors: (1) poor spatial resolution, and (2) poor biome delimitation. Poor spatial resolution strongly limits the use of some of the maps in GIS applications, especially for areas with heterogeneous landscape such as the Andes. Whilst the Land Cover Map did not suffer from poor spatial resolution, it showed poor delimitation of biomes. The results highlight that delimiting structurally heterogeneous vegetation is difficult based on remote sensed data alone. A new refined working map of South American SDTF biome is proposed, derived using the Biome Distribution Modelling (BDM) approach where georeferenced herbarium data is used in conjunction with bioclimatic data. Georeferenced specimen data play potentially an important role in biome mapping. Our study shows that herbarium data could be used as a way of ground-truthing biome maps in silico. The results also illustrate that herbarium data can be used to model vegetation maps through predictive modelling. The BDM approach is a promising new method in biome mapping, and could be particularly useful for mapping poorly known, fragmented, or degraded vegetation. We wish to highlight that biome delimitation is not an exact science, and that transparency is needed on how biomes are used as study units in macroevolutionary and ecological research.

Forgotten forests - issues and prospects in biome mapping using Seasonally Dry Tropical Forests as a case study

PubMed Central

2011-01-01

Background South America is one of the most species diverse continents in the world. Within South America diversity is not distributed evenly at both local and continental scales and this has led to the recognition of various areas with unique species assemblages. Several schemes currently exist which divide the continental-level diversity into large species assemblages referred to as biomes. Here we review five currently available biome maps for South America, including the WWF Ecoregions, the Americas basemap, the Land Cover Map of South America, Morrone's Biogeographic regions of Latin America, and the Ecological Systems Map. The comparison is performed through a case study on the Seasonally Dry Tropical Forest (SDTF) biome using herbarium data of habitat specialist species. Results Current biome maps of South America perform poorly in depicting SDTF distribution. The poor performance of the maps can be attributed to two main factors: (1) poor spatial resolution, and (2) poor biome delimitation. Poor spatial resolution strongly limits the use of some of the maps in GIS applications, especially for areas with heterogeneous landscape such as the Andes. Whilst the Land Cover Map did not suffer from poor spatial resolution, it showed poor delimitation of biomes. The results highlight that delimiting structurally heterogeneous vegetation is difficult based on remote sensed data alone. A new refined working map of South American SDTF biome is proposed, derived using the Biome Distribution Modelling (BDM) approach where georeferenced herbarium data is used in conjunction with bioclimatic data. Conclusions Georeferenced specimen data play potentially an important role in biome mapping. Our study shows that herbarium data could be used as a way of ground-truthing biome maps in silico. The results also illustrate that herbarium data can be used to model vegetation maps through predictive modelling. The BDM approach is a promising new method in biome mapping, and could be particularly useful for mapping poorly known, fragmented, or degraded vegetation. We wish to highlight that biome delimitation is not an exact science, and that transparency is needed on how biomes are used as study units in macroevolutionary and ecological research. PMID:22115315

Genomics of interaction between the brown planthopper and rice.

PubMed

Jing, Shengli; Zhao, Yan; Du, Bo; Chen, Rongzhi; Zhu, Lili; He, Guangcun

2017-02-01

Rice (Oryza sativa L.) and the brown planthopper (Nilaparvata lugens (Stål)) form a model system for dissection of the mechanism of interaction between insect pest and crop. In this review, we focus on the genomics of BPH-rice interaction. On the side of rice, a number of BPH-resistance genes have been identified genetically. Thirteen of these genes have been cloned which shed a light on the molecular basis of the interaction. On the aspect of BPH, a lot of salivary proteins have been identified using transcriptome and proteome techniques. The genetic loci of virulence were mapped in BPH genome based on the linkage map. The understanding of interaction between BPH and rice will provide novel insights into efficient control of this pest. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Genotyping by Sequencing in Almond: SNP Discovery, Linkage Mapping, and Marker Design.

