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At least 595 records · Page 33Linked to original sources
PubMed1992

Towards identification of cis-acting elements involved in the replication of enterovirus and rhinovirus RNAs: a proposal for the existence of tRNA-like terminal structures.

On the basis of a comparative analysis of published sequences, models for the secondary structure of the 3'-terminal [poly(A)-preceding] untranslated region of the entero- and rhinovirus RNAs were worked out. The models for all these viruses share a common core element, but there are an extra enterovirus-specific element and still an additional element characteristic of a subset of enterovirus RNAs. The two latter models were verified for poliovirus and coxsackievirus B genomes by testing with single-strand and double-strand specific enzymatic and chemical probes. A tRNA-like tertiary structure model for the 3'-terminal folding of enterovirus RNAs was proposed. A similar folding was proposed for the 3' termini of the negative RNA strands as well as for the 5' termini of the positive strand of all entero- and rhinovirus RNAs. Implications of these data for template recognition during negative and positive RNA strands synthesis and for the evolution of the picornavirus genomes are discussed.

Base Sequence↗
PubMed2005

Extracting single genomes from heterogenous DNA samples: a test case with Carsonella ruddii, the bacterial symbiont of psyllids (Insecta).

Analysis of many bacterial genomes is impeded by the inability to separate individual species from complex mixtures of cells or to propagate cells in pure culture. This problem is an obstacle to the study of many bacterial symbionts that live intracellularly in insects and other animals. To recover bacterial DNA from complex samples, we devised a method that facilitates the cloning of DNA fragments of distinctive G+C contents in order to generate shotgun DNA libraries enriched in inserts having a specific base composition. DNA preparations are first treated with a restriction enzyme having a common cleavage site in a particular genome and then shotgun cloned following size-fractionation. This method was applied to whole bacteriomes of the psyllid, Pachypsylla venusta, which harbors the bacterial symbiont Candidatus Carsonella ruddii. The resulting libraries were highly enriched in bacterial sequences. Through the use of alternate enzymes and partial digests, this technique can be adapted to yield virtually pure DNA libraries for individual bacterial species.

Animals↗
PubMed2005

A Bayesian genome screening of maximum number of drinks as an alcoholism phenotype with the new Haseman-Elston method.

Common human disorders, such as alcoholism, may be the result of interactions of many genes as well as environmental risk factors. Therefore, it is important to incorporate gene x gene and gene x environment interactions in complex disease gene mapping. In this study, we applied a robust Bayesian genome screening method that can incorporate interaction effects to map genes underlying alcoholism through its application to the data of the Collaborative Studies on Genetics of Alcoholism provided by Genetic Analysis Workshop 14. Our Bayesian genome screening method uses the regression-based stochastic variable selection, coupled with the new Haseman-Elston method to identify markers linked to phenotypes of interest. Compared to traditional linkage methods based on single-gene disease models, our method allows for multilocus disease models for simultaneous screening including both main and interaction (epistatic) effects. It is conceptually simple and computationally efficient through the use of Gibbs sampler. We conducted genome-wide analysis and comparison between scans based on microsatellites and single-nucleotide polymorphisms. A total of 328 microsatellites and 11,560 single-nucleotide polymorphisms (by Affymetrix) on 22 autosomal chromosomes and sex chromosome were used.

Alcoholism↗
PubMed2004

The current clinical value of genomic instability.

Clinical Oncologists have largely ignored the debate over genetic instability in human cancers. It is clear that genetic lesions themselves provide valuable information in the treatment of patients with cancer and those genetic lesions can be exploited successfully as therapeutic targets. Whether the mechanisms resulting in the accumulation of genetic lesions can be translated and used clinically remains to be seen.

Chromosome Aberrations↗
PubMed1999

Issues in genomic screening: critical values, sample sizes, and the ability to detect linkage.

