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At least 649 records · Page 36Linked to original sources
PubMed1998

Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes.

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon's site of insertion has been developed. The basic plasposon, named TnMod, consists of the Tn5 inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn5 transposase, in combination with the flexibility afforded by the modular arrangement of TnMod, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.

DNA Transposable Elements↗
PubMed2002

The Pseudomonas aeruginosa genome: how do we use it to develop strategies for the treatment of patients with cystic fibrosis and Pseudomonas infections?

In the 2 years since the complete sequence of Pseudomonas aeruginosa strain PAO1 was published, at least 200 papers have been published describing research that made use of the PAO1 genome sequence. Some of this research included genome-wide studies of gene expression or the effect of mutation on bacterial functions such as biofilm formation; this type of global analysis would not have been possible without the availability of the sequence. As a result of these and other, more traditional, research studies, there is a wealth of new knowledge about the physiology of this pathogen. This raises the possibility of new strategies for the treatment of patients with P. aeruginosa infection, either by novel antibiotics or by drugs targeting bacterial functions essential for survival and virulence in the human host.

Biofilms↗
PubMed2004

Phylogenetic analysis based on genome-scale metabolic pathway reaction content.

Phylogenetic classifications based on single genes such as rRNA genes do not provide a complete and accurate picture of evolution because they do not account for evolutionary leaps caused by gene transfer, duplication, deletion and functional replacement. Here, we present a whole-genome-scale phylogeny based on metabolic pathway reaction content. From the genome sequences of 42 microorganisms, we deduced the metabolic pathway reactions and used the relatedness of these contents to construct a phylogenetic tree that represents the similarity of metabolic profiles (relatedness) as well as the extent of metabolic pathway similarity (evolutionary distance). This method accounts for horizontal gene transfer and specific gene loss by comparison of whole metabolic subpathways, and allows evaluation of evolutionary relatedness and changes in metabolic pathways. Thus, a tree based on metabolic pathway content represents both the evolutionary time scale (changes in genetic content) and the evolutionary process (changes in metabolism).

Archaea↗
PubMed2005

Metagenomics: DNA sequencing of environmental samples.

Although genomics has classically focused on pure, easy-to-obtain samples, such as microbes that grow readily in culture or large animals and plants, these organisms represent only a fraction of the living or once-living organisms of interest. Many species are difficult to study in isolation because they fail to grow in laboratory culture, depend on other organisms for critical processes, or have become extinct. Methods that are based on DNA sequencing circumvent these obstacles, as DNA can be isolated directly from living or dead cells in various contexts. Such methods have led to the emergence of a new field, which is referred to as metagenomics.

Animals↗
PubMed2003

Proteomics of bacterial pathogens.

The rapid growth of proteomics that has been built upon the available bacterial genome sequences has opened provided new approaches to the analysis of bacterial functional genomics. In the study of pathogenic bacteria the combined technologies of genomics, proteomics and bioinformatics has provided valuable tools for the study of complex phenomena determined by the action of multiple gene sets. The review considers some of the recent developments in the establishment of proteomic databases as well as attempts to define pathogenic determinants at the level of the proteome for some of the major human pathogens. Proteomics can also provide practical applications through the identification of immunogenic proteins that may be potential vaccine targets as well as in extending our understanding of antibiotic action. There is little doubt that proteomics has provided us with new and valuable information on bacterial pathogens and will continue to be an important source of information in the coming years.

Animals↗
PubMed2003

Model systems in drug discovery: chemical genetics meets genomics.

Animal model systems are an intricate part of the discovery and development of new medicines. The sequencing of not only the human genome but also those of the various pathogenic bacteria, the nematode Caenorhabditis elegans, the fruitfly Drosophila, and the mouse has enabled the discovery of new drug targets to push forward at an unprecedented pace. The knowledge and tools in these "model" systems are allowing researchers to carry out experiments more efficiently and are uncovering previously hidden biological connections. While the history of bacteria, yeast, and mice in drug discovery are long, their roles are ever evolving. In contrast, the history of Drosophila and C. elegans at pharmaceutical companies is short. We will briefly review the historic role of each model organism in drug discovery and then update the readers as to the abilities and liabilities of each model within the context of drug development.

Animals↗
PubMed1997

Mapping the regions carrying the three contiguous antibiotic resistance genes aadE, sat4, and aphA-3 in the genomes of staphylococci.

Tn5405 (12 kb) is a staphylococcal composite transposon delimited by two inverted copies of IS1182, one of which contains IS1181. The internal part of this transposon carries three antibiotic resistance genes, aphA-3, aadE, and sat4, and three open reading frames (ORFs), orfx, orfy, and orfz, of unknown function. The dispersion of Tn5405 and the genes and ORFs included in this transposon were investigated in 50 epidemiologically unrelated staphylococci carrying aphA-3. Twenty-three maps, distinguishable by the presence or absence of the investigated genes or ORFs and/or by the sizes of the restriction fragments carrying them, were identified. Four isolates carried Tn5405, and 15 other isolates contained a Tn5405-related element. IS1182 was not detected in the aphA-3 regions mapped in 31 isolates which carried the following combinations: orfx, orfy, aadE, sat4, and aphA-3 +/- orfz; orfy, aadE, sat4, and aphA-3 +/- orfz; and aadE, sat4, aphA-3, and orfz. In all isolates, the genes and ORFs investigated were in relative positions similar to those in Tn5405. Thus, the internal part of Tn5405 appeared to be partially conserved with the maintenance, in all of the isolates, of at least the three antibiotic resistance genes.

