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At least 1,747 records · Page 97Linked to original sources
PubMed2000

Isolation of human immunodeficiency virus type 1 cores: retention of Vpr in the absence of p6(gag).

Mature human immunodeficiency virus type 1 (HIV-1) virions contain a typically cone-shaped core that encases the viral genome. In this study, we established conditions which allowed the efficient isolation of morphologically intact HIV-1 cores from virions. The isolated cores consisted mostly of cones which appeared uniformly capped at both ends but were heterogeneous with respect to the shape of the broad cap as well as the dimensions and angle of the cone. Vpr, a nonstructural virion component implicated in the nuclear import of the viral genome, was recovered in core preparations of HIV-1 and simian immunodeficiency viruses from African green monkeys. Unexpectedly, p6(gag), a structural protein required for the incorporation of Vpr, was absent from HIV-1 core preparations. Taken together, our results indicate that the incorporation of Vpr into the virion core is a conserved feature of primate lentiviruses and that the interactions required for the uptake of Vpr into assembling particles differ from those which confine Vpr within the core.

Animals↗
PubMed2004

Shaping and moving a spiroplasma.

The Mollicutes (Spiroplasma, Mycoplasma and Acholeplasma) are the most minimal cells known to exist, being the smallest and simplest free-living and self-replicating forms of life. Phylogenetically, the Mollicutes are related to gram-positive bacteria and have evolved, by regressive evolution and genome reduction, from Clostridia. The smallest genome in this group (Mycoplasma genitalium - 5.77 x 10(5) bp) is only twice that of a large virus (e.g., Entomopox viruses). The largest Mollicute genome (Spiroplasma LB12 - 2.2 x 10(6) bp) is only about half that of, e.g., Escherichia coli. Structurally, the Mollicutes lack cell walls and flagella, but have internal cytoskeletons and are motile and chemotactic. Only a cholesterol-containing unit membrane envelops the cells. No analogs to the bacterial chemotactic and motility (che, mot, fla) genes, genes for a two-component signal transduction system, genes associated with gliding, or genomic homologs for the eukaryotic cytoskeleton and motor proteins were found in the Mollicutes. The Spiroplasmas are unique amongst the Mollicutes in having a well-defined basic helical cell geometry. In this respect, the Spiroplasma cell can, essentially, be viewed as a helical dynamic membranal tube (diameter approximately 0.2 microm; equivalent to that of one eukaryotic flagellar axoneme or to a bacterial flagellar bundle). A flat cytoskeletal ribbon of parallel fibrils is attached to the inside of the cellular tube. Both tube and cytoskeleton are mutually coiled into a dynamic helix driven by differential length changes of the fibrils, which function as linear motors. The cytoskeletal ribbon follows the shortest (inner) helical line on the inner surface of the cellular tube. Being helical allows for further analytical reduction and consequent structural quantification of Spiroplasma. Of particular importance is the ability to correlate light and electron microscopy data and to calculate the fibril lengths (and corresponding molecular dimensions) in the helical and nonhelical dynamic states. The structural unit of the contractile cytoskeleton is a approximately 50-Angstrom-wide filament comprised of pairs of the 59-kD fib gene product. The monomers are arranged in pairs with opposite polarities allowing for a approximately 100-Angstrom-long axial repeat. The functional unit of the contractile cytoskeletal ribbon is a fibril comprised of an aligned pair of filaments. Neighboring repeats form a tetrameric ring with a lateral repeat of approximately 100 A. The axial length of the rings may shorten by approximately 40%, driving the changes in the fibril lengths and, consequently, helical dynamics. Local length changes result in helical symmetry breaking and nonreciprocating cell movements allowing for net directional displacement. Flexing allows for changes in swimming direction.

Chemotaxis↗
PubMed2003

Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.

Amino Acid Sequence↗
PubMed1985

Transposon-like properties of the major, long repetitive sequence family in the genome of Physarum polycephalum.

A family of long, highly-repetitive sequences, referred to previously as ;HpaII-repeats', dominates the genome of the eukaryotic slime mould Physarum polycephalum. These sequences are found exclusively in scrambled clusters. They account for about one-half of the total complement of repetitive DNA in Physarum, and represent the major sequence component found in hypermethylated, 20-50 kb segments of Physarum genomic DNA that fail to be cleaved using the restriction endonuclease HpaII. The structure of this abundant repetitive element was investigated by analysing cloned segments derived from the hypermethylated genomic DNA compartment. We show that the ;HpaII-repeat' forms part of a larger repetitive DNA structure, approximately 8.6 kb in length, with several structural features in common with recognised eukaryotic transposable genetic elements. Scrambled clusters of the sequence probably arise as a result of transposition-like events, during which the element preferentially recombines in either orientation with target sites located in other copies of the same repeated sequence. The target sites for transposition/recombination are not related in sequence but in all cases studied they are potentially capable of promoting the formation of small ;cruciforms' or ;Z-DNA' structures which might be recognised during the recombination process.