The aims of this study were to empirically investigate the ability of affected sib pairs (ASPs) to localize a gene through screening and to explore estimation of lod score critical values through resampling. To do so, we repartitioned 25 replicates of 100 simulated nuclear families into six data sets of sizes 100, 200, 300, 400, 500, and 1,000 and chose at most one mildly ASP per family. Using all marker data, we calculated maximum lod scores across the six-chromosome genome for each set. Then, we determined the cutoff value corresponding to a 5% genome-wide false positive rate using both the method of Lander and Kruglyak [1995] and a simple resampling algorithm that allows greater scan-specific flexibility. For chromosome 1, the ability of the ASPs to detect the region between markers 9 and 10 clearly increases with the sample size, and genome-wide significance is achieved for samples of size 400 or greater. Also, as expected, the critical values based on the less conservative resampling approach are generally slightly smaller than those from theoretical calculations based on the Ornstein-Uhlenbeck diffusion process of Lander and Kruglyak.

Genetic Linkage↗
PubMed1991

A plastome mutation affects processing of both chloroplast and nuclear DNA-encoded plastid proteins.

Immunoblotting of a chloroplast mutant (pm7) of Oenothera showed that three proteins, cytochrome f and the 23 kDa and 16 kDa subunits of the oxygen-evolving subcomplex of photosystem II, were larger than the corresponding mature proteins of the wild type and, thus, appear to be improperly processed in pm7. The mutant is also chlorotic and has little or no internal membrane development in the plastids. The improperly processed proteins, and other proteins that are completely missing, represent products of both the plastid and nuclear genomes. To test for linkage of these defects, a green revertant of pm7 was isolated from cultures in which the mutant plastids were maintained in a nuclear background homozygous for the plastome mutator (pm) gene. In this revertant, all proteins analyzed co-reverted to the wild-type condition, indicating that a single mutation in a plastome gene is responsible for the complex phenotype of pm7. These results suggest that the defect in pm7 lies in a gene that affects a processing protease encoded in the chloroplast genome.

Amino Acid Sequence↗
PubMed2004

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment.

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 microg-1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.

Culture Media↗
PubMed1998

Quantitative detection of Sphingomonas chlorophenolica in soil via competitive polymerase chain reaction.

The 16S ribosomal RNA gene sequence of the pentachlorophenol degrader Sphingomonas chlorophenolica strain RA2 was used to generate specific polymerase chain reaction (PCR) primers for the detection of this strain in soil, whereas a region internal to the two primers was used to provide an S. chlorophenolica strain RA2-specific oligonucleotide probe. The PCR detection system resulted in a 727 bp product detectable via gel electrophoresis and hybridization. It was specific for strain RA2 and its close relative, S. chlorophenolica ATCC 39723, as evidenced by PCR amplifications of a range of bacterial genomic DNAs. Tests of total microbial community DNA obtained from five uninoculated and two RA2-inoculated soils confirmed this specificity for introduced S. chlorophenolica RA2. Strain RA2 could be detected in soil down to a level of 10(3) cfu g-1 soil. Two strategies were followed to generate internal standard DNA for competitive PCR. First, a 479 bp MIMICS fragment was obtained based on a previously constructed gene cassette; however, this standard did not reliably quantify RA2 targets. Low stringency PCR performed with a range of bacterial genomic DNAs resulted in the generation of an amplicon with a Paenibacillus azotofixans strain that was slightly smaller than the RA2-derived product. Both products were easily separable via conventional gel electrophoresis. The use of this competitor in a threefold dilution scheme applied to the target DNA allowed for the quantitative detection of RA2-specific target DNA molecules from pure culture and from soil. The fate of strain RA2 in pentachlorophenol-contaminated soil was described using this competitive PCR approach, and the organism was shown to persist at two inoculum levels over prolonged periods of time.

DNA Primers↗
PubMed2025

ERCnet: Phylogenomic Prediction of Interaction Networks in the Presence of Gene Duplication.

