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At least 1,063 records · Page 59Linked to original sources
PubMed2002

Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae.

The understanding of the processes underlying organellar function and inheritance requires the identification and characterization of the molecular components involved. We pursued a genomic approach to define the complements of genes required for respiratory growth and inheritance of mitochondria with normal morphology in yeast. With the systematic screening of a deletion mutant library covering the nonessential genes of Saccharomyces cerevisiae the numbers of genes known to be required for respiratory function and establishment of wild-type-like mitochondrial structure have been more than doubled. In addition to the identification of novel components, the systematic screen revealed unprecedented mitochondrial phenotypes that have never been observed by conventional screens. These data provide a comprehensive picture of the cellular processes and molecular components required for mitochondrial function and structure in a simple eukaryotic cell.

Gene Deletion↗
PubMed2001

Transposition of IS10 from the host Escherichia coli genome to a plasmid may lead to cloning artefacts.

During recloning of Nicotiana tabacum L. repetitive sequence R8.3 in Escherichia coli, a modified clone that differed from the original by the insertion of an IS10 sequence was unintentionally produced. The insert was flanked by a 9-bp direct repeat derived from the R8.3 sequence, the 9-bp duplication of acceptor DNA in the site of insertion being a characteristic of IS10 transposition events. A database search using the FASTA program showed IS10 and other prokaryotic IS elements inserted into numerous eukaryotic clones. Unexpectedly, the IS10, which is not a natural component of the E. coli genome, appeared to be by far the most frequent contaminant of DNA databases among several IS sequences tested. In the GenEMBL database, the IS10 query sequence yielded positive scores with more than 500 eukaryotic clones. Insertions of shortened IS10 sequences having only one intact terminal inverted repeat were commonly found. Most full-length IS10 insertions (32 out of 40 analyzed) were flanked by 9-bp direct repeats having the consensus 5'-NPuCNN-NGPyN-3' with a strong preference for 5'-TGCTNA-GNN-3'. One insertion was flanked by an inverted repeat of more than 400 bp in length. PCR amplification and Southern analysis revealed the presence of IS10 sequences in E. coli strains commonly used for DNA cloning, including some reported to be Tn10-free. No IS10-specific PCR product was obtained with N. tabacum or human DNA. Our data suggest that transposition of IS10 elements may accompany cloning steps, particularly into large BAC vectors. This might lead to the relatively frequent contamination of DNA databases by this bacterial sequence. It is estimated that one in approximately every thousand eukaryotic clone in the databases is contaminated by IS-derived sequences. We recommend checking submitted sequences for the presence of IS10 and other IS elements. In addition, DNA databases should be corrected by removing contaminating IS sequences.

Animals↗
PubMed1997

Effect of 2',3'-dideoxycytidine on oxidative phosphorylation in the PC12 cell, a neuronal model.

Peripheral neuropathy induced by 2',3'-dideoxycytidine (ddC) could result from the previously shown inhibition of mtDNA replication by the action of ddC on the mitochondrial enzyme DNA polymerase gamma. Such inhibition would be expected to impair oxidative phosphorylation, and this was demonstrated in the present study for the PC12 cell, a model of a peripheral neuron. The dramatic rise in lactate formation upon exposure of the cell to ddC indicated that increased glycolysis was needed to produce ATP. A concomitant rise in O2 uptake indicated that oxidative phosphorylation had become uncoupled. When tested in a standard respiratory control system (isolated rat liver mitochondria), however, we found ddC not to be an uncoupler. Rather, the uncoupling most likely resulted from the failure of synthesis of one or more mitochondrial gene products necessary for oxidative phosphorylation. We also observed an important distinction between the manner in which ddC and 3'-azido-3'-deoxythymidine (AZT) act. ddC-exerted inhibition of oxidative phosphorylation was delayed for several days. This is consistent with the inhibition occurring indirectly, most likely as a result of the prior destruction of the mitochondrial genome, which encodes many of the components of the oxidative phosphorylation system. In contrast, we have shown previously that although AZT also impairs replication of the mitochondrial genome (in the Friend murine erythroleukemic cell), it also attacks directly an additional primary target leading to impairment of oxidative phosphorylation; its initial inhibition of this process is immediate, not occurring via inhibition of mitochondrial DNA replication.

Animals↗
PubMed2006

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40.

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a beta-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an alpha-neoagarobiose hydrolase to release galactose from agarose.

Alteromonadaceae↗
PubMed2002

Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro.

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.

Amino Acid Sequence↗
PubMed1988

Characterization of highly repetitive sequences of Arabidopsis thaliana.

