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At least 163 records · Page 9Linked to original sources
PubMed1996

A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp.

The lack of simple and efficient methods for extraction of DNA from Nocardia spp. has hampered molecular manipulation of the DNA for diagnostic purposes. In the present study, a method for the rapid extraction of undegraded genomic nocardial DNA was established. Briefly, 14 pathogenic Nocardia strains were grown at 37 degrees C for 3 to 5 days in Sauton broth containing 0.05% Tween 80. Subsequently, the cultures were treated for 48 h with 1.2 mg of cycloserine per ml (final concentration). Cells were then harvested by centrifugation and treated with a lysis solution containing 3 mg of lysozyme per ml. This was followed by the addition of proteinase K and sodium dodecyl sulfate to final concentrations of 0.2 mg/ml and 0.5%, respectively, and incubation for 1 h at 50 degrees C. DNA was precipitated with isopropanol after phenol-chloroform-isoamyl alcohol extractions and RNase treated before being quantitated and analyzed by agarose gel electrophoresis. The average undegraded DNA yields obtained were 101 micrograms for Nocardia brasiliensis and 121 micrograms for N. asteroides. This DNA was suitable for restriction endonuclease digestion and PCR amplification, which are methods being applied to the characterization and diagnosis of slowly growing organisms such as Nocardia spp.

DNA Restriction Enzymes↗
PubMed2005

Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial "pan-genome".

The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae, the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for approximately 80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate (Malawi LIL20/1).

The nucleotide sequence of a 55098 bp region from the right end of the genome of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) has been determined. Translation of the sequence identified 67 major open reading frames (ORFs) which are closely spaced and read from both DNA strands. At six positions intergenic tandem repeat arrays are found. Comparison of the predicted amino acid sequences of encoded proteins with protein sequence databases identified a number of homologies. These include three subunits of RNA polymerase, a protein with homology to transcription factor SII (TFSII), a DNA ligase, two subunits of mRNA capping enzyme, a DNA topoisomerase type II, a dUTPase, a protein kinase, three helicases, a ubiquitin-conjugating enzyme, a protein with homology to the nif S and nif S-like proteins identified in some bacteria and Saccharomyces cerevisiae, a protein with homology to both a myeloid differentiation primary response antigen (MyD116) and to a herpes simplex virus-encoded neurovirulence-associated protein (ICP34.5), a protein with homology to the ASFV-encoded structural protein p22, two proteins with homology to copies of the ASFV-encoded multigene family 360 and one protein with homology to the ASFV-encoded multigene family 110. Four genes encode proteins which have homology to each other and constitute a new multigene family (MGF100). Nine ORFs encode proteins which contain predicted transmembrane domains. The possible functions of these predicted ASFV-encoded proteins are discussed and the evolutionary relationship of ASFV to other viruses are considered. Despite the similarities in genome structure and replication strategy of ASFV with poxviruses, sequence similarity between them is low and the organization of ASFV-encoded genes is not colinear with that of the orthopoxviruses.

African Swine Fever Virus↗
PubMed1998

Use of representational difference analysis to identify genomic differences between pathogenic strains of Vibrio cholerae.

Representational difference analysis (RDA) is a recently developed technique used for amplifying genetic differences between two closely related genomes. We compared RDA and a modified version of RDA to examine genomic differences between the two Vibrio cholerae serogroups that cause epidemic cholera, O1 and O139, and between the two biotypes of the O1 serogroup. With both techniques, we recovered several sequences known to be found only in V. cholerae O139 but absent in its presumed progenitor, V. cholerae O1 El Tor. A greater number of unique fragments were generated in comparing the two V. cholerae O1 biotypes, consistent with the probable greater genetic differences between the two biotypes.

Blotting, Southern↗
PubMed1993

Comparative analysis of the genomes of mycobacteriophages infecting saprophytic and pathogenic mycobacteria.