Assigning gene function from genome sequences is a rate-limiting step in molecular biology research. A protein's position within an interaction network can potentially provide insights into its molecular mechanisms. Phylogenetic analysis of evolutionary rate covariation (ERC) in protein sequence has been shown to be effective for large-scale prediction of functional relationships and interactions. However, gene duplication, gene loss, and other sources of phylogenetic incongruence are barriers for analyzing ERC on a genome-wide basis. Here, we developed ERCnet, a bioinformatic program designed to overcome these challenges, facilitating efficient all-versus-all ERC analyses for large protein sequence datasets. We simulated proteome datasets and found that ERCnet achieves combined false positive and negative error rates well below 10% and that our novel "branch-by-branch" length measurements outperforms "root-to-tip" approaches in most cases, offering a valuable new strategy for performing ERC. We also compiled a sample set of 35 angiosperm genomes to test the performance of ERCnet on empirical data, including its sensitivity to user-defined analysis parameters such as input dataset size and branch-length measurement strategy. We investigated the overlap between ERCnet runs with different species samples to understand how species number and composition affect predicted interactions and to identify the protein sets that consistently exhibit ERC across angiosperms. Our systematic exploration of the performance of ERCnet provides a roadmap for design of future ERC analyses to predict functional interactions in a wide array of genomic datasets. ERCnet code is freely available at https://github.com/EvanForsythe/ERCnet.

Gene Duplication↗
PubMed2005

Large genomic deletions inactivate the BRCA2 gene in breast cancer families.

BACKGROUND: BRCA1 and BRCA2 are the two major genes responsible for the breast and ovarian cancers that cluster in families with a genetically determined predisposition. However, regardless of the mutation detection method employed, the percentage of families without identifiable alterations of these genes exceeds 50%, even when applying stringent criteria for family selection. A small but significant increase in mutation detection rate has resulted from the discovery of large genomic alterations in BRCA1. A few studies have addressed the question of whether BRCA2 might be inactivated by the same kinds of alteration, but most were either done on a relatively small number of samples or employed cumbersome mutation detection methods of variable sensitivity. OBJECTIVE: To analyse 121 highly selected families using the recently available BRCA2 multiplex ligation dependent probe amplification (MLPA) technique. RESULTS: Three different large genomic deletions were identified and confirmed by analysis of the mutant transcript and genomic characterisation of the breakpoints. CONCLUSIONS: Contrary to initial suggestions, the presence of BRCA2 genomic rearrangements is worth investigating in high risk breast or ovarian cancer families.

BRCA2 Protein↗
PubMed2007

Genome-wide linkage mapping for valve calcification susceptibility loci in hypertensive sibships: the Hypertension Genetic Epidemiology Network Study.

It remains unclear whether genetic factors contribute to the susceptibility to valve calcification. Accordingly, echocardiograms and genotyping were performed in 1871 hypertensive siblings who participated in the Hypertension Genetic Epidemiology Network Study. Genome-wide affected sibpair nonparametric linkage analysis was conducted using the allele-sharing method implemented in the Merlin computer program. A total of 1014 sibships from 858 families were evaluated for aortic valve sclerosis or mitral annular calcification. Of these, 78 sibships from 68 families contained > or =2 affected siblings with > or =1 type of valve calcification (142 affected siblings). All 3 of the traits showed a modest degree of familial aggregation, with sibling recurrence risk (SD) and sibling recurrence risk ratio (95% CI) being 0.25 (0.035) and 2.31 (1.72 to 3.11) for aortic valve sclerosis, 0.25 (0.035) and 1.78 (1.36 to 2.33) for mitral annular calcification, and 0.31 (0.030) and 1.52 (1.24 to 1.85) for aortic valve sclerosis and mitral annular calcification, respectively. Affected sibpair linkage analysis revealed the highest logarithm of odds score (3.14) in chromosome 16 at 105.6 cM for aortic valve sclerosis. Other chromosomal regions with logarithm of odds score > or =1.9 were found in chromosomes 19 (2.88), 16 (2.63), 1 (2.12), and 2 (2.03) for aortic valve sclerosis and chromosome 13 (2.12) for any valve calcification. There was no logarithm of odds score > or =1.9 for mitral annular calcification. Our study shows strong linkage of aortic valve sclerosis to chromosome 16q22.1-q22.3 and suggestive linkage to chromosome 19p13.11-p11 and identifies several other promising genomic regions that may contain specific susceptibility loci for valve calcification.

