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At least 1,117 records · Page 62Linked to original sources
PubMed2004

A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli.

Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of alpha- and beta-subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (K(m)=0.77 microM) than for 2-aminophenol (K(m)=0.89 microM) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the alpha- and beta-subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the beta- and alpha-subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.

Amino Acid Sequence↗
PubMed2006

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves.

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited approximately 98% inhibition of local lesion formation at a concentration range of approximately 30 microg ml(-1). The protein was found to be highly basic glycoprotein monomer (pI approximately 9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species.

Amaranthus↗
PubMed2003

Silent DNA: speaking RNA language?

The sequence of silent DNA in the human genome (intergenic spacers, introns and synonymous codon positions of protein-coding genes) was found here to have the higher thermostability of corresponding RNA/RNA and RNA/DNA duplexes as compared with randomized sequence. This difference increased with elevation of GC content. The revealed effect was not due to correlation of RNA/RNA and RNA/DNA thermostabilities with thermostability of the DNA/DNA duplex, which, on the contrary, was lower than in the randomized sequence and lagged behind the elevation of GC content. The same picture was observed in the genomes of other warm-blooded vertebrates but not in the lower organisms. This finding suggests that RNA-RNA and RNA-DNA interactions could be involved in the putative function of silent DNA.

Animals↗
PubMed2000

Frequency of p16(INK4A) alterations and K-ras mutations in intrahepatic cholangiocarcinoma of the liver.

BACKGROUND: Inactivation of the tumour suppressor gene p16 (CDKN2/MTS-1/INK4A) and K-ras mutations are among the most frequent genetic alterations in human malignancies. AIMS: To investigate the tumour suppressor gene p16 and its possible association with K-ras mutations in intrahepatic cholangiocarcinomas of the liver. METHODS: The status of p16 was evaluated in 41 cholangiocarcinomas by methylation specific polymerase chain reaction, microsatellite analysis, DNA sequencing, and immunohistochemical staining. K-ras mutations were determined by direct DNA sequencing analyses after microdissection. The results obtained were correlated with histopathological variables and patient survival. RESULTS: Hypermethylation of the 5' CpG island of the p16 gene was found in 34 of 41 (83%) carcinomas. Homozygous deletion at the p16 region was present in two (5%), and loss of heterozygosity (LOH) in eight cases (20%). We failed to detect p16 gene missense mutations. K-ras mutations were found in 22 of 41 (54%) cholangiocarcinomas and in two cases of tumour surrounding non-neoplastic liver tissue. All 22 cancers with K-ras mutations also exhibited methylated p16. We failed to observe a correlation between K-ras or p16 status and histopathological factors or prognosis of patients. CONCLUSION: These data suggest that inactivation of the p16 gene is a frequent event in cholangiocarcinoma. The most common somatic alteration is promotor methylation of the p16 gene which is closely associated with K-ras mutations. We failed to establish p16 or K-ras status as independent prognostic factors in these tumours.

Bile Duct Neoplasms↗
PubMed2005

Vestige: maximum likelihood phylogenetic footprinting.

BACKGROUND: Phylogenetic footprinting is the identification of functional regions of DNA by their evolutionary conservation. This is achieved by comparing orthologous regions from multiple species and identifying the DNA regions that have diverged less than neutral DNA. Vestige is a phylogenetic footprinting package built on the PyEvolve toolkit that uses probabilistic molecular evolutionary modelling to represent aspects of sequence evolution, including the conventional divergence measure employed by other footprinting approaches. In addition to measuring the divergence, Vestige allows the expansion of the definition of a phylogenetic footprint to include variation in the distribution of any molecular evolutionary processes. This is achieved by displaying the distribution of model parameters that represent partitions of molecular evolutionary substitutions. Examination of the spatial incidence of these effects across regions of the genome can identify DNA segments that differ in the nature of the evolutionary process. RESULTS: Vestige was applied to a reference dataset of the SCL locus from four species and provided clear identification of the known conserved regions in this dataset. To demonstrate the flexibility to use diverse models of molecular evolution and dissect the nature of the evolutionary process Vestige was used to footprint the Ka/Ks ratio in primate BRCA1 with a codon model of evolution. Two regions of putative adaptive evolution were identified illustrating the ability of Vestige to represent the spatial distribution of distinct molecular evolutionary processes. CONCLUSION: Vestige provides a flexible, open platform for phylogenetic footprinting. Underpinned by the PyEvolve toolkit, Vestige provides a framework for visualising the signatures of evolutionary processes across the genome of numerous organisms simultaneously. By exploiting the maximum-likelihood statistical framework, the complex interplay between mutational processes, DNA repair and selection can be evaluated both spatially (along a sequence alignment) and temporally (for each branch of the tree) providing visual indicators to the attributes and functions of DNA sequences.

