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At least 937 records · Page 52Linked to original sources
PubMed1997

Mutations in the nonstructural 5A gene of European hepatitis C virus isolates and response to interferon alfa.

The response rate to interferon alfa (IFN-alpha) in patients infected with hepatitis C virus (HCV) genotype 1 isolates is poor. A region associated with sensitivity to IFN has been identified in subtype HCV-1b isolates from Japanese patients in the carboxyterminal half of the nonstructural protein NS5A (between codon 2209 and 2248). HCV-1b isolates with at least four amino acid changes in this region compared with the HCV-1b prototype sequence were sensitive, whereas isolates identical to the prototype sequence were resistant to IFN-alpha. Patients infected with HCV-1b isolates carrying 1 to 3 mutations in NS5A(2209-2248) showed an intermediate response pattern. Because of the large geographical differences observed for HCV it is unknown whether this putative IFN-alpha sensitivity determining region is also predictive for European isolates. We analyzed 32 patients chronically infected with HCV-1a or HCV-1b isolates who were treated with 3 million units of recombinant IFN-alpha three times per week for 1 year. Before initiation, during, and after treatment serum HCV-RNA levels were assessed by a quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. The amino acid sequence of NS5A(2209-2248)was determined by direct sequencing of the PCR-amplified HCV genome and was compared with the reference sequence HCV-J. In patients chronically infected with subtype HCV-1a or HCV-1b the initial or sustained response to IFN-alpha was not related to the number of amino acid substitutions in the NS5A(2209-2248) region. In addition, the number of amino acid changes in NS5A(2209-2248) was not related to pretreatment HCV-RNA serum levels. In three patients with a pronounced initial decline of HCV-RNA levels (>3 log) sequence analyses of NS5A(2209-2248) were performed before and after therapy. Compared with the pretreatment amino acid sequence the HCV isolates of these patients revealed more mutations in the NS5A(2209-2248) region after therapy. These findings from European patients indicate that the NS5A(2209-2248) region of HCV does not represent a common interferon sensitivity determining region.

Adult↗
PubMed1994

Researches on DLA-DRB1 genotyping by PCR-RFLP. II. A study of serology and cellularly defined DLA haplotypes and their segregation.

The polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) method was used to study DLA class II gene in dogs. Genomic DNA from 11 DLA homozygous reference dogs representing 8 different haplotypes and 2 families with a total of 16 animals were amplified by the oligonucleotide primer pair (HLA-DRB-AMP-A/B) cross-hybridizing HLA-DRB specific and fit for the amplification of DLA-DRB1 gene. The corresponding amplified DNA products were 235 base pairs. Amplified DNA was digested by 32 different restriction endonucleases, which could recognize allelic variations within DLA-DRB. After digesting only with Hae III, Hha I, Hinf I, Rsa I and Sau96 I high polymorphism was revealed respectively and 9 distinct RFLP pattern were shown, which could be correlate to the DLA haplotypes studied. The 8 cellular established DLA-D specificities present in the reference panel were defined unequivocally by PCR-RFLP and correlated with DLA-Dw5 and Dw6 two subtypes. The segregation pattern of four different DLA-DRB types could be demonstrated in two families. Based on these data we conclude that PCR-RFLP typing utilizing the above mentioned primer pair and endonucleases is a valuable tool to define DLA class II types in the dog.

Alleles↗
PubMed2005

Two naturally occurring deletion mutants of 12S seed storage proteins in Arabidopsis thaliana.