PubMed

Goonetilleke, Shashi N; March, Timothy J; Wirthensohn, Michelle G; Arús, Pere; Walker, Amanda R; Mather, Diane E

2018-01-04

In crop plant genetics, linkage maps provide the basis for the mapping of loci that affect important traits and for the selection of markers to be applied in crop improvement. In outcrossing species such as almond ( Prunus dulcis Mill. D. A. Webb), application of a double pseudotestcross mapping approach to the F 1 progeny of a biparental cross leads to the construction of a linkage map for each parent. Here, we report on the application of genotyping by sequencing to discover and map single nucleotide polymorphisms in the almond cultivars "Nonpareil" and "Lauranne." Allele-specific marker assays were developed for 309 tag pairs. Application of these assays to 231 Nonpareil × Lauranne F 1 progeny provided robust linkage maps for each parent. Analysis of phenotypic data for shell hardness demonstrated the utility of these maps for quantitative trait locus mapping. Comparison of these maps to the peach genome assembly confirmed high synteny and collinearity between the peach and almond genomes. The marker assays were applied to progeny from several other Nonpareil crosses, providing the basis for a composite linkage map of Nonpareil. Applications of the assays to a panel of almond clones and a panel of rootstocks used for almond production demonstrated the broad applicability of the markers and provide subsets of markers that could be used to discriminate among accessions. The sequence-based linkage maps and single nucleotide polymorphism assays presented here could be useful resources for the genetic analysis and genetic improvement of almond. Copyright © 2018 Goonetilleke et al.

In Silico Prediction of Organ Level Toxicity: Linking Chemistry to Adverse Effects

PubMed Central

Cronin, Mark T.D.; Enoch, Steven J.; Mellor, Claire L.; Przybylak, Katarzyna R.; Richarz, Andrea-Nicole; Madden, Judith C.

2017-01-01

In silico methods to predict toxicity include the use of (Quantitative) Structure-Activity Relationships ((Q)SARs) as well as grouping (category formation) allowing for read-across. A challenging area for in silico modelling is the prediction of chronic toxicity and the No Observed (Adverse) Effect Level (NO(A)EL) in particular. A proposed solution to the prediction of chronic toxicity is to consider organ level effects, as opposed to modelling the NO(A)EL itself. This review has focussed on the use of structural alerts to identify potential liver toxicants. In silico profilers, or groups of structural alerts, have been developed based on mechanisms of action and informed by current knowledge of Adverse Outcome Pathways. These profilers are robust and can be coded computationally to allow for prediction. However, they do not cover all mechanisms or modes of liver toxicity and recommendations for the improvement of these approaches are given. PMID:28744348

In Silico Prediction of Organ Level Toxicity: Linking Chemistry to Adverse Effects.

PubMed

Cronin, Mark T D; Enoch, Steven J; Mellor, Claire L; Przybylak, Katarzyna R; Richarz, Andrea-Nicole; Madden, Judith C

2017-07-01

In silico methods to predict toxicity include the use of (Quantitative) Structure-Activity Relationships ((Q)SARs) as well as grouping (category formation) allowing for read-across. A challenging area for in silico modelling is the prediction of chronic toxicity and the No Observed (Adverse) Effect Level (NO(A)EL) in particular. A proposed solution to the prediction of chronic toxicity is to consider organ level effects, as opposed to modelling the NO(A)EL itself. This review has focussed on the use of structural alerts to identify potential liver toxicants. In silico profilers, or groups of structural alerts, have been developed based on mechanisms of action and informed by current knowledge of Adverse Outcome Pathways. These profilers are robust and can be coded computationally to allow for prediction. However, they do not cover all mechanisms or modes of liver toxicity and recommendations for the improvement of these approaches are given.

Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

PubMed Central

Sircar, Gaurab; Saha, Bodhisattwa; Mandal, Rahul Shubhra; Pandey, Naren; Saha, Sudipto; Gupta Bhattacharya, Swati

2015-01-01

Background Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation ‘Rhi o 1’. Method The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies. Results The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross-stimulate histamine release from the effector cells sensitized with anti-Rhi o 1 serum IgE. This cross-reactivity was found to be mediated by a common mAb4C3 recognizable conformational epitope. Bioinformatic studies revealed high degree of structural resemblances between the 4C3 binding sites of both the allergens. Conclusion/Significance The present study reports for the first time anew fungal aspartic protease allergen designated as Rhi o 1, which triggers IgE-mediated sensitization leading to various allergic diseases. Here we have characterized the recombinant Rhi o 1 and its immunological features including cross-reactive epitope information that will facilitate the component-resolved diagnosis of mold allergy. PMID:26672984

IC-Finder: inferring robustly the hierarchical organization of chromatin folding

PubMed Central

Haddad, Noelle

2017-01-01

Abstract The spatial organization of the genome plays a crucial role in the regulation of gene expression. Recent experimental techniques like Hi-C have emphasized the segmentation of genomes into interaction compartments that constitute conserved functional domains participating in the maintenance of a proper cell identity. Here, we propose a novel method, IC-Finder, to identify interaction compartments (IC) from experimental Hi-C maps. IC-Finder is based on a hierarchical clustering approach that we adapted to account for the polymeric nature of chromatin. Based on a benchmark of realistic in silico Hi-C maps, we show that IC-Finder is one of the best methods in terms of reliability and is the most efficient numerically. IC-Finder proposes two original options: a probabilistic description of the inferred compartments and the possibility to explore the various hierarchies of chromatin organization. Applying the method to experimental data in fly and human, we show how the predicted segmentation may depend on the normalization scheme and how 3D compartmentalization is tightly associated with epigenomic information. IC-Finder provides a robust and generic ‘all-in-one’ tool to uncover the general principles of 3D chromatin folding and their influence on gene regulation. The software is available at http://membres-timc.imag.fr/Daniel.Jost/DJ-TIMC/Software.html. PMID:28130423

A polyalanine peptide derived from polar fish with anti-infectious activities

NASA Astrophysics Data System (ADS)

Cardoso, Marlon H.; Ribeiro, Suzana M.; Nolasco, Diego O.; de La Fuente-Núñez, César; Felício, Mário R.; Gonçalves, Sónia; Matos, Carolina O.; Liao, Luciano M.; Santos, Nuno C.; Hancock, Robert E. W.; Franco, Octávio L.; Migliolo, Ludovico

2016-02-01

Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria.

Rat prostatic steroid binding protein: DNA sequence and transcript maps of the two C3 genes.

PubMed Central

Hurst, H C; Parker, M G

1983-01-01

In the rat there are two non-allelic genes C3(1) and C3(2) for the C3 polypeptide of prostatic steroid binding protein. We have cloned and sequenced both genes and show that only C3(1) is responsible for the production of authentic C3. Although there is a marked difference in their transcriptional activity, the two genes share extensive DNA sequence homology there being only one base difference from nucleotide - 235 to within the first intron. Transcript mapping has shown that there are two distinct C3 transcripts which share a unique 3' terminus but have 5' termini 38 bases apart each preceded by a 'TATA' box homology. Interestingly, an identical repetitive element is present just upstream of both genes. Both families of transcripts, which are produced in a ratio of 18:1, are coordinately regulated by testosterone. Images Fig. 3. Fig. 4. Fig. 5. PMID:6685625

Development of Genic and Genomic SSR Markers of Robusta Coffee (Coffea canephora Pierre Ex A. Froehner)

PubMed Central

Hendre, Prasad S.; Aggarwal, Ramesh K.