Algorithms↗
PubMed2004

Expression of the peptidoglycan recognition protein, PGRP, in the lactating mammary gland.

The peptidoglycan recognition protein, PGRP, known as an intracellular component of neutrophils, has been isolated from camel (Camelus dromedarius) milk by acid precipitation followed by heparin-sepharose affinity chromatography of the supernatant. The mean concentration in milk was about 120 mg/L. It decreased during lactation by 19% and increased in the event of severe mastitis by 45%. The protein bound to lactic acid bacteria and other gram-positive bacteria with an affinity similar to that reported for the human and murine orthologs, although the isoelectric point of the molecule was distinctly higher at pH 9.02. The N-terminus of mature camel PGRP was determined as NH2-ArgGluAspProPro-CO2H. Calculated and measured molecular masses were both 19.1 kDa, excluding the possibility of posttranslational modifcation or binding of cation ligands. The peptide probably builds a homotrimer at high concentration. The corresponding mRNA was isolated from lactating mammary gland tissue, and 5.3 kbp of the corresponding gene was sequenced. Similarities were found to the camel lactoferrin gene with regard to sites of expression and to the region 5' upstream to the gene.

Amino Acid Sequence↗
PubMed2000

Characterization of a cDNA encoding RP43, a CUB-domain-containing protein from the tube of Riftia pachyptila (Vestimentifera), and distribution of its transcript.

A major 43 kDa protein from the protective tube of Riftia pachyptila (Vestimentifera), named RP43, was partly microsequenced after isolation by SDS/PAGE from the protein fraction of tubes collected around the hydrothermal vents at the East Pacific Rise. On the basis of the partial peptide sequences obtained, experiments using reverse-transcriptase-mediated PCR and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of deduced amino acid sequence of RP43 showed the presence of CUB domains (100-110-residue-spanning domains first reported in the complement subcomponents C1r/C1s, epidermal-growth-factor-related sea urchin protein and bone morphogenetic protein 1) that seem to be involved in protein-protein and glycosaminoglycan-protein interactions. This peculiarity strongly suggests that RP43 might have a crucial role in tightening the different elements of the worm tube. However, the absence of chitin-binding motifs inclines us to favour a role in protein-protein interactions during assembly of the tube components. The RP43 mRNA was found to be present in specific epidermal cells from the worm body wall but never in the chitin-synthesizing gland cells. This unexpected result clearly indicates that the major tube protein is synthesized in specialized areas of the outer epithelium and that at least two different tissues are involved in the synthesis of the exoskeleton.

Amino Acid Sequence↗
PubMed2000

Virulence of the phytopathogen Pseudomonas syringae pv. maculicola is rpoN dependent.

We cloned the rpoN (ntrA and glnF) gene encoding sigma(54) from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the sigma(54) protein encoded by the Pseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation, rpoN::Km(r). In contrast to wild-type ES4326, ES4326 rpoN::Km(r) was nonmotile and could not utilize nitrate, urea, C(4)-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326 rpoN::Km(r) did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Arabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Km(r) carrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326 rpoN::Km(r) partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of hrpL in ES4326 rpoN::Km(r) did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.

Amino Acid Sequence↗
PubMed2003

Multiple endocrine neoplasia type 1 (MEN1) germline mutations in familial isolated primary hyperparathyroidism.

