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Results for “Genomic sequencing”

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At least 91 records · Page 5Linked to original sources
PubMed2001

An integrated map of Arabidopsis thaliana for functional analysis of its genome sequence.

The genome of the model plant species Arabidopsis thaliana has recently been sequenced. To accelerate its current genome research, we developed a whole-genome, BAC/BIBAC-based, integrated physical, genetic, and sequence map of the A. thaliana ecotype Columbia. This new map was constructed from the clones of a new plant-transformation-competent BIBAC library and is integrated with the existing sequence map. The clones were restriction fingerprinted by DNA sequencing gel-based electrophoresis, assembled into contigs, and anchored to an existing genetic map. The map consists of 194 BAC/BIBAC contigs, spanning 126 Mb of the 130-Mb Arabidopsis genome. A total of 120 contigs, spanning 114 Mb, were anchored to the chromosomes of Arabidopsis. Accuracy of the integrated map was verified using the existing physical and sequence maps and numerous DNA markers. Integration of the new map with the sequence map has enabled gap closure of the sequence map and will facilitate functional analysis of the genome sequence. The method used here has been demonstrated to be sufficient for whole-genome physical mapping from large-insert random bacterial clones and thus is applicable to rapid development of whole-genome physical maps for other species.

Arabidopsis↗
PubMed2005

An effective alternate cloning strategy for unstable mouse genomic sequences.

Unstable mammalian genomic sequences frequently underwent spontaneous rearrangement during the bacterial cloning process. When the flanking sequences of an INSM1 gene comprised of 3.0 and 4.5 kb were subcloned into a targeting vector for a gene deletion study, both the genomic sequences underwent spontaneous rearrangement. Neither the usage of recombinase-free Escherichia coli competent cells nor lowering the culture incubation temperature averted the recombination events. Co-transformation of a methyltransferase vector, pAIT2, with the targeting vector had little effect in preventing recombination through methylation of the plasmid DNA. Here, we show that a single-copy cloning technique is effective to clone the unstable mouse genomic DNA into the targeting vector.

Animals↗
PubMed2002

Comparative phenotypic analysis of the Bordetella parapertussis isolate chosen for genomic sequencing.

The genomes of three closely related bordetellae are currently being sequenced, thus providing an opportunity for comparative genomic approaches driven by an understanding of the comparative biology of these three bacteria. Although the other strains being sequenced are well studied, the strain of Bordetella parapertussis chosen for sequencing is a recent human clinical isolate (strain 12822) that has yet to be characterized in detail. This investigation reports the first phenotypic characterization of this strain, which will likely become the prototype for this species in comparison with the prototype strains of B. pertussis (Tohama I), B. bronchiseptica (RB50), and other isolates of B. parapertussis. Multiple in vitro and in vivo assays distinguished each species. B. parapertussis was more similar to B. bronchiseptica than to B. pertussis in many assays, including in BvgS signaling characteristics, presence of urease activity, regulation of urease expression by BvgAS, virulence in the respiratory tracts of immunocompromised mice, induction of anti-Bordetella antibodies, and serum antimicrobial resistance. In other assays, B. parapertussis was distinct from all other species (in pigment production) or more similar to B. pertussis (by lack of motility and cytotoxicity to a macrophage-like cell line). These results begin to provide phenotypes that can be related to genetic differences identified in the genomic sequences of bordetellae.

Animals↗
PubMed2003

Integration of chicken genomic resources to enable whole-genome sequencing.

Different genomic resources in chicken were integrated through the Wageningen chicken BAC library. First, a BAC anchor map was created by screening this library with two sets of markers: microsatellite markers from the consensus linkage map and markers created from BAC end sequencing in chromosome walking experiments. Second, HINdIII digestion fingerprints were created for all BACs of the Wageningen chicken BAC library. Third, cytogenetic positions of BACs were assigned by FISH. These integrated resources will facilitate further chromosome-walking experiments and whole-genome sequencing.

Animals↗
PubMed2004

Phylogenetic relationships among amphisbaenian reptiles based on complete mitochondrial genomic sequences.