The genomes of a series of mycobacteriophages have been analyzed to disclose possible relationships between genetic characteristics and host range. The percent guanine-plus-cytosine in the DNA of 14 phages was found to be 34.4 to 47.5, as determined by a double-labelling procedure, which is unaffected by the presence of modified bases. The DNA of few mycobacteriophages yielded discordant values when the G + C content was estimated by buoyant density determination and by the double labelling procedure. This observation suggests the possible presence of modified bases in these genomes. The reduced susceptibility of viral DNAs to several restriction endonucleases is suggestive of the occurrence of both methyladenine and methylcytosine in the genome of all the mycobacteriophages studied. Heterologous annealing among the 14 DNAs analyzed yielded 6 hybridization groups. Within one group, the homology level among viral genomes was estimated by comparing the electrophoretic mobilities of restriction fragments: values of 0.8 to 1.3% base substitution have thus been found. A comparison of the genomic characteristics and host range of the mycobacteriophages analyzed suggests a possible relationship between restriction pattern, G + C content, crosshybridization level and host range.

Base Composition↗
PubMed1999

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens.

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

Bacterial Proteins↗
PubMed2000

Identification of sequences encoding the detoxification metalloisomerase glyoxalase I in microbial genomes from several pathogenic organisms.

The ubiquitous glyoxalase system, which is composed of two enzymes, removes cellular cytotoxic methylglyoxal (MG). In an effort to identify critical residues conserved in the evolution of the first enzyme in this system, glyoxalase I (GlxI), as well as the structural implications of sequence alterations in this enzyme, a search of the National Center for Biotechnology Information (NCBI) database of unfinished genomes was undertaken. Eleven putative GlxI sequences from pathogenic organisms were identified and analyses of these sequences in relation to the known and previously identified GlxI enzymes were performed. Several of these sequences show a very high similarity to the Escherichia coli GlxI sequence, most notably the 79% identity of the sequence identified from Yersinia pestis, the causative agent of bubonic plague. In addition to the conservation of residues critical to binding the catalytic metal in all of the proposed GlxI enzymes, four regions in the Homo sapiens GlxI enzyme are absent in all of the bacterial GlxI sequences, with the exception of Pseudomonas putida. Removal of these regions may alter the active-site conformation of the bacterial enzymes in relation to that of the H. sapiens. These differences may be targeted for the development of inhibitors selective to the bacterial enzymes.

Amino Acid Sequence↗
PubMed2002

Investigation of Maize streak virus pathogenicity determinants using chimaeric genomes.

Genes and intergenic regions were reciprocally exchanged between a highly pathogenic Maize streak virus (MSV) isolate (MSV-MatA) and three less pathogenic isolates (MSV-Kom, MSV-R2, and MSV-VW) to determine the contribution of individual genome constituents to MSV pathogenicity in maize. Comparison of disease symptoms produced by the 54 resulting chimaeras and parental viruses enabled identification of genome constituents that are primarily responsible for the heightened pathogenicity of MSV-MatA in maize. Whereas pathogenicity determinants were detected in all of the MSV genomic regions examined, generally only chimaeras containing the MSV-MatA long intergenic region, coat protein gene, and/or movement protein gene were more pathogenic than the milder MSV isolates from which most of their genomes were derived. The pathogenicity of chimeras was strongly influenced by the relatedness of their parental viruses and evidence was found of nucleotide sequence-dependent interactions between both coding and intergenic regions.

Chimera↗
PubMed2005

Genome sequence of the non-pathogenic strain 15 of pneumonia virus of mice and comparison with the genome of the pathogenic strain J3666.

Pneumonia virus of mice (PVM) is a member of the subfamily Pneumovirinae and is the closest known relative of respiratory syncytial virus. Both viruses cause pneumonia in their respective hosts. Here, the genome sequences of two strains of PVM, non-pathogenic strain 15 and pathogenic strain J3666, are reported. Comparison of the genome sequences revealed 59 nucleotide differences between the two strains, 37 of which were coding. The nucleotide differences were spread throughout the genome, affecting cis-acting regulatory regions and seven of the ten genes. Development of a reverse-genetics system for PVM should allow further elucidation of the functional importance of the genetic differences between the two strains identified here.

Amino Acid Sequence↗