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PubMed2000

Identification of nucleocapsid binding sites within coronavirus-defective genomes.

The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N-viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3' end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949-4524), as well as a region at the 3' end of the MHV N ORF (nts 4837-5197) within MIDI-C. Binding to the full-length MIDI-C transcript (approximately 5500 nts) and to an approximately 1-kb transcript from the gene 1a region (nts 935-1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

Animals↗

PubMed2006

Construction and characterization of a highly redundant Pseudomonas aeruginosa genomic library prepared from 12 clinical isolates: application to studies of gene distribution among populations.

OBJECTIVE: To create, array, and characterize a pooled, high-coverage, genomic library composed of multiple biofilm-forming clinical strains of the opportunistic pathogen, Pseudomonas aeruginosa (PA). Twelve strains were obtained from patients with otorrhea, otitis media, and cystic fibrosis as a resource for investigating: difference in the transcriptomes of planktonic and biofilm envirovars; the size of the PA supragenome and determining the number of virulence genes available at the population level; and the distributed genome hypothesis. METHODS: High molecular weight genomic DNAs from 12 clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of approximately 1.5 kb. Equimolar amounts of the 12 sheared genomic DNAs were then pooled and used in the construction of a genomic library with approximately 250,000 clones that was arrayed and subjected to quality control analyses. RESULTS: Restriction endonuclease and sequence analyses of 686 clones picked at random from the library demonstrated that >75% of the clones contained inserts larger than 0.5 kb with the desired mean insert size of 1.4 kb. Thus, this library provides better than 4.5x coverage for each of the genomes from the 12 components clinical PA isolates. Our sequencing effort ( approximately 1 million nucleotides to date) reveals that 13% of the clones present in this library are not represented in the genome of the reference P. aeruginosa strain PA01. CONCLUSIONS: Our data suggests that reliance on a single laboratory strain, such as PA01, as being representative of a pathogenic bacterial species will fail to identify many important genes, and that to obtain a complete picture of complex phenomena, including bacterial pathogenesis and the genetics of biofilm development will require characterization of the P. aeruginosa population-based supra-genome.

Child, Preschool↗

PubMed1994

Rates and patterns of chloroplast DNA evolution.

The chloroplast genome (cpDNA) of plants has been a focus of research in plant molecular evolution and systematics. Several features of this genome have facilitated molecular evolutionary analyses. First, the genome is small and constitutes an abundant component of cellular DNA. Second, the chloroplast genome has been extensively characterized at the molecular level providing the basic information to support comparative evolutionary research. And third, rates of nucleotide substitution are relatively slow and therefore provide the appropriate window of resolution to study plant phylogeny at deep levels of evolution. Despite a conservative rate of evolution and a relatively stable gene content, comparative molecular analyses reveal complex patterns of mutational changes. Non-coding regions of cpDNA diverge through insertion/deletion changes that are sometimes site dependent. Coding genes exhibit different patterns of codon bias that appear to violate the equilibrium assumptions of some evolutionary models. Rates of molecular change often vary among plant families and orders in a manner that violates the assumption of a simple molecular clock. Finally, protein-coding genes exhibit patterns of amino acid change that appear to depend on protein structure, and these patterns may reveal subtle aspects of structure/function relationships. Only comparative studies of molecular sequences have the resolution to reveal this underlying complexity. A complete description of the complexity of molecular change is essential to a full understanding of the mechanisms of evolutionary change and in the formulation of realistic models of mutational processes.

Biological Evolution↗

PubMed1999

The BM2 protein of influenza B virus is synthesized in the late phase of infection and incorporated into virions as a subviral component.

The influenza B virus genome RNA segment 7 encodes the M1 and BM2 proteins. The BM2 protein is synthesized by a coupled translational termination-reinitiation mechanism at the overlapping stop-start pentanucleotide in a bicistronic mRNA transcribed from RNA segment 7. However, features and functions of this protein remain unclear. In this study the BM2 protein was characterized by using an antiserum raised to the BM2 protein of influenza virus strain B/Yamagata/1/73. In cells infected with B/Yamagata virus the alphaBM2 antibody specifically detected the BM2 protein with a molecular mass of 12 kDa and also a polypeptide with a molecular mass of 17 kDa. When infected cells were labelled with 32Pi and immunoprecipitated with the alphaBM2 antibody, the 32P-labelled 17 kDa polypeptide was specifically precipitated. In the presence of casein kinase inhibitor CKI-7 the synthesis of the 17 kDa and BM2 proteins was completely suppressed, although other viral proteins, except for the polymerase protein, were synthesized normally. These results suggest that the 17 kDa species is a phosphorylated form of the BM2 protein. These species were substantially synthesized in the late phase of infection and localized in the cytoplasm throughout infection. Moreover, they were transported to the plasma membrane and thereafter were incorporated into virions. These results therefore suggest that the BM2 and the 17 kDa proteins are necessary for the life-cycle of influenza B virus.