Complete mitochondrial genomic sequences are reported from 12 members in the four families of the reptile group Amphisbaenia. Analysis of 11,946 aligned nucleotide positions (5797 informative) produces a robust phylogenetic hypothesis. The family Rhineuridae is basal and Bipedidae is the sister taxon to the Amphisbaenidae plus Trogonophidae. Amphisbaenian reptiles are surprisingly old, predating the breakup of Pangaea 200 million years before present, because successive basal taxa (Rhineuridae and Bipedidae) are situated in tectonic regions of Laurasia and nested taxa (Amphisbaenidae and Trogonophidae) are found in Gondwanan regions. Thorough sampling within the Bipedidae shows that it is not tectonic movement of Baja California away from the Mexican mainland that is primary in isolating Bipes species, but rather that primary vicariance occurred between northern and southern groups. Amphisbaenian families show parallel reduction in number of limbs and Bipes species exhibit parallel reduction in number of digits. A measure is developed for comparing the phylogenetic information content of various genes. A synapomorphic trait defining the Bipedidae is a shift from the typical vertebrate mitochondrial gene arrangement to the derived state of trnE and nad6. In addition, a tandem duplication of trnT and trnP is observed in Bipes biporus with a pattern of pseudogene formation that varies among populations. The first case of convergent rearrangement of the mitochondrial genome among animals demonstrated by complete genomic sequences is reported. Relative to most vertebrates, the Rhineuridae has the block nad6, trnE switched in order with the block cob, trnT, trnP, as they are in birds.

Animals↗
PubMed2000

SELEX_DB: an activated database on selected randomized DNA/RNA sequences addressed to genomic sequence annotation.

SELEX_DB is a novel curated database on selected randomized DNA/RNA sequences designed for accumulation of experimental data on functional site sequences obtained by using SELEX and SELEX-like technologies from the pools of random sequences. This database also contains the programs for DNA/RNA functional site recognition within arbitrary nucleotide sequences. The first release of SELEX_DB has been installed under SRS and is available through the WWW at http://wwwmgs.bionet.nsc.ru/mgs/systems/selex/

DNA↗
PubMed2005

Comparative genome sequencing of Drosophila pseudoobscura: chromosomal, gene, and cis-element evolution.

We have sequenced the genome of a second Drosophila species, Drosophila pseudoobscura, and compared this to the genome sequence of Drosophila melanogaster, a primary model organism. Throughout evolution the vast majority of Drosophila genes have remained on the same chromosome arm, but within each arm gene order has been extensively reshuffled, leading to a minimum of 921 syntenic blocks shared between the species. A repetitive sequence is found in the D. pseudoobscura genome at many junctions between adjacent syntenic blocks. Analysis of this novel repetitive element family suggests that recombination between offset elements may have given rise to many paracentric inversions, thereby contributing to the shuffling of gene order in the D. pseudoobscura lineage. Based on sequence similarity and synteny, 10,516 putative orthologs have been identified as a core gene set conserved over 25-55 million years (Myr) since the pseudoobscura/melanogaster divergence. Genes expressed in the testes had higher amino acid sequence divergence than the genome-wide average, consistent with the rapid evolution of sex-specific proteins. Cis-regulatory sequences are more conserved than random and nearby sequences between the species--but the difference is slight, suggesting that the evolution of cis-regulatory elements is flexible. Overall, a pattern of repeat-mediated chromosomal rearrangement, and high coadaptation of both male genes and cis-regulatory sequences emerges as important themes of genome divergence between these species of Drosophila.

Animals↗
PubMed2006

Chromosomal and phylogenetic context for conglutin genes in Arachis based on genomic sequence.

Comparative genomic and cDNA sequence analysis of ara h 2, a major peanut allergen, and a related conglutin ara h 6 were performed in Arachis hypogaea L. and its putative progenitors, Arachis duranensis and Arachis ipaensis. The complete identity between sequences encoding Ara h 2 isoforms demonstrated that these are homeologous genes and represent orthologs from diploid ancestors. Three copies of ara h 6 were identified in A. hypogaea, one of them located in the A-genome and the other two in the B-genome. Expression analysis showed higher levels of ara h 2 transcripts compared with ara h 6. Dual-labeled genomic in situ hybridization permitted identification of two subgenomes, each of which contained one pair of ara h 2-ara h 6 signals localized by fluorescence in situ hybridization. Characterization of genomic clones showed close genetic linkage between Ara h 2.02 and one copy of ara h 6 in the B-genome. The physical linkage may have arisen by tandem duplication and divergence of an ancestral gene. A gene duplication event specific to the B-genome progenitor has resulted in ara h 6 paralogs. These data provide further evidence for progenitor relationships and genomic organization of the conglutin gene family in the genus Arachis and could contribute to the development of a hypoallergenic peanut.

Arachis↗
PubMed2002

Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus.

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.

Amino Acid Sequence↗
PubMed2006

Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.

BACKGROUND: Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls. RESULTS: All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR. CONCLUSION: Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.

Bias↗
PubMed2003

A proteomic view of cell physiology of Bacillus subtilis--bringing the genome sequence to life.