BACKGROUND: Familial isolated hyperparathyroidism (FIHP) is an autosomal dominant disorder characterized by uniglandular or multiglandular parathyroid tumours that occur in the absence of other endocrine tumours. The disorder may represent either an early stage of multiple endocrine neoplasia type 1 (MEN1), or an allelic variant of MEN1, or a distinct entity involving another locus. We have explored these possibilities in seven families in whom primary hyperparathyroidism occurred as the sole endocrinopathy. METHODS: Seven FIHP families were ascertained and venous blood samples obtained from 35 members (17 affected and 18 unaffected) for DNA sequence analysis of the MEN1 gene. The mean (+/- SD) follow-up period in the 17 affected members was 15.06 (+/- 8.83) years. RESULTS: Four heterozygous germline mutations of the MEN1 gene were identified. These consisted of two 4-bp intragenic deletions that would result in prematurely truncated proteins, and two missense (Asp153Val and Ala411Pro) mutations. Furthermore, analysis of parathyroid tumour DNA from one individual revealed a loss of the wild-type allele and retention of the mutant allele, consistent with Knudson's 'two-hit' model of hereditary cancer and a tumour suppressor role for MEN1 in FIHP. CONCLUSIONS: Our results provide further support for FIHP being a distinct allelic variant of MEN1, and an analysis of the 16 mutations reported to date indicate that FIHP is associated with a higher frequency of missense MEN1 mutations.

Adenoma↗
PubMed2000

Sequence analysis and clinical significance of the iceA gene from Helicobacter pylori strains in Japan.

The Helicobacter pylori iceA gene was recently identified as a genetic marker for the development of peptic ulcer in a Western population. To assess the significance of iceA subtypes of H. pylori in relation to peptic ulcer, 140 Japanese clinical isolates (88 from Fukui and 52 from Okinawa) were characterized. Sequence analysis of the iceA1 gene from 25 representative Japanese strains was also carried out to identify the differences in iceA between the ulcer group and the gastritis group. The iceA1 genotype was not correlated with the presence of peptic ulceration in either area. In addition, sequence analysis led to identification of five deletions and five point mutations (a nonsense mutation or a 1-bp insertion) within the iceA1 open reading frame corresponding to previously published sequences. These mutations were identified in both clinical groups (ulcer and gastritis groups) in each area. Local DNA sequence analysis revealed that the endpoints of all five deletions coincided with direct repeats. We also found four strains that carried longer iceA1 open reading frames compared with that for strain 60190. In conclusion, carriage of an iceA1 strain does not seem to be a risk factor for peptic ulcer in Japanese subjects. The critical mutations in the iceA1 gene in some isolates from patients with peptic ulcers suggested that IceA does not participate in the pathogenesis of peptic ulcer in Japan. We also found deletion hot spots that were associated with direct repeats in iceA1 and that favored a small-deletion model of slipped mispairing events during replication. We showed that iceA1 sequence variations may be useful tools for analysis of the population genetics of H. pylori.

Bacterial Proteins↗
PubMed2006

Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2.

Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non-synonymous single nucleotide polymorphisms (NS-SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site-directed mutagenesis showed that the TM-12 NS-SNPs, and principally the G1473A NS-SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele-specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy-Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.

ATP-Binding Cassette Transporters↗
PubMed1996

Substrate properties of fluorescent ribonucleotides in the terminal transferase-catalyzed labeling of DNA sequencing primers.

Terminal deoxynucleotidyltransferase (terminal transferase, E.C. 2.7.7.31) has been used to add a single fluorescent ribonucleotide to the 3' terminus of DNA sequencing primers, thereby creating primers suitable for automated DNA sequence analysis. The previously introduced procedure using fluorescein-UTP for the postsynthetic labeling of primers can, under appropriate reaction conditions, now be extended to commercially available fluorescein-ATP and fluorescein-CTP permitting greater flexibility in primer design. The products of these addition reactions have been shown to provide sequence data qualitatively and quantitatively identical to those obtained with conventional 5'-terminally labeled primers using cycle sequencing conditions in conjunction with an automated sequencer. Ribonucleotide derivatives of four other dyes (coumarin, tetramethylrhodamine, lissamine and Texas Red) were also examined for their potential in the terminal transferase-catalyzed reaction. Whereas coumarin-UTP was efficiently incorporated giving a monoaddition product, the conjugates of all other dyes with ATP, CTP and UTP were extremely poor substrates under all conditions tested.

Coumarins↗
PubMed2005

Genetic and antigenic analysis of Bordetella pertussis isolates recovered from clinical cases in Ontario, Canada, before and after the introduction of the acellular pertussis vaccine.