Two naturally occurring Arabidopsis mutants, Cape Verde Islands and Monte (Mr-0), with aberrant 12S seed storage protein (SSP) profiles have been identified by SDS-PAGE. In both mutants, one of the 12S globulin bands is missing while a new band of lower molecular mass is present. Tandem mass spectrometry-mass spectrometry (MS/MS) analyses of the mutant peptides have revealed that both are shorter variants of 12S globulin with deletion sites detected within the alpha-subunits of 12S globulin cruciferin B (CRB) and C (CRC), respectively. Sequence analyses of the genomic DNA flanking the deletion sites have demonstrated that both deletions occurred at the genomic level. These two mutants are referred to as CRBDelta12 and CRCDelta13 with the delta sign indicating a deletion and the number indicating amino acids deleted. Alignment of these two mutant sequences with that of soybean A3B4 subunit, for which the crystal structure was determined recently, have revealed that the CRCDelta13 deletion is located in a hypervariable/disordered region, and will probably not affect the structure of the hexameric globulin. The CRBDelta12 deletion, however, is located in a binding region that is thought to be important for the hexamer formation. However, CRBDelta12 appears to accumulate normally as judged by its band intensity relative to the other SSP subunits on the protein gels. Thus it seems that the seed can, to a certain extent, tolerate some mutations in its storage proteins.

Amino Acid Sequence↗
PubMed1983

A thermodynamic theory of codon bias in viral genes.

The relationship between degeneracy in the genetic code and the occurrence of a strong codon bias is examined, with particular reference to a group of viral genomes. The present paper shows how codon bias may have been imposed by thermodynamic considerations at the time the primitive DNA first formed in the primordial soup. Using a four-state Ising-like model with stacking interactions between successive base pairs, we show how primeval periodic DNA polymers could have arisen the remnants of which are still observed in codon biases today.

Base Composition↗
PubMed1993

Application of the mini-mu phage for the isolation of lac transcriptional fusions in Bacillus subtilis genes.

A cassette containing a selectable cat gene and the lacZ gene without its own promoter has been incorporated into the mini-Mu bacteriophage genome. This mini-Mu derivative, referred to as mMu-Bs, can be used in Escherichia coli for the generation of lacZ transcriptional fusions to Bacillus subtilis genes cloned into plasmids. The resultant fusions can be analyzed in B. subtilis either as multicopy plasmids or as a single copy integrated via a Campbell-like recombination into the wild-type locus of the cloned fragment.

Bacillus subtilis↗
PubMed2002

A high frequency of mtDNA polymorphisms in HeLa cell sublines.

The complete mtDNA sequences from the uncloned "founder" HeLa cells and from five sublines have been determined. These sequences all carry a common "core" of 38 single basepair alterations relative to the revised Cambridge Reference Sequence (CRS). The HeLa mitochondrial genome is of African descent and it is a member of the African L3 haplogroup. The sequence of the HeLa mtDNA resolves the uncertainty surrounding the mosaic composition of the original CRS for human mtDNA. Most importantly, we detected a total of eight polymorphisms that have arisen in the mtDNA coding region of different HeLa sublines. These observations suggest that HeLa mtDNA has a high rate of sequence divergence, relative to the phylogenetically-derived divergence rate for mtDNAs in the human population, which results from a relaxation of negative selection against the fixation of deleterious mutations. Furthermore, this high frequency of polymorphisms in HeLa mtDNA may reflect a process similar to the accumulation of somatic mtDNA mutations in human cancers. Preliminary analysis of single-cell derived subclone lines revealed the occurrence of another polymorphism and provided evidence for a large number of mtDNA segregation units.

Base Sequence↗
PubMed2001

A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment.

A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% (n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1--3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.

Animals↗
PubMed2003

High-resolution comparative genomic hybridisation yields a high detection rate of chromosomal aberrations in childhood acute lymphoblastic leukaemia.

BACKGROUND: Cytogenetic aberrations are of prognostic significance in childhood acute lymphoblastic leukaemias and a high detection rate could improve the biological understanding and classification of these diseases. METHODS: Bone-marrow samples from 92 children with acute lymphoblastic leukaemia were studied by high-resolution comparative genomic hybridisation (HRCGH) using dynamic standard reference intervals that enhance both specificity and sensitivity in the detection of aberrations. RESULTS: In 80 patients (87%) HRCGH revealed a total of 405 aberrations, mostly whole chromosome gains (n = 265) and partial losses (n = 80). The 25 leukaemias with a gain of more than five whole chromosomes by HRCGH harboured only 7% of all losses. With G-band karyotyping 59 patients (64%) had aberrations. HRCGH revealed more aberrations per patient than did G-band karyotyping (median: 3 vs. 1, P = 0.005), revealed aberrations in 27 of the 34 patients for whom the G-band karyotyping failed or was found to be normal, and specifically revealed more 9p losses (21% vs. 5%, P < 0.005), 12p losses (12% vs. 2%, P < 0.05) and 17q gains (11% vs. 1%, P < 0.01). Compared to the present study, the frequency of patients with aberrant karyotypes was significantly lower in previous conventional CGH studies (64% vs. 87%, P < 0.0001), as was the rate of partial aberrations per patient (1.1% vs. 1.7, P < 0.001), particularly with fewer 6q losses, 9p losses and 17q gains detected. CONCLUSION: HRCGH is superior to conventional CGH as an adjunct to G-band karyotyping as it detects recurrent aberrations at a significantly higher rate than both these techniques.