We have analyzed three classes of highly repetitive DNA sequences of Arabidopsis thaliana, composed of tamdemly repeated units of 180 bp, 500 bp, and 160 bp, respectively. The three families comprise approximately 2% of the Arabidopsis genome and are the major component of the highly repetitive DNA. The 500-bp element arose by duplication of one half of a 180-bp ancestor and insertion of a foreign segment between the two duplicated parts followed by amplification. The repeat elements contain occasionally palindromes and other motifs but none are significantly conserved. There is no significant similarity with previously published repetitive elements. Heterogeneity between monomers ranges from 6% to 17%. Monomers derived from different clusters in the genome are more diverged than monomers of the same array.

Base Sequence↗
PubMed2003

Genome-wide linkage scan reveals multiple susceptibility loci influencing lipid and lipoprotein levels in the Quebec Family Study.

A genome-wide linkage study was performed to identify chromosomal regions harboring genes influencing lipid and lipoprotein levels. Linkage analyses were conducted for four quantitative lipoprotein/lipid traits, i.e., total cholesterol, triglyceride, HDL-cholesterol (HDL-C), and LDL-C concentrations, in 930 subjects enrolled in the Québec Family Study. A maximum of 534 pairs of siblings from 292 nuclear families were available. Linkage was tested using both allele-sharing and variance-component linkage methods. The strongest evidence of linkage was found on chromosome 12q14.1 at marker D12S334 for HDL-C, with a logarithm of the odds (LOD) score of 4.06. Chromosomal regions harboring quantitative trait loci (QTLs) for LDL-C included 1q43 (LOD = 2.50), 11q23.2 (LOD = 3.22), 15q26.1 (LOD = 3.11), and 19q13.32 (LOD = 3.59). In the case of triglycerides, three markers located on 2p14, 11p13, and 11q24.1 provided suggestive evidence of linkage (LOD > 1.75). Tests for total cholesterol levels yielded significant evidence of linkage at 15q26.1 and 18q22.3 with the allele-sharing linkage method, but the results were nonsignificant with the variance-component method. In conclusion, this genome scan provides evidence for several QTLs influencing lipid and lipoprotein levels. Promising candidate genes were located in the vicinity of the genomic regions showing evidence of linkage.

Adult↗
PubMed2004

The absence of a functional relationship between ATM and BLM, the components of BASC, in DT40 cells.

Bloom syndrome (BS) and ataxia-telangiectasia (A-T) are rare autosomal recessive diseases associated with chromosomal instability. The genes responsible for BS and A-T have been identified as BLM and ATM, respectively, whose products were recently found to be components of BRCA1-associated genome surveillance complex (BASC), a supercomplex possibly involved in the recognition and repair of aberrant DNA structures. Based on experiments using BLM(-/-) DT40 cells and BLM(-/-)/RAD54(-/-) DT40 cells, we previously suggested that BLM functions to reduce the formation of double-strand breaks (DSBs) during DNA replication. To examine whether ATM is involved in the recognition and/or repair of DSBs generated in BLM(-/-) DT40 cells and to address the functional relationship between the two BASC components, we generated BLM(-/-)/ATM(-/-) DT40 cells and characterized their properties as well as those of ATM(-/-) and BLM(-/-) DT40 cells. BLM(-/-)/ATM(-/-) cells proliferated slightly more slowly than either BLM(-/-) or ATM(-/-) cells. The sensitivity of BLM(-/-)/ATM(-/-) cells to gamma-irradiation was similar to that of ATM(-/-) cells, while BLM(-/-) cells were slightly resistant to gamma-irradiation compared with wild-type cells. BLM(-/-) cells showed sensitivity to methyl methanesulfonate (MMS) and UV irradiation while ATM(-/-) cells did not show sensitivity to either agent. The sensitivity of BLM(-/-)/ATM(-/-) cells to MMS and UV was similar to that of BLM(-/-) cells. Disrupting the function of ATM reduced the targeted integration frequency in BLM(-/-) DT40 cells. However, a defect in ATM only slightly reduced the increased sister chromatid exchanges (SCEs) in BLM(-/-) DT40 cells.

Adenosine Triphosphatases↗
PubMed1994

Pulsed-field fingerprinting of listeriae: identification of genomic divisions for Listeria monocytogenes and their correlation with serovar.

