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PubMed1995

Detection of novel marine methanotrophs using phylogenetic and functional gene probes after methane enrichment.

A major limitation of rRNA-targeted group-specific probes is that they may cross-react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences. We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology. Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients. The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries. Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected. A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas. Organisms containing this sequence were observed in enrichments by in situ hybridization. The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes.

Alcohol Oxidoreductases↗
PubMed2006

Application of random matrix theory to microarray data for discovering functional gene modules.

We show that spectral fluctuation of coexpression correlation matrices of yeast gene microarray profiles follows the description of the Gaussian orthogonal ensemble (GOE) of the random matrix theory (RMT) and removal of small values of the correlation coefficients results in a transition from the GOE statistics to the Poisson statistics of the RMT. This transition is directly related to the structural change of the gene expression network from a global network to a network of isolated modules.

Algorithms↗
PubMed1993

Cloning of the fourth functional gene for protein phosphatase 1 in Drosophila melanogaster from its chromosomal location.

Complementary DNA encoding a catalytic subunit of protein phosphatase 1, PP1 87B, hybridises at four positions (87B, 9C, 13C and 96A) to Drosophila melanogaster polytene chromosomes, three of which are known to be expressed [Dombrádi, V., Axton, J.M., Brewis, N.D., Da Cruz e Silva, E.F., Alphey, L. & Cohen, P.T.W. (1990) Eur. J. Biochem. 194, 739-745]. The fourth gene has been isolated by screening a genomic library of cosmid clones, representing division 13 of the X-chromosome of D. melanogaster, with a PP1 87B probe. This library was constructed as part of the Drosophila genome mapping project [Sidén-Kiamos, I., Saunders, R.D.C., Spanos, L., Majerus, T., Trenear, J., Savakis, C., Louis, C., Glover, D.M., Ashburner, M. & Kafatos, F.C. (1990) Nucleic Acids Res. 18, 6261-6270]. The 5' non-coding region of the isolated gene hybridised to cytological position 13C1-2. By combining reverse transcription and the polymerase chain reaction, the gene was shown to be expressed at a very low level. The PP1 13C gene encodes a protein of 302 amino acids with a predicted molecular mass of 34.5 kDa. It shows 85-94% amino acid identity to the other three protein phosphatase 1 catalytic subunits (PP1 87B, PP1 96A and PP1 9C) described previously, being most closely related to the isoform PP1 87B, which is involved in the control of chromosome separation at cell division and the regulation of chromosome condensation at interphase.

Amino Acid Sequence↗
PubMed2005

Association of functional gene polymorphisms of matrix metalloproteinase (MMP)-1, MMP-3 and MMP-9 with the progression of chronic liver disease.

BACKGROUND AND AIMS: Matrix metalloproteinases (MMP) play an important role in the progression of liver fibrosis in addition to fibrogenesis. MMP-1, MMP-3, and MMP-9 gene polymorphisms have been shown to influence the transcriptional activity of their respective gene promoter in an allele-specific manner. The aim of this study was to examine the possible association of MMP-1, MMP-3, and MMP-9 gene polymorphisms with the progression of chronic liver disease in the Japanese population. METHODS: We examined 91 patients with HCV-related chronic hepatitis and 89 patients with HCV-related liver cirrhosis. We determined MMP-1 1G/2G, MMP-3 5A/6A, and MMP-9 C/T polymorphisms using polymerase-chain reaction based assays. RESULTS: In MMP-1 genotypes, the 2G homozygotes were significantly more in cirrhotic group than in chronic hepatitis group. In MMP-3 genotypes, there were no significant differences in genotype distributions and allele frequencies between chronic hepatitis and liver cirrhosis groups. However, 5A carriers had a significantly lower age at liver cirrhosis diagnosis and a higher Child-Pugh score compared with the 6A homozygotes. In MMP-9 genotypes, the C homozygotes and C allele frequencies were significantly more in liver cirrhosis group than in chronic hepatitis group. CONCLUSION: These findings suggest that MMP-1, MMP-3, and MMP-9 gene polymorphisms account for some of the variability in the progression of HCV-related chronic liver diseases.