Animals↗

PubMed1983

Closely related species of Drosophila can contain different libraries of middle repetitive DNA sequences.

Very few of the middle repetitive DNA sequences found in Drosophila melanogaster are present in all of the species of the D. melanogaster subgroup. One member of the subgroup, D. erecta, lacks most of the families of repetitive elements from D. melanogaster (including copia and 412) but possesses many other families not present in the D. melanogaster genome. Other species in the subgroup possess some families in these two species and lack others. From this we conclude that most individual middle repetitive families are highly unstable components of the Drosophila genome over short periods of evolutionary time.

Animals↗

PubMed2003

Genomic instability and endoreduplication triggered by RAD17 deletion.

Cell cycle checkpoints are critical for genomic stability. Rad17, a component of the checkpoint clamp loader complex (Rad17/Rfc2-5), is required for the response to DNA damage and replication stress. To explore the role of Rad17 in the maintenance of genomic integrity, we established somatic conditional alleles of RAD17 in human cells. We find that RAD17 is not only important for the Atr-mediated checkpoint but is also essential for cell viability. Cells lacking RAD17 exhibited acute chromosomal aberrations and underwent endoreduplication at a high rate. Therefore, RAD17 links the checkpoint to ploidy control and is essential for the maintenance of chromosomal stability.

Blotting, Southern↗

PubMed2026

Mutation in THO2, a component of THO/TREX complex, causes transcriptional gene silencing and genome-wide DNA methylation changes.

DNA methylation plays important roles in silencing of transgenes, endogenous genes, and transposable elements (TEs). To identify genes involved in antagonizing transcriptional or DNA hypermethylation-induced gene silencing, a genetic screening was conducted and thus a tho2-8 mutant was recovered. THO2 is a major component of the THO/TREX (Transcription-Export) complex, which plays essential roles in mRNA export. The tho2-8 mutation caused overaccumulation of DNA methylation on a d35S promoter ahead of LUC, suggesting its roles in antisilencing of transgenes. This mutation also resulted in significant genome-wide alterations in DNA methylation in a locus-specific manner, including 2513 hyper-DMRs and 1717 hypo-DMRs. The hyper-DMRs in the tho2-8 mutant not only exhibited a considerable overlap with those in DNA demethylation mutants (like ros1-7), but also with hypo-DMRs from nrpd1-3 and nrpe1-11 mutants, demonstrating that THO2 is able to protect those loci targeted by DNA demethylation and/or RdDM pathways from hypermethylation. The tho2-8 mutant also contained a plethora of CHH hypo-DMRs, which overlapped in large numbers with those from the nrpd1-3 and nrpe1-11 mutants, indicating that THO2 is required for the establishment/maintenance of DNA methylation at many loci. Additionally, the tho2-8 mutation caused an increase in overall 24-nt siRNA levels and many upregulated and downregulated DEGs/DETEs. The effects of THO2 on DNA methylation patterns appeared to be associated with the functioning of Pol IV and Pol V because THO2 physically interacted with NRPD7 and was necessary for normal accumulation levels of several Pol V-dependent IGNs' transcripts. Thus, this study provided valuable insights into new roles of THO2 in DNA methylation patterning.

DNA Methylation↗

PubMed1996

Domains of the measles virus N protein required for binding to P protein and self-assembly.