DNA, Bacterial↗
PubMed1998

Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

Chromosome Mapping↗
PubMed2000

HOBACGEN: database system for comparative genomics in bacteria.

We present here HOBACGEN, a database system devoted to comparative genomics in bacteria. HOBACGEN contains all available protein genes from bacteria, archaea, and yeast, taken from SWISS-PROT/TrEMBL and classified into families. It also includes multiple alignments and phylogenetic trees built from these families. The database is organized under a client/server architecture with a client written in Java, which may run on any platform. This client integrates a graphical interface allowing users to select families according to various criteria and notably to select homologs common to a given set of taxa. This interface also allows users to visualize multiple alignments and trees associated to families. In tree displays, protein gene names are colored according to the taxonomy of the corresponding organisms. Users may access all information associated to sequences and multiple alignments by clicking on genes. This graphic tool thus gives a rapid and simple access to all data required to interpret homology relationships between genes and distinguish orthologs from paralogs. Instructions for installation of the client or the server are available at http://pbil.univ-lyon1. fr/databases/hobacgen.html.

Amino Acid Sequence↗
PubMed2001

Exploitation of the low fidelity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the nucleotide composition bias in the HIV-1 genome to alter the drug resistance development of HIV.

The RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G-->A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G-->A) to an adenine-to-guanine (A-->G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development.

Anti-HIV Agents↗
PubMed2000

Antisense PNA effects in Escherichia coli are limited by the outer-membrane LPS layer.

Antisense peptide nucleic acids (PNAs) can inhibit Escherichia coli gene expression and cell growth through sequence-specific RNA binding, and this opens possibilities for novel anti-infective agents and tools for microbial functional genomics. However, the cellular effects of PNAs are limited relative to effects in cell extracts, presumably because of cell barrier components such as the outer-membrane lipopolysaccharide (LPS) layer or drug efflux pumps, both of which function to exclude antibiotics and other foreign molecules. To evaluate the importance of such cellular factors on PNA effects, the authors developed a positive assay for antisense inhibition by targeting the lac operon repressor and compared PNA susceptibilities in mutant and wild-type E. coli by assessing lacZ induction. Strains with defective LPS (AS19 and D22) were more permeable to the antibiotic nitrocefin and more susceptible to PNA than the wild-type. Also, PNA potency was improved in wild-type cells grown in the presence of certain cell-wall-permeabilizing agents. In contrast, the activities of the Acr and Emr drug efflux pumps were not found to affect PNA susceptibility. The results show that the LPS layer is a major barrier against cell entry, but PNAs that can enter E. coli are likely to remain active inside cells.

Bacterial Proteins↗
PubMed2003

A general strategy to uncover transcription factor properties identifies a new regulator of drug resistance in yeast.

We demonstrate a genomewide approach to determine the physiological role of a putative transcription factor, Ylr266, identified through yeast genome sequencing program. We constructed activated forms of the zinc finger (Zn(2)Cys(6)) protein Ylr266, and we analyzed the corresponding transcriptomes with DNA microarrays to characterize the up-regulated genes. The direct target genes of Ylr266 were further identified by in vivo chromatin immunoprecipitation procedure. The functions of the genes directly controlled by YLR266c are in agreement with the observed drug-resistance phenotype of the cell expressing an activated form of Ylr266. These target genes code for ATP-binding cassette or major facilitator superfamily transporters such as PDR15, YOR1, or AZR1 or for other proteins such as SNG1, YJL216c, or YLL056c which are already known to be involved in the yeast pleiotropic drug resistance (PDR) phenomenon. YLR266c could thus be named PDR8. Overlaps with the other PDR networks argue in favor of a new specific role for PDR8 in connection with the well known PDR regulators PDR1/PDR3 and YRR1. This strategy to identify the regulatory properties of an anonymous transcription factor is likely to be generalized to all the Zn(2)Cys(6) transcription factors from Saccharomyces cerevisiae and related yeasts.

Drug Resistance, Microbial↗
PubMed1994

[New information on the structure of mycobacteria].