Aged↗
PubMed2006

Environmental survey for four pathogenic bacteria and closely related species using phylogenetic and functional genes.

Bacterial species with high DNA sequence similarity to pathogens could affect the specificity of assays designed to detect biological threat agents in environmental samples. The natural presence of four pathogenic bacteria, Bacillus anthracis, Clostridium perfringens, Francisella tularensis, and Yersinia pestis and their closely related species, was determined for a large collection of soil and aerosol samples. Polymerase chain reaction (PCR) and gene sequencing were used using group-specific 16S rRNA primers to identify pathogens and related species, and pathogen-specific virulence genes. Close relatives of B. anthracis (B. cereus group species) were detected in 37% of the soils and 25% of the aerosol samples. The B. anthracis protective antigen (pag) gene or a close homolog was detected in 16 of these samples. For the other three pathogen groups, the frequency of detection was much lower, and none of the samples were positive with both the phylogenetic and virulence gene primer sets.

Aerosols↗
PubMed2005

Identification of four novel exon 5 splice variants of the mouse mu-opioid receptor gene: functional consequences of C-terminal splicing.

The rat mu-opioid receptor clone in which novel exon 5 was found in the place of exon 4 (MOR-1B) was one of the first MOR-1 variants described. We now have identified the mouse homolog of the rat MOR-1B as well as four additional variants derived from splicing from exon 3 into different sites within exon 5. The sequences of all of the variants were identical except for the intracellular tip of the C terminus encoded by exon 5, where each variant predicted a unique amino acid sequence ranging from 2 to 39 amino acids. All of the mMOR-1B variants were selective for mu-opioids in receptor-binding assays, as anticipated, because they all have identical binding pockets defined by the transmembrane domains. However, the relative potency and efficacy of mu-agonists to each other varied from variant to variant in guanosine 5'-O-(3-[35S]thio)triphosphate-binding studies, as shown by morphine-6beta-glucuronide, which was the most efficacious agent against mouse MOR-1B1 (mMOR-1B1) and the least efficacious agent against mMOR-1B2. mMOR-1B4 was quite unusual. Although mMOR-1B4 was mu-selective in receptor-binding studies and antagonists labeled mMOR-1B4 well, the binding affinities of most of the mu-agonists were far lower than those seen with mMOR-1, suggesting that the 39 amino acids at the C terminus of mMOR-1B4 influences the conformation of the receptor and its ligand recognition site itself either directly or through its interactions with other proteins. In conclusion, alterations in the amino acid sequence of the C terminus do not alter the mu-specificity of the receptor but they can influence the binding characteristics, efficacy, and potency of mu-opioids.

Animals↗
PubMed1984

Antigens encoded by the 3'-terminal region of human T-cell leukemia virus: evidence for a functional gene.

Antibodies in sera from patients with adult T-cell leukemia-lymphoma or from healthy carriers of type I human T-cell leukemia virus (HTLV) recognize an antigen of approximately 42 kilodaltons (p42) in cell lines infected with HTLV-I. Radiolabel sequence analysis of cyanogen bromide fragments of p42 led to the conclusion that this antigen is encoded in part by LOR, a conserved portion of the "X" region that is flanked by the envelope gene and the 3' long terminal repeat of HTLV-I. It is possible that this novel product mediates the unique transformation properties of the HTLV family.

Amino Acid Sequence↗
PubMed2003

Evaluation of broiler litter with reference to the microbial composition as assessed by using 16S rRNA and functional gene markers.

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10(9) CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.

Animals↗
PubMed2004

Coupling of functional gene diversity and geochemical data from environmental samples.