Adolescent↗
PubMed2002

Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50.

Porphyromonas gingivalis is an anaerobic, asaccharolytic Gram-negative rod associated with chronic periodontitis. We have undertaken a proteomic study of the outer membrane of P. gingivalis strain W50 using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteins were identified by reference to the pre-release genomic sequence of P. gingivalis available from The Institute for Genomic Research. Out of 39 proteins identified, five were TonB-linked outer membrane receptors, ten others were putative integral outer membrane proteins and four were putative lipoproteins. Pyroglutamate was found to be the N-terminal residue of seven of the proteins, and was predicted to be the N-terminal residue of 13 additional proteins. The RgpA, Kgp and HagA polyproteins were identified as fully processed domains in outer membranes prepared in the presence of proteinase inhibitors. Several domains were found to be C-terminally truncated 16-57 residues upstream from the N-terminus of the following domain, at a residue penultimate to a lysine. This pattern of C-terminal processing was not detected in a W50 strain isogenic mutant lacking the lysine-specific proteinase Kgp. Construction of another W50 isogenic mutant lacking the arginine-specific proteinases indicated that RgpB and/or RgpA were also involved in domain processing. The C-terminal adhesin of RgpA, designated RgpA27, together with RgpB and two newly identified proteins designated P27 and P59 were found to migrate on two-dimensional gels as vertical streaks at a molecular mass 13-42 kDa higher than that calculated from their gene sequences. The electrophoretic behaviour of these proteins, together with their immunoreactivity with a monoclonal antibody that recognizes lipopolysaccharide, is consistent with a modification that could anchor the proteins to the outer membrane.

Adhesins, Bacterial↗
PubMed2005

Estimation of the nuclear DNA content of gossypium species.

BACKGROUND AND AIMS: Gossypium is an economically important, globally distributed taxon comprising more than 50 species. DNA content estimates from about half of the species indicate over a 3-fold variation exists. However, the nine DNA content estimates for G. hirsutum reveal over a 2-fold difference for this species alone. Recent reports have shown that several plant compounds can bias DNA content estimates obtained by commonly used methods. The purpose of this research was to examine the standardization procedures used for DNA content determinations with flow cytometry as applied to Gossypium, and generate revised DNA content estimates for all available Gossypium species using best-standard practices. METHODS: Flow cytometry was used to measure fluorescence of isolated Gossypium nuclei stained with propidium iodide. Fluorescence values were converted to DNA content estimates based on the nuclear fluorescence of standard genotypes of barley, corn and rice. Various combinations of nuclei preparations relative to the standards were evaluated for their influence on the estimates. KEY RESULTS: Both external standardization and internal standardization with Oryza sativa 'IR36' yielded statistically similar DNA content estimates for Gossypium. Internal standardization with Hordeum vulgare 'Sultan' resulted in a high estimate of DNA content. Nuclear DNA content estimates were generated for 37 Gossypium species using external standardization. Estimates of ancestral genome sizes reveal that both increases and decreases in nuclear DNA content have occurred. Variation in intraspecific and intragenomic DNA content was low, and the allopolyploid AD-genome size was nearly the additive of its progenitor genomes. CONCLUSIONS: Due to unknown factors, internal standardization with H. vulgare 'Sultan' may not be appropriate for DNA content determinations of Gossypium. The current DNA content estimates support accepted cytogenetic divisions of the genus. Gossypium is a genus that exhibits genome constancy both through speciation within genomic groups and allopolyploidization.