The nucleocapsid protein (N, 525 amino acids) of measles virus plays a central role in the replication of the viral genomic RNA. Its functions require interactions with itself and with other viral components. The N protein encapsidates genomic RNA, a function reflected in its ability to self-assemble into nucleocapsid-like particles in the absence of other viral proteins. The substrate for the packaging of nascent RNA during RNA replication is a complex between the N and phosphoprotein (P). The domains on the N protein that promote binding to P protein and self-assembly have been identified utilizing a series of N protein deletions. Two noncontiguous regions, amino acids 4-188 and 304-373 of N protein, are required for the formation of the soluble N-P complex, while deletion of amino acids 189-239 did not affect N-P binding. Amino acids 240-303 appear to be necessary for the stability of the protein. The N-terminal 398 amino acids are all required for the formation of organized nucleocapsid-like particles, since deletion of the central region from amino acids 189-373 completely abolished N-N interaction, and deletion of amino acids 4-188 and 374-492 caused the formation of unstructured aggregates.

Animals↗

PubMed2000

Functional genomics of Neisseria meningitidis pathogenesis.

The pathogenic bacterium Neisseria meningitidis is an important cause of septicemia and meningitis, especially in childhood. The establishment and maintenance of bacteremic infection is a pre-requisite for all the pathological sequelae of meningococcal infection. To further understand the genetic basis of this essential step in pathogenesis, we analyzed a library of 2,850 insertional mutants of N. meningitidis for their capacity to cause systemic infection in an infant rat model. The library was constructed by in vitro modification of Neisseria genomic DNA with the purified components of Tn10 transposition. We identified 73 genes in the N. meningitidis genome that are essential for bacteremic disease. Eight insertions were in genes encoding known pathogenicity factors. Involvement of the remaining 65 genes in meningocoocal pathogenesis has not been demonstrated previously, and the identification of these genes provides insights into the pathogenic mechanisms that underlie meningococcal infection. Our results provide a genome-wide analysis of the attributes of N. meningitidis required for disseminated infection, and may lead to new interventions to prevent and treat meningococcal infection.

Animals↗

PubMed1999

Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved.

Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro.

Binding Sites↗

PubMed2005

The organization and inheritance of the mitochondrial genome.

Mitochondrial DNA (mtDNA) encodes essential components of the cellular energy-producing apparatus, and lesions in mtDNA and mitochondrial dysfunction contribute to numerous human diseases. Understanding mtDNA organization and inheritance is therefore an important goal. Recent studies have revealed that mitochondria use diverse metabolic enzymes to organize and protect mtDNA, drive the segregation of the organellar genome, and couple the inheritance of mtDNA with cellular metabolism. In addition, components of a membrane-associated mtDNA segregation apparatus that might link mtDNA transmission to mitochondrial movements are beginning to be identified. These findings provide new insights into the mechanisms of mtDNA maintenance and inheritance.

Animals↗

PubMed2003

Molecular characterization of Enterococcus faecalis two-component signal transduction pathways related to environmental stresses.

A key issue in the comprehension of the Enterococcus faecalis stress response is to understand how this bacterium is able to modulate its gene expression in accordance to environmental conditions. Through bioinformatic analysis of the E. faecalis V583 genome, nine two-component systems and a single orphan response regulator were identified. A transcriptional study gave evidence of four systems whose expression is induced by at least one environmental stress. In addition, gene disruption experiments allowed the isolation of eight response regulator mutants. Insertional inactivation of the response regulator gene err 05 resulted in growth default and cell morphology alterations; and also in expression default of the sagA gene, this latter being recently shown involved in E. faecalis stress resistance toward numerous lethal treatments (Y. Le Breton, A. Mazé, A. Hartke, S. Lemarinier, Y. Auffray and A. Rincé (2002) Current Microbiol 45: 434-439). Disruption of the err 04, err 08 or err 18 genes resulted in sensitivity towards heat. Finally, the err 10 mutant was shown to be more sensitive to acid pH and NaCl whereas its growth was less affected by bile salts or heat. We also demonstrated that the heat resistance phenotype of the err 10 mutant was correlated with an increase of the heat shock proteins DnaK and GroEL level.

Amino Acid Sequence↗

PubMed2005

Miniaturization in functional genomics and proteomics.

Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future.

Biological Assay↗

PubMed2004

Mapping disease-susceptibility genes in admixed populations using interval principal component tests.