2014-01-01

Coffee breeding and improvement efforts can be greatly facilitated by availability of a large repository of simple sequence repeats (SSRs) based microsatellite markers, which provides efficiency and high-resolution in genetic analyses. This study was aimed to improve SSR availability in coffee by developing new genic−/genomic-SSR markers using in-silico bioinformatics and streptavidin-biotin based enrichment approach, respectively. The expressed sequence tag (EST) based genic microsatellite markers (EST-SSRs) were developed using the publicly available dataset of 13,175 unigene ESTs, which showed a distribution of 1 SSR/3.4 kb of coffee transcriptome. Genomic SSRs, on the other hand, were developed from an SSR-enriched small-insert partial genomic library of robusta coffee. In total, 69 new SSRs (44 EST-SSRs and 25 genomic SSRs) were developed and validated as suitable genetic markers. Diversity analysis of selected coffee genotypes revealed these to be highly informative in terms of allelic diversity and PIC values, and eighteen of these markers (∼27%) could be mapped on a robusta linkage map. Notably, the markers described here also revealed a very high cross-species transferability. In addition to the validated markers, we have also designed primer pairs for 270 putative EST-SSRs, which are expected to provide another ca. 200 useful genetic markers considering the high success rate (88%) of marker conversion of similar pairs tested/validated in this study. PMID:25461752

Discovery of Pod Shatter-Resistant Associated SNPs by Deep Sequencing of a Representative Library Followed by Bulk Segregant Analysis in Rapeseed

PubMed Central

Huang, Shunmou; Yang, Hongli; Zhan, Gaomiao; Wang, Xinfa; Liu, Guihua; Wang, Hanzhong

2012-01-01

Background Single nucleotide polymorphisms (SNPs) are an important class of genetic marker for target gene mapping. As of yet, there is no rapid and effective method to identify SNPs linked with agronomic traits in rapeseed and other crop species. Methodology/Principal Findings We demonstrate a novel method for identifying SNP markers in rapeseed by deep sequencing a representative library and performing bulk segregant analysis. With this method, SNPs associated with rapeseed pod shatter-resistance were discovered. Firstly, a reduced representation of the rapeseed genome was used. Genomic fragments ranging from 450–550 bp were prepared from the susceptible bulk (ten F2 plants with the silique shattering resistance index, SSRI <0.10) and the resistance bulk (ten F2 plants with SSRI >0.90), and also Solexa sequencing-produced 90 bp reads. Approximately 50 million of these sequence reads were assembled into contigs to a depth of 20-fold coverage. Secondly, 60,396 ‘simple SNPs’ were identified, and the statistical significance was evaluated using Fisher's exact test. There were 70 associated SNPs whose –log10 p value over 16 were selected to be further analyzed. The distribution of these SNPs appeared a tight cluster, which consisted of 14 associated SNPs within a 396 kb region on chromosome A09. Our evidence indicates that this region contains a major quantitative trait locus (QTL). Finally, two associated SNPs from this region were mapped on a major QTL region. Conclusions/Significance 70 associated SNPs were discovered and a major QTL for rapeseed pod shatter-resistance was found on chromosome A09 using our novel method. The associated SNP markers were used for mapping of the QTL, and may be useful for improving pod shatter-resistance in rapeseed through marker-assisted selection and map-based cloning. This approach will accelerate the discovery of major QTLs and the cloning of functional genes for important agronomic traits in rapeseed and other crop species. PMID:22529909

Human In Silico Drug Trials Demonstrate Higher Accuracy than Animal Models in Predicting Clinical Pro-Arrhythmic Cardiotoxicity.

PubMed

Passini, Elisa; Britton, Oliver J; Lu, Hua Rong; Rohrbacher, Jutta; Hermans, An N; Gallacher, David J; Greig, Robert J H; Bueno-Orovio, Alfonso; Rodriguez, Blanca