Cell Nucleus↗
PubMed2006

The UCSC Known Genes.

The University of California Santa Cruz (UCSC) Known Genes dataset is constructed by a fully automated process, based on protein data from Swiss-Prot/TrEMBL (UniProt) and the associated mRNA data from Genbank. The detailed steps of this process are described. Extensive cross-references from this dataset to other genomic and proteomic data were constructed. For each known gene, a details page is provided containing rich information about the gene, together with extensive links to other relevant genomic, proteomic and pathway data. As of July 2005, the UCSC Known Genes are available for human, mouse and rat genomes. The Known Genes serves as a foundation to support several key programs: the Genome Browser, Proteome Browser, Gene Sorter and Table Browser offered at the UCSC website. All the associated data files and program source code are also available. They can be accessed at http://genome.ucsc.edu. The genomic coverage of UCSC Known Genes, RefSeq, Ensembl Genes, H-Invitational and CCDS is analyzed. Although UCSC Known Genes offers the highest genomic and CDS coverage among major human and mouse gene sets, more detailed analysis suggests all of them could be further improved.

Base Sequence↗
PubMed1997

PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing.

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.

Animals↗
PubMed2004

Feline immunodeficiency virus subtypes A, B and C and intersubtype recombinants in Ontario, Canada.

Knowledge of the geographical distribution of feline immunodeficiency virus (FIV) subtypes is important for understanding different disease courses and for vaccine design. Intersubtype recombination may develop in areas where more than one subtype is prevalent and has the potential to create new transmittable variants with novel pathogenic properties. In this study, 40 FIV-positive DNA samples were classified by sequence analysis of the LTR-gag region. Phylogenetic analysis indicated that 32 Canadian FIV isolates clustered with previously identified subtypes A, B and C and that subtype A was most frequent in Ontario. Four strains with inconsistent clade assignment were further analysed by sequencing of the env-LTR regions. Comparisons of phylogenetic trees constructed from the two different regions of the genome and analysis of similarities to reference sequences yielded classification of three samples as A/B and one as A/C intersubtype recombinants. Although the A/B recombinant samples were obtained from unrelated cats in geographically disparate regions, a common breakpoint was consistently identified within gag. In addition, there was no evidence of co-infection with parental strains of subtypes A and B as indicated by PCR-based limiting dilution assays, although these assays allowed for the identification of two different recombinant viruses co-existing in one sample. Both sequences contained the same breakpoint. These findings suggested that a new circulating recombinant FIV may be enzootic in Ontario.

Animals↗
PubMed2002

Identification of Brachyspira hyodysenteriae-specific DNA fragments using representational difference analysis.

Two novel Brachyspira hyodysenteriae-specific DNA fragments, designated as Bh100 and Bh400, were identified using representational difference analysis. To isolate the fragments the combined DNA of the Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira murdochii and Brachyspira innocens reference strains was subtracted from the genome of B. hyodysenteriae strain B204. Both fragments were present in a single copy and mapped to different positions on the genome of B. hyodysenteriae B78(T). Larger fragments encompassing the continuous open reading frames (ORF) of Bh100 and Bh400 were cloned and analysed. Whereas the ORF of 2130 bp encompassing Bh100 did not show homology to any known bacterial protein, Bh400 was part of a putative operon with significant homology to the phosphotransferase system of Bacillus subtilis.

Blotting, Southern↗
PubMed2005

Improvement of galactose uptake in Saccharomyces cerevisiae through overexpression of phosphoglucomutase: example of transcript analysis as a tool in inverse metabolic engineering.