Journal Article↗
PubMedDate not supplied

[The relation of enzymatic methylation of inverted DNA repeats to transcription in mouse P815 mastocytoma cells (author's transl)].

The isolation of transcribed DNA sequences of P815 cells and the partial characterization with respect to their sequence composition and relative rates of enzymatic DNA methylation are reported in this paper. Transcribed regions were purified by affinity chromatography using immobilized heterogenous nuclear RNA of P815 cells. About 10% of total genome was found in this fraction. Reassociation analyses showed differences in sequence composition of transcribed versus non-transcribed DNA fractions. The relative proportion of inverted repeats was doubled in the transcribed fraction whereas ordinary highly repetitive sequences comprising mainly of satellite DNA were found almost exclusively in the non-transcribed regions of the P815 genome. About 70% of transcribed portions corresponds to unique and intermediary DNA sequences. After labelling of cells with L-[Methyl-3H]methionine and [14C]deoxycytidine relative rates of enzymatic DNA methylation were computed for different kinetic components of transcribed and non-transcribed portions of P815 genome. No difference was found except in inverted repeats. In transcribed DNA the relative rate of enzymatic DNA methylation was only about 40% of that of the non-transcribed ones. We have quantitated this hypomethyltion and found that there is, in average, about one 5-methylcytosine residue in 100 nucleotides of transcribed inverted repeats, compared to about 2.5 5-methylcytosines in non-transcribed fractions. In view of these data we propose that the enzymatic methylation of inverted DNA repeats negatively controls the transcriptional process in a given genomic region.

Animals↗
PubMed2004

Predicted ATP-binding cassette systems in the phytopathogenic mollicute Spiroplasma kunkelii.

Spiroplasma kunkelii is a cell wall-free, helical, and motile mycoplasma-like organism that causes corn stunt disease in maize. The bacterium has a compact genome with a gene set approaching the minimal complement necessary for cellular life and pathogenesis. A set of 21 ATP-binding cassette (ABC) domains was identified during the annotation of a draft S. kunkelii genome sequence. These 21 ABC domains are present in 18 predicted proteins, and are components of 16 functional systems, which account for 5% of the protein coding capacity of the S. kunkelii genome. Of the 16 systems, 11 are membrane-bound transporters, and two are cytosolic systems involved in DNA repair and the oxidative stress response; the genes for the remaining three hypothetical systems harbor nonsense and/or frameshift mutations, so their functional status is doubtful. Assembly of the 11 multicomponent transporters, and comparisons with other known systems permitted functional predictions for the S. kunkelii ABC transporter systems. These transporters convey a wide variety of substrates, and are critical for nutrient uptake, multidrug resistance, and perhaps virulence. Our findings provide a framework for functional characterization of the ABC systems in S. kunkelii.

ATP-Binding Cassette Transporters↗
PubMed1978

Cae I: an endonuclease isolated from the African green monkey with properties indicating site-specific cleavage of homologous and heterologous mammalian DNA.

Component alpha DNA is a highly repetitive sequence that comprises nearly a quarter of the African green monkey (Cercopithecus aethiops) genome. A previous microbial restriction enzyme analysis showed that the repeat structure of component alpha DNA is based upon a monomeric unit of 176 +/- 4 base-pairs. An endonuclease, provisionally termed Case I, has been isolated from African green monkey testes that cleaves component alpha DNA into multimeric segments based upon the same repeat periodicity as that revealed by microbial restriction enzymes. The primary sites of Cae I cleavage in the component alpha sequence appear to be 120 +/- 6 base-pairs distant from the Hind III sites and 73 +/- 6 base-pairs distant from the Eco RI* sites. Cae I has been partially characterized with special reference to the effects of ATP and S-adenosylmethionine on the cleavage of component alpha DNA. Cae I may be a member of a class of similar site-specific nucleases present in mammalian cells. Cae I also cleaves mouse satellite DNA into a multimeric series of discrete segments: the periodicity of this series is shorter than that revealed by Eco RII retriction analysis of mouse satellite DNA.

Adenosine Triphosphate↗
PubMed2017

Drug target ontology to classify and integrate drug discovery data.

