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At least 145 records · Page 8Linked to original sources
PubMed2004

Harnessing the catabolic diversity of rhodococci for environmental and biotechnological applications.

The field of Rhodococcus cell engineering is rapidly advancing because of the availability of improved genetic tools and increased insights in their broad catabolic and biochemical diversity. Rhodococci harbor large linear plasmids that may contribute to their catabolic diversity. In addition, multiple pathways and gene homologs are often present, thus further increasing Rhodococcus catabolic versatility and efficiency. The recent development of effective genetic tools for Rhodococcus, such as unmarked gene deletion, transposon-based mutagenesis, and gene expression systems, now allows the construction of biocatalysts with desirable properties for industrial purposes. This is exemplified here by a description of cell engineering of biocatalysts for improved desulphurization and steroid biotransformation.

Androstenedione↗
PubMed2006

Expression of Drosophila BarH1-H2 homeoproteins in developing dopaminergic cells and segmental nerve a (SNa) motoneurons.

Barh1/h2 genes encode two related homeobox transcription factors (B-H1 and B-H2) previously shown to play essential roles in the formation and specification of the distal leg segments and in retinal neurogenesis. Here we describe the restricted expression pattern of the B-H1/-H2 homeoprotein within the embryonic ventral nerve cord of Drosophila. We show that B-H1/-H2 are specifically expressed in a subset of dopaminergic neurons, namely the unpaired ventral midline dopaminergic neuron, and in a subpopulation of laterally projecting motoneurons, i.e. the five motoneurons forming the segmental nerve a (SNa) branch. Using the GAL4-UAS system we show that B-H1/-H2(Gal4) in combination with a membrane-targeted enhanced green fluorescent protein reporter line provides a powerful genetic tool reproducibly to label SNa motoneuron projections and terminals at the periphery, and their dendritic tree in the ventral nerve cord. Thus, the highly restricted expression pattern of the B-H1/-H2 homeoproteins and notably the related Gal4 driver represent powerful genetic tools to identify and study genes that control axon guidance, synaptogenesis or dendritic arborization within a small subpopulation of motoneurons identifiable from embryogenesis to late larval stages.

Animals↗
PubMed2004

[Alu elements in the human genome].

BACKGROUND: The sequencing of the human genome has revealed that almost half of the genome consists of interspersed repeats. Among the different types of interspersed repeats in humans, Alu elements are the most frequent ones, and new Alu elements are still integrated into new positions in the humane genome. In this review some consequences of this Alu activity are discussed. The first part of the review is focused on Alu element activity that causes genetic disease while the use of Alu elements as genetic tools for several purposes is discussed in the last part. MATERIALS AND METHODS: This review article is based on a literature search on Medline. RESULTS AND INTERPRETATION: Alu elements cause disease by two different mechanisms. A de novo insertion of an Alu element into, or close to, a gene may disrupt its normal function, while homologous recombination between two Alu elements may result in deletions or duplications that cause genetic disease. The high density of Alu elements in the human genome combined with their absence in non-primate species make them useful as genetic tools. Alu elements also represent "molecular fossils" that give phylogenetic information about primates as well as information about historical DNA rearrangements in the human genome.

Alu Elements↗
PubMed2004

ANDI-03: a genetic algorithm tool for the analysis of activation detector data to unfold high-energy neutron spectra.

The thresholds of (n,xn) reactions in various activation detectors are commonly used to unfold the neutron spectra covering a broad energy span, i.e. from thermal to several hundreds of MeV. The saturation activities of the daughter nuclides (i.e. reaction products) serve as the input data of specific spectra unfolding codes, such as SAND-II and LOUHI-83. However, most spectra unfolding codes, including the above, require an a priori (guess) spectrum to starting up the unfolding procedure of an unknown spectrum. The accuracy and exactness of the resulting spectrum primarily depends on the subjectively chosen guess spectrum. On the other hand, the Genetic Algorithm (GA)-based spectra unfolding technique ANDI-03 (Activation-detector Neutron DIfferentiation) presented in this report does not require a specific starting parameter. The GA is a robust problem-solving tool, which emulates the Darwinian Theory of Evolution prevailing in the realm of biological world and is ideally suited to optimise complex objective functions globally in a large multidimensional solution space. The activation data of the 27Al(n,alpha)24Na, 116In(n,gamma)116mIn, 12C(n,2n)11C and 209Bi(n,xn)(210-x)Bi reactions recorded at the high-energy neutron field of the ISIS Spallation source (Rutherford Appleton Laboratory, UK) was obtained from literature and by applying the ANDI-03 GA tool, these data were used to unfold the neutron spectra. The total neutron fluence derived from the neutron spectrum unfolded using GA technique (ANDI-03) agreed within +/-6.9% (at shield top level) and +/-27.2% (behind a 60 cm thick concrete shield) with the same unfolded with the SAND-II code.