Through genome-wide transcript analysis of a reference strain and two recombinant Saccharomyces cerevisiae strains with different rates of galactose uptake, we obtained information about the global transcriptional response to metabolic engineering of the GAL gene regulatory network. One of the recombinant strains overexpressed the gene encoding the transcriptional activator Gal4, and in the other strain the genes encoding Gal80, Gal6, and Mig1, which are negative regulators of the GAL system, were deleted. Even though the galactose uptake rates were significantly different in the three strains, we surprisingly did not find any significant changes in the expression of the genes encoding the enzymes catalyzing the first steps of the pathway (i.e., the genes encoding Gal2, Gal1, Gal7, and Gal10). We did, however, find that PGM2, encoding the major isoenzyme of phosphoglucomutase, was slightly up-regulated in the two recombinant strains with higher galactose uptake rates. This indicated that PGM2 is a target for overexpression in terms of increasing the flux through the Leloir pathway, and through overexpression of PGM2 the galactose uptake rate could be increased by 70% compared to that of the reference strain. Based on our findings, we concluded that phosphoglucomutase plays a key role in controlling the flux through the Leloir pathway, probably due to increased conversion of glucose-1-phosphate to glucose-6-phosphate. This conclusion was supported by measurements of sugar phosphates, which showed that there were increased concentrations of glucose-6-phosphate, galactose-6-phosphate, and fructose-6-phosphate in the strain construct overexpressing PGM2.

Culture Media↗
PubMed1990

Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization.

Genomic DNA was prepared from four reference strains of Mycobacterium paratuberculosis and 46 isolates of this organism from New Zealand, Australia, Canada, and Norway and also from two mycobactin-dependent "wood pigeon" strains. The DNA was characterized by restriction endonuclease analysis, both with and without DNA hybridization, with a probe specific to a repetitive DNA sequence in M. paratuberculosis. Both techniques differentiated M. paratuberculosis strains into two groups, but DNA hybridization revealed more differences between strains within the larger group. All the strains from cattle and many strains from other animals belonged to this group. The second group of nine strains included the Faroe Islands strain, all New Zealand sheep strains, and one New Zealand goat strain. Primary isolation of strains belonging to this group was difficult to achieve. DNA from acid-fast organisms harvested directly from intestinal tissues of sheep with Johne's disease was shown to have restriction and hybridization patterns identical to those of DNA obtained from M. paratuberculosis isolates cultured from the same tissues. Two Norwegian goat strains and the wood pigeon strains did not hybridize to the M. paratuberculosis probe and had restriction patterns very different from those of other M. paratuberculosis strains. The wood pigeon strains had restriction patterns very similar to those of strains of Mycobacterium avium, indicating that they should be classified as that species. The presence of two distinct groups of M. paratuberculosis strains and their predominant distribution in different host animals may be significant in management of mixed-animal farming operations.

Animals↗
PubMed1994

Analysis of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum by multilocus enzyme electrophoresis.

The genetic diversity of 74 Australian field isolates of Erysipelothrix rhusiopathiae and 22 reference strains for serovars of E. rhusiopathiae or Erysipelothrix tonsillarum was examined by multilocus enzyme electrophoresis. Four serovar reference strains of E. tonsillarum (strains KS 20 A, Wittling, Lengyel-P, and Bano 107 for serovars 25, 3, 10, and 22, respectively) were genetically distinct from E. rhusiopathiae. However, the E. tonsillarum reference strain for serovar 14 (Iszap-4) and the reference strain for serovar 13 (Pecs-56), which has been said to represent a new genomic species, were found to cluster with typical isolates and reference strains of E. rhusiopathiae. Our reference strain for serovar 7 (Rotzunge) was also genetically typical of E. rhusiopathiae, thus indicating that these serotype reactivities cannot be relied upon as a means of identifying isolates as E. tonsillarum. Australian field isolates of E. rhusiopathiae were genetically diverse. Those recovered from sheep or birds were more diverse than those isolated from pigs, and isolates of serovar 1 were more diverse than those of serovar 2. The diversity found among isolates of the same serovar and the presence of isolates of different serovars in the same electrophoretic types (ETs) indicated that serotyping of E. rhusiopathiae was unreliable for use as an epidemiological tool. Some ETs contained isolates recovered from different animal species. ET 41 contained 32.2% of the field isolates and two reference strains, indicating that this clone of E. rhusiopathiae is both widespread and commonly associated with disease in various species of animals.

Animals↗