BACKGROUND: One of the most successful approaches to develop new small molecule therapeutics has been to start from a validated druggable protein target. However, only a small subset of potentially druggable targets has attracted significant research and development resources. The Illuminating the Druggable Genome (IDG) project develops resources to catalyze the development of likely targetable, yet currently understudied prospective drug targets. A central component of the IDG program is a comprehensive knowledge resource of the druggable genome. RESULTS: As part of that effort, we have developed a framework to integrate, navigate, and analyze drug discovery data based on formalized and standardized classifications and annotations of druggable protein targets, the Drug Target Ontology (DTO). DTO was constructed by extensive curation and consolidation of various resources. DTO classifies the four major drug target protein families, GPCRs, kinases, ion channels and nuclear receptors, based on phylogenecity, function, target development level, disease association, tissue expression, chemical ligand and substrate characteristics, and target-family specific characteristics. The formal ontology was built using a new software tool to auto-generate most axioms from a database while supporting manual knowledge acquisition. A modular, hierarchical implementation facilitate ontology development and maintenance and makes use of various external ontologies, thus integrating the DTO into the ecosystem of biomedical ontologies. As a formal OWL-DL ontology, DTO contains asserted and inferred axioms. Modeling data from the Library of Integrated Network-based Cellular Signatures (LINCS) program illustrates the potential of DTO for contextual data integration and nuanced definition of important drug target characteristics. DTO has been implemented in the IDG user interface Portal, Pharos and the TIN-X explorer of protein target disease relationships. CONCLUSIONS: DTO was built based on the need for a formal semantic model for druggable targets including various related information such as protein, gene, protein domain, protein structure, binding site, small molecule drug, mechanism of action, protein tissue localization, disease association, and many other types of information. DTO will further facilitate the otherwise challenging integration and formal linking to biological assays, phenotypes, disease models, drug poly-pharmacology, binding kinetics and many other processes, functions and qualities that are at the core of drug discovery. The first version of DTO is publically available via the website http://drugtargetontology.org/ , Github ( http://github.com/DrugTargetOntology/DTO ), and the NCBO Bioportal ( http://bioportal.bioontology.org/ontologies/DTO ). The long-term goal of DTO is to provide such an integrative framework and to populate the ontology with this information as a community resource.

Biological Ontologies↗
PubMed1976

DNA sequence repetition in the genome of the American oyster.

The genome of Crassostrea virignica, the American oyster, has been studied by reassociation kinetics in order to construct a profile of DNA sequence frequency components. Oyster DNA has been shown to contain at least 51% single copy DNA sequences and two classes of middle repetitive DNA. The major repetitive class contains sequences which are repeated on the average 20 times and comprise 29% of oyster DNA. The other class represents 10% of oyster DNA and contains sequences repeated approx. 3000 times. In addition the DNA of oyster contains at least 1% foldback sequences. The spectrum of DNA repetition components in the American oyster is similar to that found in the genomes of other mollusks.

Animals↗
PubMed1995

Overexpression of the XPA repair gene increases resistance to ultraviolet radiation in human cells by selective repair of DNA damage.

Overexpression of XPA genes, both wild type and a missense mutant, which code for a damage-specific, DNA-binding protein, increased the survival of repair-deficient and -competent human cells to levels above that of normal cells that did not overexpress XPA. The first 3 h after cells were damaged were most critical to achieving this increased survival. The dose at which 37% of the irradiated population survives could be restored to about one-half that of normal cells, with no detectable genome-wide repair of pyrimidine dimers or (6-4) photoproducts, suggesting that intermediate levels of XPA gene expression can direct repair to restricted critical regions of the genome. Current views of repair implicate transcriptionally active genes as a major component of such critical regions. Consistent with this interpretation, the repair of a transfected, actively expressed luciferase gene was higher than that of genomic DNA at intermediate and higher levels of XPA expression. High levels of XPA expression resulted in increased repair at early times after irradiation and extensive repair of (6-4) photoproducts but little, if any, pyrimidine dimer repair in the whole genome. At the highest level of expression, some clonal cell lines acquired resistance to radiation that corresponded to a dose at which 37% of the irradiated population survives that was about 1.5 to 2 times that of normal cells. The XPA gene product, therefore, can influence levels of DNA repair and radiation sensitivity quantitatively by contributing to selective repair at certain sites in the genome.

Cells, Cultured↗
PubMed2005

Conditioning the genome identifies additional diabetes resistance loci in Type I diabetes resistant NOR/Lt mice.