Clamped homogeneous electric field (CHEF) electrophoresis was optimized for genomic analyses of Listeria monocytogenes. Various human, animal, food, and environmental isolates, as well as strains representing other Listeria species, were separately digested with rarely cutting endonucleases. Of 176 L. monocytogenes strains analyzed, the enzymes AscI and ApaI established 63 and 72 unique restriction endonuclease digestion profiles (REDP), respectively. The 22 non-L. monocytogenes strains exhibited 18 AscI and 19 ApaI unique REDP. Statistical analyses of REDP information using the Dice coincidence index and principal component analysis revealed two distinct genomic divisions of L. monocytogenes that also correlated with the flagellar (H) antigen type: division I contained serovar 1/2a, 1/2c, 3a, and 3c stains and division II contained serovar 1/2b, 3b, 4b, 4d, and 4e strains. Division I isolates digested with ApaI were further grouped into cluster IA (serovar 1/2c and 3c) and cluster IB (serovar 1/2a and 3a) strains. Likewise, division II isolates digested with ApaI were further grouped into cluster IIA (serovar 1/2b and 3b) and cluster IIB (serovar 4b, 4d, and 4e) strains. These data indicate that genotypic data generated by CHEF can be directly related to phenotypic data generated by serotyping for establishing the overall relatedness of isolates. Moreover, these data further substantiate that CHEF analysis is a reproducible and highly discriminating method for characterizing L. monocytogenes strains at the molecular level.

Animals↗
PubMed2000

Mechanism of genome transcription in segmented dsRNA viruses.

Genome transcription is a critical stage in the life cycle of a virus, as this is the process by which the viral genetic information is presented to the host cell protein synthesis machinery for the production of the viral proteins needed for genome replication and progeny virion assembly. Viruses with dsRNA genomes face a particular challenge in that host cells do not produce proteins which can transcribe from a dsRNA template. Therefore, dsRNA viruses contain all of the necessary enzymatic machinery to synthesize complete mRNA transcripts within the core without the need for disassembly. Indeed one of the more striking observations about genome transcription in dsRNA viruses is that this process occurs efficiently only when the transcriptionally competent particle is fully intact. This observation suggests that all of the components of the TCP, including the viral genome, the transcription enzymes, and the viral capsid, function together to produce and release mRNA transcripts and that each component has a specific and critical role to play in promoting the efficiency of this process. This review has examined the process of genome transcription in dsRNA viruses from the perspective of rotavirus as a model system. However, despite numerous architectural and organizational differences among the families of dsRNA viruses, numerous studies suggest that the basic mechanism of mRNA production may be similar in most, if not all, viruses having dsRNA genomes. Important functional similarities include (1) the presence of a capsid-bound RNA-dependent RNA polymerase, which produces single-stranded mRNA transcripts from the dsRNA genome and regenerates the dsRNA genome from single-stranded RNA templates; (2) in viruses infecting eukaryotic hosts, the presence of all the enzymatic activities needed to generate the 5' cap required by the eukaryotic translation machinery; (3) the high degree of structural order present in the packaged genome, suggesting the requirement for organization in the viral core; (4) the role of the innermost capsid protein as a scaffold on which the core components of the transcription apparatus are assembled; and (5) the release of nascent mRNA transcripts through channels at the icosahedral vertices. The process of genome transcription in dsRNA viruses will become better understood as structural studies progress to higher resolution and as more viruses become amenable to study using site-directed mutagenesis coupled with viral reconstitution to generate recombinant particles having precise functional and structural changes. Future studies will dissect important intermolecular interactions required for efficient mRNA synthesis and will shed further light on the reasons for which the viral core must be structurally intact in order for transcription to occur efficiently. Structural studies of the capping enzymes at atomic resolution will reveal how multiple enzyme activities reside within a single polypeptide and how they act in concert to synthesize the 5' cap on the end of each mature transcript. Perhaps most interestingly, high resolution structural studies of actively transcribing virions will provide insight into the conformational changes that occur within the core during mRNA synthesis. Together, these studies will clarify the function of this complex macromolecular machine and will also shed additional light on the basic principles of virus architecture and assembly, as well as provide avenues for the design of antiviral therapies.

Animals↗
PubMed2003

A genome-wide scan for loci affecting normal adult height in the Framingham Heart Study.

OBJECTIVE: To map loci influencing normal adult height in 335 families from the Framingham Heart Study. METHODS: We analyzed data consisting of 1,702 genotyped individuals who have been followed over time. The first height measurement for individuals between the ages 20-55 years was analyzed in a genome-wide scan using variance component linkage analysis. Sex, age, and cohort effects were removed before analysis. RESULTS: Two regions (18pter-p11, 22q11.2) with multipoint LOD scores >1.0 (-log p values >2.0) were detected: we obtained LOD scores of 1.38 at D18S1364, and of 1.10 at D22S345. Analysis of height as a sex-limited phenotype revealed a peak in the 9p21 region near D9S319 with a maximum LOD score of 1.65 (-log p value >3.0) when only male height phenotypes were used. When only female phenotypes were used, a peak with a maximum LOD score of 1.85 (-log p value of 2.70) was observed in the 11q25-qter region near D11S2359. CONCLUSIONS: Our region of interest on chromosome 9 has been implicated by two prior studies. Variance components analysis appeared to be sensitive to pedigree structures as well as the method of IBD computation used.