Algorithms↗
PubMed1999

The Dictyostelium genome project: an invitation to species hopping.

Dictyostelium allows some of the general problems of eukaryotic biology to be addressed by using molecular genetic tools that are more normally associated with yeast. The genome project, now underway, marks an important increase in the attractiveness of Dictyostelium as an experimental organism and will invite increased 'species hopping' by experimenters. We provide a brief guide to the problems that are being addressed in Dictyostelium, to the genome project itself and to the molecular genetic tools available for its exploitation.

Animals↗
PubMed2026

Development of an arabinose-inducible gene expression system for nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) are emerging pathogens for which genetic tools remain limited. Here, we developed an arabinose-inducible gene expression system based on a modified pBAD24 vector adapted for mycobacterial hosts. The vector carries replication origins for mycobacteria and Escherichia coli, as well as selectable markers compatible with NTM. In Mycobacterium abscessus (Mycobacteroides abscessus), the system enabled dose-dependent induction of target gene expression by arabinose, as demonstrated by increased antibiotic resistance and quantitative RT-PCR analysis. Although basal expression was observed in the absence of arabinose, expression levels were tunable across arabinose concentrations. The system was also functional in Mycobacterium smegmatis (Mycolicibacterium smegmatis) and Mycobacterium bovis BCG, although the degree of basal expression varied among host species. These results establish a tunable inducible expression system for mycobacteria and provide a useful genetic tool for studies of NTM biology.

Arabinose↗
PubMed2002

Molecular pathogenesis of mycoplasma animal respiratory pathogens.

Mycoplasmas contain the smallest genomes and are the smallest known free-living organisms, yet little is known about the molecular details of their pathogenic mechanisms. This review focuses on pathogens of production animals, but related species colonize and cause disease in humans, fish and other animals, plants and insects. The general lack of genetic tools and the inability to apply the few that are available to some mycoplasma-host systems has hindered studies of this nature. During the last decade, which was characterized by unparalleled advances in the understanding of bacterial virulence, studies of mycoplasma pathogenesis has languished behind other experimental systems. The one exception has been studies on mycoplasma antigenic variation. The explosion of studies in this area has been due primarily to the fact that they can be performed in vitro without genetic tools and with simple well developed biochemical approaches. Not withstanding that antigenic variation may play an important role in disease, there have been few studies establishing the importance of this phenomenon in vivo for a variety of reasons. The same is true for cell invasion as it has been defined in cell culture systems, which if it occurs in vivo may change the way we think about mycoplasma disease. These advances give insight to an extraordinary group of organisms that interact with their hosts in unique and intriguing ways.

Animals↗
PubMed2004

Arsenic hyperaccumulation in gametophytes of Pteris vittata. A new model system for analysis of arsenic hyperaccumulation.

The sporophyte of the fern Pteris vittata is known to hyperaccumulate arsenic (As) in its fronds to >1% of its dry weight. Hyperaccumulation of As by plants has been identified as a valuable trait for the development of a practical phytoremediation processes for removal of this potentially toxic trace element from the environment. However, because the sporophyte of P. vittata is a slow growing perennial plant, with a large genome and no developed genetics tools, it is not ideal for investigations into the basic mechanisms underlying As hyperaccumulation in plants. However, like other homosporous ferns, P. vittata produces and releases abundant haploid spores from the parent sporophyte plant which upon germination develop as free-living, autotrophic haploid gametophyte consisting of a small (<1 mm) single-layered sheet of cells. Its small size, rapid growth rate, ease of culture, and haploid genome make the gametophyte a potentially ideal system for the application of both forward and reverse genetics for the study of As hyperaccumulation. Here we report that gametophytes of P. vittata hyperaccumulate As in a similar manner to that previously observed in the sporophyte. Gametophytes are able to grow normally in medium containing 20 mm arsenate and accumulate >2.5% of their dry weight as As. This contrasts with gametophytes of the related nonaccumulating fern Ceratopteris richardii, which die at even low (0.1 mm) As concentrations. Interestingly, gametophytes of the related As accumulator Pityrogramma calomelanos appear to tolerate and accumulate As to intermediate levels compared to P. vittata and C. richardii. Analysis of gametophyte populations from 40 different P. vittata sporophyte plants collected at different sites in Florida also revealed the existence of natural variability in As tolerance but not accumulation. Such observations should open the door to the application of new and powerful genetic tools for the dissection of the molecular mechanisms involved in As hyperaccumulation in P. vittata using gametophytes as an easily manipulated model system.