2017-01-01

Early prediction of cardiotoxicity is critical for drug development. Current animal models raise ethical and translational questions, and have limited accuracy in clinical risk prediction. Human-based computer models constitute a fast, cheap and potentially effective alternative to experimental assays, also facilitating translation to human. Key challenges include consideration of inter-cellular variability in drug responses and integration of computational and experimental methods in safety pharmacology. Our aim is to evaluate the ability of in silico drug trials in populations of human action potential (AP) models to predict clinical risk of drug-induced arrhythmias based on ion channel information, and to compare simulation results against experimental assays commonly used for drug testing. A control population of 1,213 human ventricular AP models in agreement with experimental recordings was constructed. In silico drug trials were performed for 62 reference compounds at multiple concentrations, using pore-block drug models (IC 50 /Hill coefficient). Drug-induced changes in AP biomarkers were quantified, together with occurrence of repolarization/depolarization abnormalities. Simulation results were used to predict clinical risk based on reports of Torsade de Pointes arrhythmias, and further evaluated in a subset of compounds through comparison with electrocardiograms from rabbit wedge preparations and Ca 2+ -transient recordings in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). Drug-induced changes in silico vary in magnitude depending on the specific ionic profile of each model in the population, thus allowing to identify cell sub-populations at higher risk of developing abnormal AP phenotypes. Models with low repolarization reserve (increased Ca 2+ /late Na + currents and Na + /Ca 2+ -exchanger, reduced Na + /K + -pump) are highly vulnerable to drug-induced repolarization abnormalities, while those with reduced inward current density (fast/late Na + and Ca 2+ currents) exhibit high susceptibility to depolarization abnormalities. Repolarization abnormalities in silico predict clinical risk for all compounds with 89% accuracy. Drug-induced changes in biomarkers are in overall agreement across different assays: in silico AP duration changes reflect the ones observed in rabbit QT interval and hiPS-CMs Ca 2+ -transient, and simulated upstroke velocity captures variations in rabbit QRS complex. Our results demonstrate that human in silico drug trials constitute a powerful methodology for prediction of clinical pro-arrhythmic cardiotoxicity, ready for integration in the existing drug safety assessment pipelines.

Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

PubMed

Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

2016-10-01

This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

Genic Microsatellite Markers in Brassica rapa: Development, Characterization, Mapping, and Their Utility in Other Cultivated and Wild Brassica Relatives

PubMed Central

Ramchiary, Nirala; Nguyen, Van Dan; Li, Xiaonan; Hong, Chang Pyo; Dhandapani, Vignesh; Choi, Su Ryun; Yu, Ge; Piao, Zhong Yun; Lim, Yong Pyo

2011-01-01

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species. PMID:21768136

Regional gene mapping using mixed radiation hybrids and reverse chromosome painting.

PubMed

Lin, J Y; Bedford, J S

1997-11-01

We describe a new approach for low-resolution physical mapping using pooled DNA probe from mixed (non-clonal) populations of human-CHO cell hybrids and reverse chromosome painting. This mapping method is based on a process in which the human chromosome fragments bearing a complementing gene were selectively retained in a large non-clonal population of CHO-human hybrid cells during a series of 12- to 15-Gy gamma irradiations each followed by continuous growth selection. The location of the gene could then be identified by reverse chromosome painting on normal human metaphase spreads using biotinylated DNA from this population of "enriched" hybrid cells. We tested the validity of this method by correctly mapping the complementing human HPRT gene, whose location is well established. We then demonstrated the method's usefulness by mapping the chromosome location of a human gene which complemented the defect responsible for the hypersensitivity to ionizing radiation in CHO irs-20 cells. This method represents an efficient alternative to conventional concordance analysis in somatic cell hybrids where detailed chromosome analysis of numerous hybrid clones is necessary. Using this approach, it is possible to localize a gene for which there is no prior sequence or linkage information to a subchromosomal region, thus facilitating association with known mapping landmarks (e.g. RFLP, YAC or STS contigs) for higher-resolution mapping.

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction*

PubMed Central

Rahman, Kh. Shamsur; Chowdhury, Erfan Ullah; Sachse, Konrad; Kaltenboeck, Bernhard

2016-01-01

X-ray crystallography has shown that an antibody paratope typically binds 15–22 amino acids (aa) of an epitope, of which 2–5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6–11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7–12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16–30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences. PMID:27189949

Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3

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

Haiming Chen; Lalioti, M.D.; Perrin, G.

1996-07-01

In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to 2 genes from other species) and tomore » a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3{prime} UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown. 56 refs., 8 figs., 1 tab.« less

A Segment of the Apospory-Specific Genomic Region Is Highly Microsyntenic Not Only between the Apomicts Pennisetum squamulatum and Buffelgrass, But Also with a Rice Chromosome 11 Centromeric-Proximal Genomic Region1[W

PubMed Central

Gualtieri, Gustavo; Conner, Joann A.; Morishige, Daryl T.; Moore, L. David; Mullet, John E.; Ozias-Akins, Peggy

2006-01-01

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory. PMID:16415213

A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region.