Adolescent↗
PubMed1992

A P element chimera containing captured genomic sequences was recovered at the vestigial locus in Drosophila following targeted transposition.

A P element carrying the Dopa decarboxylase gene, P[Ddc], was targeted into vg21, a cryptic P element induced mutant allele of the vestigial (vg) locus. The resulting allele, vg28w, contained the expected P[Ddc] plus an additional 9.5 kb of DNA, captured from elsewhere on chromosome II. Reversion of the vg28w mutant allele demonstrated that the entire insert can excise but cannot reinsert at an appreciable frequency. We explain the targeted transposition as the repair of a double stranded gap, created by the excision of the P element at vg21, and suggest that the formation of chimeric elements may be an important component of P element dependent genomic instability.

Alleles↗
PubMed2002

HF2: a double-stranded DNA tailed haloarchaeal virus with a mosaic genome.

HF2 is a haloarchaeal virus infecting two Halorubrum species (Family Halobacteriaceae). It is lytic, has a head-and-tail morphology and belongs to the Myoviridae (contractile tails). The linear double-stranded DNA genome was sequenced and found to be 77 670 bp in length, with a mol% G+C of 55.8. A total of 121 likely open reading frames (ORFs) were identified, of which 37 overlapped at start and stop codons. The predicted proteins were usually acidic (average pI of 4.8), and less than about 12% of them had homologues in the sequence databases. Four complete tRNA-like sequences (tRNA-Arg, -Asx, -Pro and -Tyr) and an incomplete tRNA-Thr were detected. A transcription map showed that most of the genome was transcribed and that the synthesis of transcripts occurred in a highly organized and reproducible pattern over a 5 h infection cycle. Transcripts often spanned multiple ORFs, suggesting that viral genes were organized into operons. The predicted ORF and observed transcript directions matched well and showed that transcription is mainly directed inwards from the genome termini, meeting at about 45-48 kb, and this was also a turning point in a cumulative GC-skew plot. The low point in cumulative GC-skew, near the left end, was a region rich in short repeats and lacking ORFs, which is likely to be an origin of replication. The HF2 genome is a mosaic of components from widely different sources, demonstrating clearly that viruses of haloarchaea, like their bacteriophage counterparts, are vectors for the exchange and transmission of genetic material between wide taxonomic distances, even across domains.

Archaea↗
PubMed1992

[The role of mobile genetic elements (MGE) in microevolution].

The MGEs of Drosophila and other objects contain open reading frames (ORFs) encoding transposition enzymes, and "motifs" similar to functional sites: promoters, enhancers, heat shock regulatory sites, those of reception of different stress external signals and hormones, recombination sites, etc. In other words, MGE play a role of "movable cassettes of regulatory elements" in the genomes. The patterns of genome MGE localization are the important components of polygenic systems of character expression, the subjects of variation and evolution. The summed up density of MGE localization along the genome has probably the upper value corresponding to approx. 1 MGE copy per gene. The transpositional variability of MGE patterns could be random, non-random, self-related and inducible. The MGE patterns are important subjects of microevolution and reconstruction of trees of the pattern similarity is an effective method for its description. The patterns could be changed by selection of limited quantitative characters. The stress induction (temperature, treatment, dysgenic cross, etc.) stimulated the MGE transpositions and excisions, mass in population and multiple in individuals. The temperature induction acts probably through the system of response to heat shock treatment. The totality of MGE patterns make up the genomic system capable of quick reorganizations after stress external and genomic influences. The stress external influences are often correlated with passing of the population through the "bottle-neck" stage. The rate of transpositions has an upper limiting border, so named "boundary of regulation error catastrophe", that corresponds to approx. 1 transposition per genome, per generation. After the stress induction of transpositions this boundary could be exceeded, the state of the population norm becoming disrupted. The changes in MGE patterns are also supposed to accompany the changes of characters of isolation, i.e. to accompany the speciation.

Animals↗
PubMed1995

Analysis of a Coxiella burnetti gene product that activates capsule synthesis in Escherichia coli: requirement for the heat shock chaperone DnaK and the two-component regulator RcsC.