The genome sequence is the "blue-print of life", and the proteomic approach brings this genome sequence to life. Simple model systems are urgently required to "train" this transformation of the genome sequence into life: why not Bacillus subtilis, the model organism for gram-positive bacteria and of functional genomics? By combination of the highly sensitive 2D protein gel electrophoresis with the identification of the protein spots by microsequencing or mass spectrometry we established a 2D protein index of Bacillus subtilis. In order to depict the entire proteome of a B. subtilis cell, alkaline, cell-wall associated, or extracellular proteins were also included. The proteins of this database (see http://microbio2.biologie.uni-greifswald.de:8880/sub2d.htm) were allocated to proteins with house-keeping functions typical of growing cells and to proteins synthesized particularly in non-growing cells. A computer-aided evaluation of the 2D gels loaded with radioactively-labeled proteins from growing or stressed/starved cells proved to be a powerful tool for the analysis of global regulation of the expression of the entire genome. This is shown for the analysis of glycolysis/TCA cycle (house keeping proteins) and for the analysis of the heat stress stimulon. For the heat stress stimulon it is demonstrated how the proteomic approach can be used: (i) to define the structure of a stimulon, (ii) to dissect stimulons into regulons, (iii) to analyze the regulation, structure, and function of unknown regulons, (iv) to define overlapping reguIons or modulons, and finally (v) to explore complex adaptational networks. Furthermore, it will be demonstrated how the "dual channel pattern comparison" or "proteomics signature" (R. VanBogelen) can be used for a comprehensive understanding or prediction of the physiological state of growing or starving cell populations. This is shown for glucose-starved cells. In order to describe the structure and function of gene regulation groups it is generally recommended to complement the proteomics approach with DNA array technologies. Further studies will focus on the analysis of the global regulation of gene expression by the proteomic approach that cannot be addressed by the application of DNA array techniques: the phosphoproteome and its implications in signal transduction; the global control of protein stability; protein targeting and protein secretion.

Adaptation, Physiological↗
PubMed2004

Integration of the rat recombination and EST maps in the rat genomic sequence and comparative mapping analysis with the mouse genome.

Inbred strains of the laboratory rat are widely used for identifying genetic regions involved in the control of complex quantitative phenotypes of biomedical importance. The draft genomic sequence of the rat now provides essential information for annotating rat quantitative trait locus (QTL) maps. Following the survey of unique rat microsatellite (11,585 including 1648 new markers) and EST (10,067) markers currently available, we have incorporated a selection of 7952 rat EST sequences in an improved version of the integrated linkage-radiation hybrid map of the rat containing 2058 microsatellite markers which provided over 10,000 potential anchor points between rat QTL and the genomic sequence of the rat. A total of 996 genetic positions were resolved (avg. spacing 1.77 cM) in a single large intercross and anchored in the rat genomic sequence (avg. spacing 1.62 Mb). Comparative genome maps between rat and mouse were constructed by successful computational alignment of 6108 mapped rat ESTs in the mouse genome. The integration of rat linkage maps in the draft genomic sequence of the rat and that of other species represents an essential step for translating rat QTL intervals into human chromosomal targets.

Animals↗
PubMed2000

From microbial genome sequence to applications.

Whole genome sequences of microbial pathogens present new opportunities for clinical applications. Chief among these are development of antimicrobials, diagnostics, and vaccines. While antimicrobial development is a more difficult, long-term prospect, new diagnostics and vaccines are likely to be the first products of microbial genomics. To take advantage of whole genome sequences, methods for production of gene products in surrogate hosts (heterologous expression) are required that will work for large-scale, high-throughput gene expression. This will allow genomic information from even the most experimentally difficult pathogens to be mined for applications. In addition, screening methods to test gene products for their potential as vaccine candidates are needed for large-scale screening. These areas for technological development should be stimulated by the potential for converting genomic sequence information into applications.

Animals↗
PubMed2004

The Arabidopsis genome sequence as a tool for genome analysis in Brassicaceae. A comparison of the Arabidopsis and Capsella rubella genomes.

The annotated Arabidopsis genome sequence was exploited as a tool for carrying out comparative analyses of the Arabidopsis and Capsella rubella genomes. Comparison of a set of random, short C. rubella sequences with the corresponding sequences in Arabidopsis revealed that aligned protein-coding exon sequences differ from aligned intron or intergenic sequences in respect to the degree of sequence identity and the frequency of small insertions/deletions. Molecular-mapped markers and expressed sequence tags derived from Arabidopsis were used for genetic mapping in a population derived from an interspecific cross between Capsella grandiflora and C. rubella. The resulting eight Capsella linkage groups were compared to the sequence maps of the five Arabidopsis chromosomes. Fourteen colinear segments spanning approximately 85% of the Arabidopsis chromosome sequence maps and 92% of the Capsella genetic linkage map were detected. Several fusions and fissions of chromosomal segments as well as large inversions account for the observed arrangement of the 14 colinear blocks in the analyzed genomes. In addition, evidence for small-scale deviations from genome colinearity was found. Colinearity between the Arabidopsis and Capsella genomes is more pronounced than has been previously reported for comparisons between Arabidopsis and different Brassica species.

Arabidopsis↗