Family-based association approach for mapping disease-susceptibility genes of complex human diseases is a topical issue in genetic epidemiology. It is well known that admixture between genetically differentiated populations can result in high levels of linkage disequilibrium at loci separated far apart. This property has been capitalized upon to reduce the burden of genotyping in a genomewide association scan. The authors describe a new approach for admixture mapping--the "interval principal component test" (IPCT). The genome is divided into a multitude of non-overlapping "intervals" (with interval length of 10-20 cM) and the information of the markers in the same interval is integrated using the principal component analysis. Monte-Carlo simulation shows that an interval-by-interval scan using IPCT has much better performances than a conventional marker-by-marker scan using the transmission/disequilibrium test (TDT).

Computer Simulation↗

PubMed2006

Are genome-wide association studies all that we need to dissect the genetic component of complex human diseases?

With the availability of dense maps of anonymous and frequent SNPs spanning the whole human genome, genome-wide association studies are now becoming a reality. In this paper, we discuss the utility of these approaches to detect genetic risk variants involved in complex disease susceptibility and, in the best case scenario where a signal is detected, how helpful it will be to the understanding of the pathological process.

Genetic Diseases, Inborn↗

PubMed1982

Physical parameters affecting the rate and completion of RNA driven hybridization of DNA: new measurements relevant to quantitation based on kinetics.

Differences in the RNA-driven hybridization kinetics of genomic DNA and cDNA probes led us to examine physical parameters affecting these reactions. Cloned cDNA complementary to serum albumin (SA) mRNA hybridized in accordance with single component kinetics, whereas cloned SA genomic DNA hybridized more slowly and with multiple component kinetics. This difference is largely attributable to the relatively short and variable lengths of the mRNA complementary regions in the cloned genomic DNA. The rate of mRNA driven hybridization is affected to about half the extent observed for DNA renaturation as Na+ is increased or decreased from 0.18M. In the annealing of nucleic acids of high sequence complexity, after approximately 70% of reaction has been reached, the rate of the reaction is slowed and completion is not reached under "static" conditions. In practical terms, this is not the case for systems of low sequence complexity. This problem can be largely overcome by continuous or frequent mixing of the reactants, so that complex cDNA probes are hybridized essentially to completion, and kinetics can therefore be more readily compared to simple complexity standards.

Animals↗

PubMed1999

Chromosomal organization of a sequence related to LTR-like elements of Ty1-copia retrotransposons in Avena species.

A repetitive sequence, pAs17, was isolated from Avena strigosa (As genome) and characterized. The insert was 646 bp in length and showed 54% AT content. Databank searches revealed its high homology to the long terminal repeat (LTR) sequences of the specific family of Ty1-copia retrotransposons represented by WIS2-1A and Bare. It was also found to be 70% identical to the LTR domain of the WIS2-1A retroelement of wheat and 67% identical to the Bare-1 retroelement of barley. Southern hybridizations of pAs17 to diploid (A or C genomes), tetraploid (AC genomes), and hexaploid (ACD genomes) oat species revealed that it was absent in the C diploid species. Slot-blot analysis suggested that both diploid and tetraploid oat species contained 1.3 x 10(4) copies, indicating that they are a component of the A-genome chromosomes. The hexaploid species contained 2.4 x 10(4) copies, indicating that they are a component of both A- and D-genome chromosomes. This was confirmed by fluorescent in situ hybridization analyses using pAs17, two ribosomal sequences, and a C-genome specific sequence as probes. Further, the chromosomes involved in three C-A and three C-D intergenomic translocations in Avena murphyi (AC genomes) and Avena sativa cv. Extra Klock (ACD genomes), respectively, were identified. Based on its physical distribution and Southern hybridization patterns, a parental retrotransposon represented by pAs17 appears to have been active at least once during the evolution of the A genome in species of the Avena genus.

Avena↗

PubMed1996

The gene distribution of the human genome.

Linear correlations exist between the GC levels of third codon positions (GC3) of individual human genes and the GC levels of long genomic sequences and DNA molecules (50-100 kb in size) embedding the genes. These linear relationships allow the positioning of the GC3 histogram of cDNA sequences from the databases relative to the CsCl profile of human DNA. In turn, this allows an estimate of the relative concentrations of genes in genomic regions of different GC content. An estimate obtained by using current sequence data and Gaussian decompositions of the GC3 histogram and of the CsCl profile indicates that the GC-richest (non-ribosomal) component of the human genome is at least 17 times as gene-rich as the GC-poor regions. Moreover, our results suggest that the most recent physical maps of the human genome consisting of overlapping YACs cover less than 50% of the genes.

Chromosome Mapping↗