While sharing the H2g7 MHC and many other important Type I diabetes susceptibility (Idd) genes with NOD mice, the NOR strain remains disease free due to resistance alleles within the approximately 12% portion of their genome that is of C57BLKS/J origin. Previous F2 segregation analyses indicated multiple genes within the 'Idd13' locus on Chromosome 2 provide the primary component of NOR diabetes resistance. However, it was clear other genes also contribute to NOR diabetes resistance, but were difficult to detect in the original segregation analyses because they were relatively weak compared to the strong Idd13 protection component. To identify these further genetic components of diabetes resistance, we performed a new F2 segregation analyses in which NOD mice were outcrossed to a 'genome-conditioned' NOR stock in which a large component of Idd13-mediated resistance was replaced with NOD alleles. These F2 segregation studies combined with subsequent congenic analyses confirmed the presence of additional NOR resistance genes on Chr. 1 and Chr. 4, and also potentially on Chr. 11. These findings emphasize the value for diabetes gene discovery of stratifying not only MHC loci conferring the highest relative risk but also as many as possible of the non-MHC loci presumed to contribute significantly.

Alleles↗
PubMed2000

Large-scale SNP scoring from unamplified genomic DNA.

Discoveries from the Human Genome Project (HGP) continue to spur changes in medical technology that will lead to new diagnostic procedures in the clinical lab. As more single nucleotide polymorphisms (SNPs) are discovered and correlated to human diseases, demands for genetic tests will increase. The enormity of the number of SNPs makes developing inexpensive and reliable high-throughput methods for SNP scoring imperative. High-throughput screening (HTS) means, at a minimum, a production rate of thousands of assays per day. Ideally, the technology will be easy, inexpensive and amenable to automation. The Invader assay offers a simple diagnostic platform to detect single nucleotide changes with high specificity and sensitivity from unamplified, genomic DNA. The Invader assay uses a structure-specific 5' nuclease (or flap endonuclease) to cleave sequence-specific structures in each of two cascading reactions. The cleavage structure forms when two synthetic oligonucleotide probes hybridise in tandem to a target. One of the probes cycles on and off the target and is cut by the nuclease only when the appropriate structure forms. These cleaved probes then participate in a second Invader reaction involving a dye-labelled fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe generates a signal, which can be readily analysed by fluorescence microtitre plate readers. The two cascading reactions amplify the signal significantly; each original target molecule can lead to more than 10(6) cleaved signal probes in one hour. This signal amplification permits identification of single base changes directly from genomic DNA without prior target amplification. The sequences of the oligonucleotide components of the secondary reaction are independent of the target of interest and permit the development of universal secondary reaction components useful to identify any target.

DNA↗
PubMed2005

High-diversity genes in the Arabidopsis genome.

High-diversity genes represent an important class of loci in organismal genomes. Since elevated levels of nucleotide variation are a key component of the molecular signature for balancing selection or local adaptation, high-diversity genes may represent loci whose alleles are selectively maintained as balanced polymorphisms. Comparison of 4300 random shotgun sequence fragments of the Arabidopsis thaliana Ler ecotype genome with the whole genomic sequence of the Col-0 ecotype identified 60 genes with putatively high levels of intraspecific variability. Eleven of these genes were sequenced in multiple A. thaliana accessions, 3 of which were found to display elevated levels of nucleotide polymorphism. These genes encode the myb-like transcription factor MYB103, a putative soluble starch synthase I, and a homeodomain-leucine zipper transcription factor. Analysis of these genes and 4-7 flanking genes in 14-20 A. thaliana ecotypes revealed that two of these loci show other characteristics of balanced polymorphisms, including broad peaks of nucleotide diversity spanning multiple linked genes and an excess of intermediate-frequency polymorphisms. Scanning genomes for high-diversity genomic regions may be useful in approaches to adaptive trait locus mapping for uncovering candidate balanced polymorphisms.

Alleles↗
PubMed1994

Packaging of hepatitis delta virus RNA via the RNA-binding domain of hepatitis delta antigens: different roles for the small and large delta antigens.

Hepatitis delta virus (HDV) is composed of four specific components. The first component is envelope protein which contains hepatitis B surface antigens. The second and third components are nucleocapsid proteins, referred to as small and large hepatitis delta antigens (HDAgs). The final component is a single-stranded circular RNA molecule known as the viral genome. In order to study the mechanism of HDV RNA packaging, a four-plasmid cotransfection system in which each viral component was provided by a separate plasmid was employed. Virus-like particles released from Huh-7 cells receiving such a cotransfection were found to contain HDV RNA along with three proteins. Therefore, the four-plasmid cotransfection system could lead to successful HDV RNA packaging in vitro. The system was then used to show that the large HDAg alone was able to achieve a low level of HDV RNA packaging. Analysis of a variety of large HDAg mutants revealed that the RNA-binding domain was essential for viral RNA packaging. By increasing the incorporation of small HDAg into virus-like particles, we found a three- to fourfold enhancement of HDV RNA packaging. This effect was probably through a direct binding of HDV RNA, independent from that of large HDAg, with the small HDAg. The subsequent RNA-protein complex was packaged into particles. The results provided insight into the roles and functional domains of small and large HDAgs in HDV RNA packaging.