Sixty-eight Bordetella pertussis isolates (obtained between 1994 and 2004 from the province of Ontario in Canada) were compared by the following phenotypic and genetic analyses: serotyping; pulsed-field gel electrophoresis; and partial DNA sequence analysis of their pertactin, pertussis toxin, and fimbriae genes. Although temporal genetic variations were observed among the isolates, which is consistent with the current view that B. pertussis evolves over time, no specific antigenic or genetic type was detected in 48 isolates collected shortly after the introduction of the acellular pertussis vaccine. Further surveillance with clinical data and isolates collected periodically will be required to ensure that any genetic divergence that could affect vaccine efficacy will not be occurring.

Antigenic Variation↗
PubMed2001

Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11.

Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.

Amino Acid Sequence↗
PubMed2004

Cloning and characterization of a mosquito larvicidal toxin produced during vegetative stage of Bacillus sphaericus 2297.

The mosquitocidal toxin 1 (mtx1) gene from genomic DNA of B. sphaericus strain 2297 was cloned and expressed in E. coli. DNA sequencing analysis of the cloned gene revealed a single open reading frame encoding an 870-amino acid polypeptide. Expression level of the full-length gene in E. coli was very low even though strong promoter was used or the gene was expressed as a fusion protein. Expression level was highly improved after the putative leader sequence was deleted, and the truncated gene was expressed as a fusion protein with glutathione S-transferase (GST-tMtx1). E. coli cells expressing GST-tMtx1 was highly toxic to Culex quinquefasciatus larvae and showed lower toxicity against Anopheles dirus and Aedes aegypti larvae. Enterobacter amnigenus An11, a mosquito larval gut colonizable bacteria, transformed with the cloned gene exhibited mosquito larvicidal activity. Result suggested that there is a potential to develop this protein to be used as an alternative mosquito control agent.

Animals↗
PubMed1995

An aromatase pseudogene is transcribed in the bovine placenta.

The present investigation is focused on a new, truncated bovine aromatase cytochrome P-450-encoding transcript (bCyp19) which was repeatedly isolated from a placental lambda gt10 cDNA library. Different cDNA probes derived from bCyp19 were used during screenings. The deviant transcript contains areas of considerable sequence homology (89-98%) corresponding to exons II, III, V, VIII and IX of bCyp19. Exons VI and VII are missing. Exon IV is replaced by a bovine repeat sequence motif. Numerous translation stop codons occur within all reading frames, thus suggesting that the transcript does not encode a functional protein. The gene was detected by PCR in the genomes of all animals investigated (n = 38). The experiments demonstrate that the bovine genome contains a non-functional copy (pseudogene, Cyp19 phi) of bCyp19 that is transcribed together with the bCyp19 messenger in the bovine post-parturition placenta.

Animals↗
PubMed2005

An algorithm for the determination and quantification of components of nucleic acid mixtures based on single sequencing reactions.

BACKGROUND: Determination and quantification of nucleic acid components in a mixture is usually accomplished by microarray approaches, where the mixtures are hybridized against specific probes. As an alternative, we propose here that a single sequencing reaction from a mixture of nucleic acids holds enough information to potentially distinguish the different components, provided it is known which components can occur in the mixture. RESULTS: We describe an algorithm that is based on a set of linear equations which can be solved when the sequencing profiles of the individual components are known and when the number of sequenced nucleotides is larger than the number of components in the mixture. We have implemented the procedure for one type of sequencing approach, pyrosequencing, which produces a stepwise output of peaks that is particularly suitable for the procedure. As an example we use signature sequences from ribosomal RNA to distinguish and quantify several different species in a mixture. Using simulations, we show that the procedure may also be applicable for dideoxy sequencing on capillary sequencers, requiring only some instrument specific adaptations of protocols and software. CONCLUSION: The parallel sequencing approach described here may become a simple and cheap alternative to microarray experiments which aim at routine re-determination and quantification of known nucleic acid components from environmental samples or tissue samples.

Algorithms↗
PubMedDate not supplied

An overview on the distribution of word counts in Markov chains.

In this paper, we give an overview about the different results existing on the statistical distribution of word counts in a Markovian sequence of letters. Results concerning the number of overlapping occurrences, the number of renewals and the number of clumps will be presented. Counts of single words and also multiple words are considered. Most of the results are approximations as the length of the sequence tends to infinity. We will see that Gaussian approximations switch to (compound) Poisson approximations for rare words. Modeling DNA sequences or proteins by stationary Markov chains, these results can be used to study the statistical frequency of motifs in a given sequence.

Biometry↗