A 1.2-kb EcoRI genomic DNA fragment of Coxiella burnetti, when cloned onto a multicopy plasmid, was found to induce capsule synthesis (mucoidy) in Escherichia coli. Nucleotide sequence analysis revealed the presence of an open reading frame that could encode a protein of 270 amino acids. Insertion of a tet cassette into a unique NruI restriction site resulted in the loss of induction of mucoidy. Because of its ability to induce mucoidy, we designated this gene mucZ. Computer search for homologies to mucZ revealed 42% identity to an open reading frame located at 1 min of the E. coli chromosome. Interestingly, the C-terminal amino acid residues of MucZ share significant homology with the J domain of the DnaJ protein and its homologs, suggesting potential interactions between MucZ and components of the DnaK-chaperone machinery. Results presented in this paper suggest that E. coli requires DnaK-chaperone machinery for Lon-RcsA-mediated induction of capsule synthesis, as noticed first by S. Gottesman (personal communication). The induction caused by MucZ is independent of Lon-RcsA and is mediated through the two-component regulators RcsC and RcsB. DnaK and GrpE but not DnaJ are also required for the RcsB-mediated MucZ induction, and we propose that MucZ is a DnaJ-like chaperone protein that might be required for the formation of an active RcsA-RcsB complex and for the RcsC-dependent phosphorylation of RcsB. Discussions are presented that suggest three different roles for alternative forms of the DnaK-chaperone machinery in capsule production.

ATP-Dependent Proteases↗
PubMed2004

GenePalette: a universal software tool for genome sequence visualization and analysis.

To make effective use of the growing host of complete genome sequences, biologists must have easy-to-use software tools that allow them to visualize, analyze, and modify genome data in an interactive and generalized manner. In an effort to bridge the gap between genome and researcher, we have created GenePalette (www.genepalette.org), a desktop application that can access any genome sequence and display the positions of various features [e.g., transcription factor binding sites (TFBSs)] relative to the introns and exons of annotated genes. Written in Java, GenePalette can run on all Java-supporting operating systems (Mac, PC, Unix, Linux). Annotated sequence encompassing the majority of public genome data is rapidly retrieved from GenBank or Ensembl. The software provides intuitive access to the selected genomic region through three interface components: a colorful graphical display showing a schematic of genes and features; an annotated sequence view in which features and genes are highlighted directly on the sequence; and the selectable raw sequence. The three interface components are fully integrated and presented on one page, permitting the user to move easily between representations at different levels of resolution, ranging from kilobases to individual nucleotides. GenePalette is a particularly powerful platform for analyzing the organization of cis-regulatory elements and designing wet-lab experiments to investigate them.

Computational Biology↗
PubMed2002

Two-component signal transduction pathways in Arabidopsis.

The two-component system, consisting of a histidine (His) protein kinase that senses a signal input and a response regulator that mediates the output, is an ancient and evolutionarily conserved signaling mechanism in prokaryotes and eukaryotes. The identification of 54 His protein kinases, His-containing phosphotransfer proteins, response regulators, and related proteins in Arabidopsis suggests an important role of two-component phosphorelay in plant signal transduction. Recent studies indicate that two-component elements are involved in plant hormone, stress, and light signaling. In this review, we present a genome analysis of the Arabidopsis two-component elements and summarize the major advances in our understanding of Arabidopsis two-component signaling.

Amino Acid Sequence↗
PubMed1996

Genomic structure and chromosomal mapping of the mouse VE-cadherin gene (Cdh5).

Vascular endothelial cadherin (VE-cadherin) is located strictly at endothelial junctions and appears to be a major adhesive component of cell to cell contacts. Genomic clones spanning 36 kb and encompassing the mouse VE-cadherin gene have been isolated and characterized. The gene is composed of 12 exons that exhibit conventional vertebrate splicing. The first exon is entirely untranslated, and both exons 2 and 12 contain untranslated regions. A single major transcriptional start site was identified and located 75 bases upstream of the translation initiation codon in the cDNA sequence. The proximal 5'-flanking domain lacks consensus TATA and CAAT boxes at the usual positions. Exon-intron boundaries are similar to those of other cadherin genes, with some exceptions that may have a functional significance in VE-cadherin behavior. The VE-cadherin gene (locus Cdh5) maps to mouse chromosome 8, where it colocalizes with E-cadherin (locus Cdh1), P-cadherin (locus Cdh3), and M-cadherin (locus Cdh14) genes, suggesting that it might be part of a larger cluster of cadherin sequences.

Amino Acid Sequence↗