Antigens, Viral↗
PubMedDate not supplied

[Assays on comparing the local concentration of HU protein in the different regions of Escherichia coli genomic DNA].

HU, a nonspecific histone-like DNA binding protein is a major component of the bacterial nucleoid. HU is referred to as an accessory factor for complex protein-DNA assembly and as a protein involved in DNA compaction. In this study we investigated in vivo HU binding along the different regions of E. coli genome. For this purpose we used ChIP--in vivo formaldehyde crosslinking and immunoprecipitation of protein-DNA complexes with antiHU-antibodies. This technique allows to compare the local concentration of HU protein in the different regions of E. coli genomic DNA. In this study we analysed the HU-DNA crosslinking both in exponentially growing and stationary phase of bacteria in the following regions of E. coli genome: oriC region, promoter and structural regions of hupA and hupB genes coding two different subunits of HU, and structural parts of dps and glgS genes which are active only in stationary phase. Our results indicate that in exponentially growing E. coli cells the local concentration of HU protein is uniform for all analysed regions of genome and does not depend on their transcriptional status. The twofold increase of local concentration of HU protein was also shown for all analysed genome regions in the stationary phase cells.

Base Sequence↗
PubMed2004

DNMT3B interacts with hSNF2H chromatin remodeling enzyme, HDACs 1 and 2, and components of the histone methylation system.

The non-random pattern of genome-wide DNA methylation in mammalian cells is established and maintained by DNA methyltransferases DNMT1, 3A, and 3B. De novo DNA methyltransferase DNMT3B is critical for embryonic development and is mutated in ICF syndrome. Despite its importance in normal cellular functioning, little is known about how DNMT3B operates in the context of chromatin. Here we demonstrate that DNMT3B associates with four chromatin-associated enzymatic activities common to transcriptionally repressed, heterochromatic regions of the genome: DNA methyltransferase, histone deacetylase, ATPase, and histone methylase activities. By immunoprecipitation and GST pull-down, we show that DNMT3B interacts with HDAC1, HDAC2, HP1 proteins, Suv39h1, and the ATP-dependent chromatin remodeling enzyme hSNF2H. Endogenous hSNF2H is also associated with DNA methyltransferase activity. These proteins co-localize extensively with DNMT3B in heterochromatic regions. Our results therefore link DNMT3B to three other components of the epigenetic machinery and provide important insights into how DNA methylation patterns may be established within the chromatin environment.

Adenosine Triphosphatases↗
PubMed2001

The genomic complexity of micro B chromosomes of Brachycome dichromosomatica.

A major sequence component of the micro B chromosome of Brachycome dichromosomatica (2 n=4) is the tandem repeat Bdm29, which was found by in situ hybridisation to be distributed along the entire length of the chromosome. A high copy number of this sequence does not occur as a regular feature of the A chromosomes in this species but it was found in infrequent individuals in two wild populations that were analysed. In these instances Bdm29 is localised within heterochromatic, polymorphic segments on the long arm of chromosome 1. The origin of the micro B chromosomes was investigated by determining whether they are related to this A chromosome polymorphism by simple excision and/or integration. Results obtained by using Bdm29, together with a newly isolated repeat sequence, Bdm54, and a number of other sequences known to occur on the micro B chromosome, as probes in in situ hybridisation and Southern analysis demonstrated that the formation of micro B chromosomes is a complex multistep process. The observation that the genomic organisation of the micro B chromosome is unlike anything found on the A chromosomes precludes their origin by simple excision and also indicates that micro Bs do not integrate directly into the A complement to form polymorphic heterochomatic segments.

Asteraceae↗
PubMed2000

Retrotransposon evolution in diverse plant genomes.

Retrotransposon or retrotransposon-like sequences have been reported to be conserved components of cereal centromeres. Here we show that the published sequences are derived from a single conventional Ty3-gypsy family or a nonautonomous derivative. Both autonomous and nonautonomous elements are likely to have colonized Poaceae centromeres at the time of a common ancestor but have been maintained since by active retrotransposition. The retrotransposon family is also present at a lower copy number in the Arabidopsis genome, where it shows less pronounced localization. The history of the family in the two types of genome provides an interesting contrast between "boom and bust" and persistent evolutionary patterns.

Amino Acid Sequence↗