Genomic techniques commonly used for assessing distributions of microorganisms in the environment often produce small sample sizes. We investigated artificial neural networks for analyzing the distributions of nitrite reductase genes (nirS and nirK) and two sets of dissimilatory sulfite reductase genes (dsrAB1 and dsrAB2) in small sample sets. Data reduction (to reduce the number of input parameters), cross-validation (to measure the generalization error), weight decay (to adjust model parameters to reduce generalization error), and importance analysis (to determine which variables had the most influence) were useful in developing and interpreting neural network models that could be used to infer relationships between geochemistry and gene distributions. A robust relationship was observed between geochemistry and the frequencies of genes that were not closely related to known dissimilatory sulfite reductase genes (dsrAB2). Uranium and sulfate appeared to be the most related to distribution of two groups of these unusual dsrAB-related genes. For the other three groups, the distributions appeared to be related to pH, nickel, nonpurgeable organic carbon, and total organic carbon. The models relating the geochemical parameters to the distributions of the nirS, nirK, and dsrAB1 genes did not generalize as well as the models for dsrAB2. The data also illustrate the danger (generating a model that has a high generalization error) of not using a validation approach in evaluating the meaningfulness of the fit of linear or nonlinear models to such small sample sizes.

Bacteria↗
PubMed2004

Microarray and functional gene analyses of sulfate-reducing prokaryotes in low-sulfate, acidic fens reveal cooccurrence of recognized genera and novel lineages.

Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 microM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family "Syntrophobacteraceae" were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.

Bacterial Proteins↗
PubMed1987

uvrC gene function has no specific role in repair of N-2-aminofluorene adducts.

In Escherichia coli, plasmid DNA modified with N-2-aminofluorene adducts survived equally well in wild-type, uvrA, or uvrB strains. Increased sensitivity was found in uvrC and uvrD strains. Moreover, N-2-aminofluorene-mediated toxicity in the uvrC background was reversed when an additional uvrA mutation was introduced into the strain.

DNA Repair↗
PubMed1973

Requirement of a functional gene 32 product of bacteriophage T4 in UV, repair.

A temperature-sensitive mutation in gene 32 was used to study the role of gene 32 protein in the repair of UV-damaged DNA of bacteriophage T4. It was possible to distinguish between repair and replication of DNA at 33 C. At this temperature, DNA replication continued, and the intracellular DNA was stable. In contrast, no significant repair of UV-damaged DNA was observed even 40 min after the irradiation. Therefore, it was concluded that the defect in the repair mechanism at this temperature is not a simple consequence of the defect in DNA replication but that gene 32 apparently has an independent role for DNA repair. It was reported previously that gene 32 product is required for both T4 DNA replication and genetic recombination. In addition to these findings, this study has given direct evidence that, in vivo, this protein is also essential for the UV repair mechanism.

Centrifugation, Density Gradient↗
PubMed1971

Reversion of frameshift mutations stimulated by lesions in early function genes of bacteriophage T4.

Temperature-sensitive (ts) mutants representative of a number of genes of phage T4 were crossed with rII mutants to allow isolation of ts, rII double-mutant recombinants. The rII mutations used were characterized as frameshift mutations primarily on the basis of their revertability by proflavine. For each ts, rII double mutant, the effect of the ts mutation on spontaneous reversion of the rII mutation was determined over a range of incubation temperatures. A strong enhancement in reversion of two different rII mutants was detected when they were combined with tsL56, a mutation in gene 43 [deoxyribonucleic acid (DNA) polymerase]. Three other mutants defective in gene 43 enhanced reversion about fourfold. Two mutations in gene 32, which specifies a protein necessary for DNA replication, enhanced reversion about 5-fold and 18-fold, respectively. Two additional mutations in gene 43 and two in gene 32 had no effect. Fivefold and threefold enhancements in reversion were also found with mutations in genes 44 (DNA synthesis) and 47 (deoxyribonuclease), respectively. No significant effect was found with mutations in seven additional genes. The results of other workers suggest that frameshift mutations arise from errors in strand alignment during repair synthesis occurring at chromosome tips. Our results show that such errors can be enhanced by mutations in the DNA polymerase, the gene 32 protein, and the enzymes specified by genes 44 and 47. This implies that these proteins are employed in the repair process occurring at chromosome tips and that mutational errors in these proteins can lead to loss of ability to recognize and reject strand misalignments.

Adenine↗