PubMed

Gualtieri, Gustavo; Conner, Joann A; Morishige, Daryl T; Moore, L David; Mullet, John E; Ozias-Akins, Peggy

2006-03-01

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.

Identification and Characterization of Crr1a, a Gene for Resistance to Clubroot Disease (Plasmodiophora brassicae Woronin) in Brassica rapa L.

PubMed Central

Hatakeyama, Katsunori; Suwabe, Keita; Tomita, Rubens Norio; Kato, Takeyuki; Nunome, Tsukasa; Fukuoka, Hiroyuki; Matsumoto, Satoru

2013-01-01

Clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae Woronin, is one of the most economically important diseases of Brassica crops in the world. Although many clubroot resistance (CR) loci have been identified through genetic analysis and QTL mapping, the molecular mechanisms of defense responses against P. brassicae remain unknown. Fine mapping of the Crr1 locus, which was originally identified as a single locus, revealed that it comprises two gene loci, Crr1a and Crr1b. Here we report the map-based cloning and characterization of Crr1a, which confers resistance to clubroot in Brassica rapa. Crr1aG004, cloned from the resistant line G004, encodes a Toll-Interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) protein expressed in the stele and cortex of hypocotyl and roots, where secondary infection of the pathogen occurs, but not in root hairs, where primary infection occurs. Gain-of-function analysis proved that Crr1aG004 alone conferred resistance to isolate Ano-01 in susceptible Arabidopsis and B. rapa. In comparison, the susceptible allele Crr1aA9709 encodes a truncated NB-LRR protein, which lacked more than half of the TIR domain on account of the insertion of a solo-long terminal repeat (LTR) in exon 1 and included several substitutions and insertion-deletions in the LRR domain. This study provides a basis for further molecular analysis of defense mechanisms against P. brassicae and will contribute to the breeding of resistant cultivars of Brassica vegetables by marker-assisted selection. Data deposition The sequence reported in this paper has been deposited in the GenBank database (accession no. AB605024). PMID:23382954

Discovery and mapping of a new expressed sequence tag-single nucleotide polymorphism and simple sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L.

PubMed Central

Allegre, Mathilde; Argout, Xavier; Boccara, Michel; Fouet, Olivier; Roguet, Yolande; Bérard, Aurélie; Thévenin, Jean Marc; Chauveau, Aurélie; Rivallan, Ronan; Clement, Didier; Courtois, Brigitte; Gramacho, Karina; Boland-Augé, Anne; Tahi, Mathias; Umaharan, Pathmanathan; Brunel, Dominique; Lanaud, Claire

2012-01-01

Theobroma cacao is an economically important tree of several tropical countries. Its genetic improvement is essential to provide protection against major diseases and improve chocolate quality. We discovered and mapped new expressed sequence tag-single nucleotide polymorphism (EST-SNP) and simple sequence repeat (SSR) markers and constructed a high-density genetic map. By screening 149 650 ESTs, 5246 SNPs were detected in silico, of which 1536 corresponded to genes with a putative function, while 851 had a clear polymorphic pattern across a collection of genetic resources. In addition, 409 new SSR markers were detected on the Criollo genome. Lastly, 681 new EST-SNPs and 163 new SSRs were added to the pre-existing 418 co-dominant markers to construct a large consensus genetic map. This high-density map and the set of new genetic markers identified in this study are a milestone in cocoa genomics and for marker-assisted breeding. The data are available at http://tropgenedb.cirad.fr. PMID:22210604

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias.

PubMed

Clarke, Laurence J; Soubrier, Julien; Weyrich, Laura S; Cooper, Alan

2014-11-01

Studies of insect assemblages are suited to the simultaneous DNA-based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR-amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR-amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR-amplified the blend using five primer sets, targeting either COI or 16S, with high-throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers. © 2014 John Wiley & Sons Ltd.