Adult↗
PubMed1980

Two species of full-length cDNA are synthesized in high yield by melittin-treated avian retrovirus particles.

A method of activating endogenous cDNA synthesis in avian retroviruses that results in the formation of two species of full-length cDNA in high yield is described. Tests of biological activity show infectivity of at least the same order of magnitude as for full-length cDNA made by other procedures. Melittin, the major component of bee venom, is used as an alternative to nonionic detergents to make the viral envelope permeable and thus activate the endogenous RNA-dependent DNA polymerase. This compound is a toxic peptide known to interact with phospholipid membranes. It appears to be less disruptive to the viral structure than detergents, resulting in a more efficient transcription of the viral genome. Preliminary tests indicate that this method will also prove useful for studying enzymatic activities associated with other enveloped viruses.

Alpharetrovirus↗
PubMed2000

Mismatch repair is required for O(6)-methylguanine-induced homologous recombination in human fibroblasts.

O:(6)-methylguanine is responsible for homologous recombination induced by N:-methyl-N:'-nitro-N:-nitrosoguanidine (MNNG) [H. Zhang et al. (1996) CARCINOGENESIS:, 17, 2229]. To test the hypothesis that mismatch repair is causally involved in this process, we generated mismatch repair-deficient strains from a human fibroblast line containing a substrate for detecting intrachromosomal homologous recombination. The four strains selected for study exhibited greatly increased resistance to the cytotoxic effects of MNNG, which was not affected by depletion of O:(6)-alkylguanine-DNA alkyltransferase, and greatly increased sensitivity to the mutagenic effect of MNNG, suggesting that the mutagenic base modifications induced in these four cell strains by MNNG persist in their genomic DNA. Tests showed that their extracts are deficient in the repair of G:T mismatches. The frequency of homologous recombination induced by MNNG in three of these strains was significantly (5-7-fold) lower than that induced in the parental cell strain. This was not the result of a generalized defect in recombination, because when (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epox y-7,8,9, 10-tetrahydrobenzo[a]pyrene was used to induce recombination, all three lines responded with a normal or even a somewhat higher frequency than that observed in the parental strain. The lack of recombination displayed by the fourth strain was shown to result from the loss of part of the recombination substrate. The results strongly suggest that functional mismatch repair is required for MNNG-induced homologous recombination.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Polymerase chain reaction based genotyping for characterization of SLA-DQB and SLA-DRB alleles in domestic pigs.

Molecular genotyping of swine major histocompatibility complex SLA-DQB and SLA-DRB genes using polymerase chain reaction (PCR)-based amplification is described. Locus-specific oligonucleotide primers were designed for the analysis of expressed SLA genes by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products were sequenced, and the information gained was used to design primers for PCR genotyping of the exon 2 (beta 1) region from genomic DNA templates. A single segregating amplification product was detected for both DQB and DRB in all animals. PCR products were digested with restriction enzymes. Seven SLA-DQB PCR-restriction fragment length polymorphism (RFLP) pattern types were observed for both HaeIII and RsaI that defined 14 SLA-DQB alleles. A total of seven SLA-DRB PCR-RFLP pattern types were defined using MspI (3 RFLP pattern types) and RsaI (6 RFLP pattern types). In order to demonstrate their universal utility, the primers were tested on genomic DNA samples from 10 different swine breeds. No breed-specific alleles were observed. These results show that locus-specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA-DQB and SLA-DRB from genomic DNA.

Alleles↗
PubMed2003

Functional genomic analysis of the 61D-61F region of the third chromosome of Drosophila melanogaster.

Assigning functional significance to completed genome sequences is one of the next challenges in biological science. Conventional genetic tools such as deficiency chromosomes help assign essential complementation groups to their corresponding genes. We describe an F2 genetic screen to identify lethal mutations within cytogenetic region 61D-61F of the third chromosome of Drosophila melanogaster. One hundred sixteen mutations were identified by their failure to complement both Df(3L)bab-PG and Df(3L)3C7. These alleles were assigned to 14 complementation groups and 9 deficiency intervals. Complementation groups were ordered using existing deficiencies, as well as new deficiencies generated in this study. With the aid of the genomic sequence, genetic and physical maps in the region were correlated by use of PCR to localize the breakpoints of deficiencies within a 268-kb genomic contig (GenBank accession No. AC005847). Six essential complementation groups were assigned to specific genes, including genes encoding a porphobilinogen deaminase and a Sac1-like protein.