Recent progress about the mycobacterial structures have been realized and two major structures have been concerned: the genome and the cell wall. From these acquired new knowledge several lines of clinical research and diagnosis application emerged. Cloning and sequencing of several mycobacterial genes led to the development of diagnostic tools (DNA probes, PCR, finger printings of indated mycobacterial strains) and the potential detection of multiding resistant strains of M. tuberculosis. Genetic manipulations involving various mycobacterial genes do open the way for more precise molecular approaches concerning virulence factors involved in the pathophysiological understanding of mycobacterial diseases. Comparative physico-chemical and ultra-structural analysis of the mycobacterial cell wall evoked a highly complexed cell wall structure, constituted of a double lipidic layer linked to the peptidoglycan (PG). The first layer is constituted of mycolic acids that are linked to the PG by arabinogalactan, and to the superficial layer by hydrophobic interactions of glycolipids. The superficial layer is constituted of amphiphatic glycolipids, having a lipidic banal pole and a polysaccharidic apical pole. The knowledge of the mycobacterial cell wall structure opened the way of: the development of immunological diagnostic tools, being now days in clinical evaluation phase, a better approach for host-bacteria relationship study at the cellular level (macrophage, lymphocytes), and the understanding of the mode of action of antimycobacterial drugs such as isoniazid and ethambutol.

Animals↗
PubMed2001

The human genome, implications for oral health and diseases, and dental education.

We are living in an extraordinary time in human history punctuated by the convergence of major scientific and technological progress in the physical, chemical, and biological ways of knowing. Equally extraordinary are the sparkling intellectual developments at the interface between fields of study. One major example of an emerging influence on the future of oral health education is at the interface between the human genome, information technology, and biotechnology with miniaturizations (nanotechnology), suggesting new oral health professional competencies for a new century. A great deal has recently been learned from human and non-human genomics. Genome databases are being "mined" to prompt hypothesis-driven "postgenomic" or functional genomic science in microbial models such as Candida albicans related to oral candidiasis and in human genomics related to biological processes found in craniofacial, oral, and dental diseases and disorders. This growing body of knowledge is already providing the gene content of many oral microbial and human genomes and the knowledge of genetic variants or polymorphisms related to disease, disease progression, and disease response to therapeutics (pharmacogenomics). The knowledge base from human and non-human genomics, functional genomics, biotechnology, and associated information technologies is serving to revolutionize oral health promotion, risk assessment using biomarkers and disease prevention, diagnostics, treatments, and the full range of therapeutics for craniofacial, oral, and dental diseases and disorders. Education, training, and research opportunities are already transforming the curriculum and pedagogy for undergraduate science majors, predoctoral health professional programs, residency and specialty programs, and graduate programs within the health professions. In the words of Bob Dylan, "the times they are a-changing."

Biotechnology↗
PubMed1999

Transposon Tn21, flagship of the floating genome.

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.

Base Sequence↗
PubMed1999

Comparison of line probe assay (LIPA) and sequence analysis for detection of HIV-1 drug resistance.

The identification of HIV strains that are resistant to antiretroviral drugs, which emerge during a patient's therapy or are already present in infected individuals prior to treatment, is of increasing importance for the clinical management of HIV-infected persons. Two different methods were compared for the genotypic detection of resistance development in the HIV-1 reverse transcriptase (RT) gene, the commonly used sequence analysis, and the commercially available RT-line immunoprobe assay (LIPA), which can detect mutations at six separate codons of the RT gene, which are known to confer resistance to certain nucleoside inhibitors. Eighty serum samples from HIV-1-infected persons, some of whom were receiving antiretroviral therapy, were investigated in parallel by sequencing as well as by LIPA. LIPA results agreed with sequence data in the vast majority of the cases. However, in 40% of the samples, LIPA failed to yield evaluable results for one or more of the codon positions. In particular, LIPA detection rate was low at codon 41 (75%), whereas at codons 69/70, 74, 184, and 215 results were obtained from 90%-95% of the samples. A number of mutations in the vicinity of the respective codons were detected by sequencing, and these may have been responsible for the LIPA hybridization failure. There remained a number of samples, however, where no explanation for the lack of hybridization could be derived from sequence data. Our results indicate that the use of the LIPA does not eliminate the need for sequence analysis for detection of drug-resistant HIV strains.

Adult↗
PubMed2005

Utilizing the power of microbial genetics to bridge the gap between the promise and the application of marine natural products.

Marine organisms are a rich source of secondary metabolites. They have yielded thousands of compounds with a broad range of biomedical applications. Thus far, samples required for preclinical and clinical studies have been obtained by collection from the wild, by mariculture, and by total chemical synthesis. However, for a number of complex marine metabolites, none of these options is feasible for either economic or environmental reasons. In order to proceed with the development of many of these promising therapeutic compounds, a reliable and renewable source must be found. Over the last twenty years, the study of microbial secondary metabolites has greatly advanced our understanding of how nature utilizes simple starting materials to yield complex small molecules. Much of this work has focused on polyketides and nonribosomal peptides, two classes of molecules that are prevalent in marine micro- and macroorganisms. The lessons learned from the study of terrestrial metabolite biosynthesis are now being applied to the marine world. As techniques for cloning and heterologous expression of biosynthetic pathways continue to improve, they may provide our greatest hope for bridging the gap between the promise and application of many marine natural products.

Animals↗