Arsenates↗
PubMedDate not supplied

[Development of genetic and molecular approaches for the diagnosis and study of the pathogenicity of Helicobacter pylori, agent of gastric inflammatory diseases].

Helicobacter pylori (H. pylori) is a small gram-negative bacillus, recently discovered, found in the stomach of patients with active chronic gastritis and duodenal ulcers. Production of a potent urease has been described as a trait common to all H. pylori so far isolated. To clarify the role of urease in the pathogenic process, as well as to engineer genetic tools useful for the diagnosis of H. pylori, we cloned the genes responsible for urease activity. A genomic library was constructed in Escherichia coli (E. coli) from the chromosomal DNA of the H. pylori strain 85P using a shuttle cosmind vector that we constructed in vitro capable of replicating both in E. coli and Campylobacter jejuni (C. jejuni). The genes responsible for the urease biosynthesis were cloned into E. coli host, then mobilized into C. jejuni where they were expressed. At least six different genes were shown to be required for the expression of the synthesis of an active enzyme; these genes belong to the same cluster and are regulated at the transcriptional level. The two genes encoding the two subunits of the urease enzyme were identified and sequenced; the products of these genes were compared to the other bacterial ureases. The genetic approach allowed to determine the amino-acid sequence of the most immunogenic antigens of H. pylori. In addition, it provides us with genetic tools: a 294-base pairs (bp) DNA fragment internal to one of the urease genes, was shown to be specific of H. pylori strains. This fragment was selectively amplified by polymerase chain reaction (P.C.R.) using two primers designed to target the urease region of all H. pylori isolates present in biological specimen. In addition, P.C.R. followed by direct DNA sequencing of the 294-bp amplified product was shown to be useful to identify and to distinguish between different H. pylori isolates.

Bacterial Typing Techniques↗
PubMedDate not supplied

[Development of genetic and molecular approaches for the diagnosis and the study of the pathogenic power of Helicobacter pylori, agent of inflammatory gastric diseases].

Helicobacter pylori (H. pylori) is a small Gram negative bacillus, recently discovered, found in the stomach of patients with active chronic gastritis and duodenal ulcers. Production of a potent urease has been described as a trait common to all H. pylori so far isolated. To clarify the role of urease in the pathogenic process, as well as to engineer genetic tools useful for the diagnosis of H. pylori, we cloned the genes responsible for urease activity. A genomic library was constructed in Escherichia coli (E. coli) from the chromosomal DNA of the H. pylori stain 85P using a shuttle cosmid vector that we constructed in vitro capable of replicating both in E. coli and Campylobacter jejuni (C. jejuni). The genes responsible for the urease biosynthesis were cloned into E. coli host, then mobilized into C. jejuni where they were expressed. At least six different genes were shown to be required for the expression of the synthesis of an active enzyme: these genes belong to the same cluster and are regulated at the transcriptional level. The two genes encoding the two subunits of the urease enzyme were identified and sequenced; the products of these genes were compared to be other bacterial ureases. The genetic approach allowed to determine the amino-acid sequence of the most immunogenic antigens of H. pylori. In addition, it provides us with genetic tools: a 294-base pairs (bp) DNA fragment internal to one of the urease genes, was shown to be specific of H. pylori strains.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗
PubMed2003

High-throughput selection of effective RNAi probes for gene silencing.

RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNAi has recently emerged as a powerful genetic tool to analyze gene function in mammalian cells. The power of this method is limited however, by the uncertainty in predicting the efficacy of small interfering RNAs (siRNAs) in silencing a gene. This has imposed serious limitations not only for small-scale but also for high-throughput RNAi screening initiatives in mammalian systems. We have developed a reliable and quantitative approach for the rapid and efficient identification of the most effective siRNA against any gene. The efficacy of siRNA sequences is monitored by their ability to reduce the expression of cognate target-reporter fusions with easily quantified readouts. Finally, using micro array-based cell transfections, we demonstrate an unlimited potential of this approach in high-throughput screens for identifying effective siRNA probes for silencing genes in mammalian systems. This approach is likely to have implications in the use of RNAi as a reverse genetic tool for analyzing mammalian gene function on a genome-wide scale.

Animals↗
PubMed2002

Real-time quantitative polymerase chain reaction: a potential tool for genetic analysis in neuropathology.

Since its introduction in the early- to mid-1980s, the polymerase chain reaction (PCR) has been modified and optimized for an increasing number of applications. Early on, the focus was on the amplification of a specific nucleic acid template into quantities amenable to identification and experimental manipulation. While this remains an important application, recent technology has allowed the use of PCR to accurately quantitate the amount of a specific nucleic acid template present in a complex sample. Rather than simply analyzing the final product amount following the course of sequential cycles of amplification, quantitative PCR allows one to measure the accumulation of PCR product during the course of the reaction ("real-time PCR"). Under the appropriate conditions the number of PCR cycles required for the accumulation of a specific amount of product (during the exponential phase of the reaction) is a reflection of the relative amount of nucleic acid template present in the sample under analysis. Real-time quantitative PCR allows one to analyze a relatively large number of samples in a short period of time, potentially allowing multiple markers to be applied on a sample within a time frame consistent with clinical settings. In this overview, we will highlight the uses of real-time quantitative PCR as a potential diagnostic tool in neuropathology, focusing on the analysis of CNS tumors.

Brain Neoplasms↗
PubMed2002

Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I.

The obligate aerobic yeast Yarrowia lipolytica is introduced as a powerful new model for the structural and functional analysis of mitochondrial complex I. A brief introduction into the biology and the genetics of this nonconventional yeast is given and the relevant genetic tools that have been developed in recent years are summarized. The respiratory chain of Y. lipolytica contains complexes I-IV, one "alternative" NADH-dehydrogenase (NDH2) and a non-heme alternative oxidase (AOX). Because the NADH binding site of NDH2 faces the mitochondrial intermembrane space rather than the matrix, complex I is an essential enzyme in Y. lipolytica. Nevertheless, complex I deletion strains could be generated by attaching the targeting sequence of a matrix protein, thereby redirecting NDH2 to the matrix side. Deletion strains for several complex I subunits have been constructed that can be complemented by shuttle plasmids carrying the deleted gene. Attachment of a hexa-histidine tag to the NUGM (30 kDa) subunit allows fast and efficient purification of complex I from Y. lipolytica by affinity-chromatography. The purified complex has lost most of its NADH:ubiquinone oxidoreductase activity, but is almost fully reactivated by adding 400-500 molecules of phosphatidylcholine per complex I. The established set of genetic tools has proven useful for the site-directed mutagenesis of individual subunits of Y. lipolytica complex I. Characterization of a number of mutations already allowed for the identification of several functionally important amino acids, demonstrating the usefulness of this approach.

Electron Spin Resonance Spectroscopy↗
PubMed2005

A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae.

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.

Base Sequence↗
PubMed1998

Cre expression in primary spermatocytes: a tool for genetic engineering of the germ line.

Transgenic mice were generated expressing a testicular Cre recombinase driven by promoter sequences derived from the gene encoding Synaptonemal Complex Protein 1 (Sycp1), expressed at an early stage of the male meiosis (leptotene to zygotene). Recombination at target LoxP sites was examined during germinal differentiation in mice harboring Sycp1-Cre and a second transgene where LoxP sites flank either the beta geo coding region, the Pgk1 promoter, or a tk-neo cassette inserted into the Rxr alpha locus. The LoxP-flanked transgenes were stably maintained in the somatic tissues of the double transgenic animals, as well as in the progeny of the females. Mice born after mating the double-transgenic males with normal females showed extensive deletions of the LoxP-flanked sequences. When the males were hemizygous for the Sycp1-Cre transgene, the deletions were observed even in the fraction of the offspring which had not inherited the Cre gene, thus demonstrating that expression occurred in the male parent during spermatogenesis. The high efficiency of excision at the LoxP sites makes the Sycp1-Cre transgenic males suitable for evaluating the role of defined gene functions in the germinal differentiation process.

Animals↗