Alleles↗
PubMed2006

A genome-wide DNA microsatellite association screen to identify chromosomal regions harboring candidate genes in diabetic nephropathy.

In an effort to accelerate the identification of susceptibility genes in diabetic nephropathy, the first genome-wide fluorescence-based DNA microsatellite (n=6000) association screen was performed, using pools of genomic DNA derived from Irish patients with (cases; n=200) and without (controls; n=200) type 1 diabetic nephropathy. Allele image profiles were generated for 5353 (89.2%) microsatellite markers for both case and control pools. Allele counts (estimated from allele image profiles) were compared in case versus control groups, and empirical P values were generated. Markers then were ranked on the basis of their empirical P values (lowest to highest). Repeat PCR amplification and electrophoresis of pooled samples were performed systematically on ranked markers until the 50 most associated markers with consistent results were identified. DNA samples that composed the pools then were genotyped individually for these markers. Two markers on chromosome 10, D10S558 (Pcorrected=0.005) and D10S1435 (Pcorrected=0.016), revealed statistically significant associations with diabetic nephropathy. An additional four markers (D6S281, D4S2937, D2S291, and D17S515) also are worthy of further investigation. Relevant functional candidate genes have been identified in the vicinity of these markers, demonstrating the feasibility of low-resolution genome-wide microsatellite association screening to identify possible candidate genes for diabetic nephropathy.

Adolescent↗
PubMed1994

Distribution of targets for avian retrovirus DNA integration in vivo.

The targeting of DNA integration in retrovirus-infected cells is a central yet very poorly understood aspect of the biology of the virus. To investigate this problem, we have assessed the use of specific sites for integration targets of avian leukosis virus (ALV) DNA within defined regions of turkey embryo fibroblast (TEF) cellular DNA. For this purpose, we developed an assay of sufficient sensitivity and specificity to allow detection and location of single integration events in a population of 5 million cells. Targets selected for study were either regions cloned by virtue of a previous integration event or clones chosen at random from cellular DNA. By use of this approach, we found that all genomic regions tested contained integration targets, with a frequency that varied from approximately 0.2 to 4 times that expected for random integration. Within regions, the frequency of use of specific sites varied considerably, with some sites used up to 280 times random frequency. When one region was introduced into cells at moderately high copy number by transfection, it provided integration targets in a pattern very much like that seen with the same sequence in vitro. On the basis of our sampling, we conclude that most or all regions of the TEF genome are accessible to ALV retroviral integration. As with integration in vitro, integration specificity seems to be determined largely by local structural features rather than accessibility of specific regions.

Animals↗
PubMed2003

The gene encoding pyolysin, the pore-forming toxin of Arcanobacterium pyogenes, resides within a genomic islet flanked by essential genes.

The plo gene, encoding the Arcanobacterium pyogenes cholesterol-dependent cytolysin, pyolysin (PLO), was localized to a 2.7-kb genomic islet of reduced %G+C content and alternate codon usage frequency. This islet, conserved among isolates from diverse hosts and geographical locations, separated the housekeeping genes smc and ftsY, which are found adjacent in many prokaryotes. The ftsY and ffh genes, located downstream of the plo islet, encode components of the signal recognition particle. Mutational analysis suggested that these genes were essential for viability in A. pyogenes. The A. pyogenes ffh gene was unable to complement a conditional ffh mutant of Escherichia coli and its overexpression was toxic in E. coli. Mutagenesis of the islet-encoded orf121 did not affect plo expression, indicating that it may not be involved directly in the regulation of plo expression. Regardless, the presence of the plo gene as part of a genomic islet inserted between genes essential for normal growth may provide selective pressure for the retention of this important virulence factor.

Bacterial Proteins↗