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G Wegrzyn

Publications and source records attributed to G Wegrzyn.

At least 19 recordsLinked to original sources
PubMed2006

A modified Vibrio harveyi mutagenicity assay based on bioluminescence induction.

AIMS: Mutagenic pollution of natural environment is currently one of the most serious ecological problems. Therefore, rapid detection of the presence of mutagens is a very important issue. Although many mutagenicity assays have already been described, only a few are suitable for testing samples from natural environment. One of such assays is a microbiological mutagenicity test based on genetically modified Vibrio harveyi strains. The aim of this work was to modify and improve the V. harveyi assay. METHODS AND RESULTS: A series of V. harveyi dark and dim mutants were tested for reversion of their phenotype towards efficient light emission in response to incubation with known mutagens. Luminescence of the A16 strain (luxE mutant) increased significantly after a few hours of such a treatment with various mutagenic agents, revealing a dose-response correlation. Sensitivity of the assay has been determined for different mutagens. CONCLUSIONS: The luminescence-based V. harveyi mutagenicity assay is rapid, sensitive and reveals a dose-response correlation for various mutagens. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in this study is a potentially useful tool in studies on mutagenic pollution of environment, especially marine water.

Aminacrine↗
PubMed2005

Rapid detection of viruses using electrical biochips and anti-virion sera.

AIMS: Rapid detection and quantification of viruses is crucial in clinical practice, veterinary medicine, agriculture, basic research as well as in biotechnological factories. However, although various techniques were described and are currently used, development of more rapid, more sensitive and quantitative methods seems to be still important. METHODS AND RESULTS: Here we describe a method for rapid detection of viruses (using bacteriophages as model viruses), based on electrical biochip array technology with the use of antibodies against capsid proteins. CONCLUSIONS: Using the procedure developed in this work, we were able to detect 2 x 10(4) virions on the chip. The whole assay procedure takes c. 50 min and the assay is quantitative. SIGNIFICANCE AND IMPACT OF THE STUDY: This procedure may be useful in various approaches, including detection of bacteriophage contamination in bioreactors and possibly detection of toxin gene-bearing phages or other viruses in food samples.

Antibodies, Viral↗
PubMed2004

Electric chips for rapid detection and quantification of nucleic acids.

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

Biosensing Techniques↗
PubMed2004

A general model for genetic regulation of turnover of glycosaminoglycans suggests a possible procedure for prediction of severity and clinical progress of mucopolysaccharidoses.

Mucopolysaccharidoses are rare genetic diseases from the group of lysosomal storage disorders caused by deficiency of enzymes involved in degradation of mucopolysaccharides (glycosaminoglycans, GAGs). Within each mucopolysaccharidosis, there is a continuous spectrum of clinical features from the very severe to the more mildly affected individuals. Surprisingly, in most cases, it is not possible to predict severity and clinical progress (i.e., the natural history) of the disease on the basis of detection of particular mutations or residual activity of the deficient enzyme. In this article, the reasons for such an unexpected difficulty are discussed. A model for the correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate(s) has been proposed previously by others, however, in that model it was assumed that substrate concentration in the lysosome is not regulated, thus the residual activity of a hydrolase would be the only determinant of the rate of substrate accumulation. On the other hand, both a general model for genetic regulation of turnover of GAGs and results of very recent studies strongly suggest that expression of genes coding for enzymes involved in GAG synthesis is precisely regulated and may vary between individuals. Therefore, we propose that apart from measurement of residual activity of the enzyme involved in degradation of GAGs, the efficiency of synthesis of these compounds should also be estimated. If the hypothesis presented in this article is true, the ratio of the synthesis of glycosaminoglycans to the residual activity of the deficient enzyme should be of considerable prognostic value.

Gene Expression Regulation↗
PubMed2003

Detection of mutagenic pollution of natural environment using microbiological assays.

One of the most important and serious ecological problems is mutagenic pollution of the natural environment. Therefore, detection of mutagenic compounds in samples taken from natural habitats is of special interest. Microbiological mutagenicity tests seem to be very useful tools for such detection. In this review article, a general view on the tests employing genetically modified bacterial strains designed for detection of low concentrations of mutagenic compounds is presented. Moreover, a comparison of advantages and disadvantages of selected assays, developed early on and more recently, and features of these assays are discussed. It appears that none of the currently available mutagenicity tests is perfect or optimal for all purposes. Thus, a choice for the particular assay must depend on the nature of studies and specific tasks of the experiments to be performed.

Bacteria↗
PubMed2001

Inhibition of spontaneous induction of lambdoid prophages in Escherichia coli cultures: simple procedures with possible biotechnological applications.

BACKGROUND: Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage. RESULTS: Here we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction. Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E. coli cells. A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development. Overproduction of the cI repressor abolished spontaneous induction of the lambda(imm434) prophage. CONCLUSIONS: Simple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E. coli strains by these phages are described. Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains.

Bacteriophage lambda↗
PubMed2001

Inheritance of the replication complex: a unique or common phenomenon in the control of DNA replication?

Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage lambda demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for lambda plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.

Bacteriophage lambda↗
PubMed2001

Rapid degradation of bacteriophage lambda O protein by ClpP/ClpX protease influences the lysis-versus-lysogenization decision of the phage under certain growth conditions of the host cells.

The initiator of bacteriophage lambda DNA replication, the O protein, is rapidly degraded in Escherichia coli by the ClpP/ClpX protease encoded by the host. Although the biochemical mechanism of this degradation has been investigated intensively, a physiological role for this process remained unknown since little effect of dysfunction of clpP and clpX genes on the lytic development of the phage was observed. Here we demonstrate that activities of clpP and clpX genes influence the lysis-versus-lysogenization decision of bacteriophage lambda under certain growth conditions of the host cells. This decision is influenced specifically by ClpP/ClpX-mediated O degradation and resultant inhibition of early lambda DNA replication because mutations in clpP and clpX genes have little effect on stability of other lambda proteins involved in the regulation of the phage developmental switch.

Adenosine Triphosphatases↗
PubMed2001

Construction and use of a broad-host-range plasmid expressing the lamB gene for utilization of bacteriophage lambda vectors in the marine bacterium Vibrio harveyi.

The remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation. Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage lambda. However, this bacteriophage is specific for E. coli, and thus lambda-based techniques have been restricted to this bacterium. Plasmids expressing the E. coli gene coding for bacteriophage lambda receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage lambda infection. However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria. Here we describe construction of a broad-host-range plasmid expressing the lamB gene. Introduction of this plasmid to V. harveyi cells and expression of lamB made this strain susceptible to bacteriophage lambda adsorption and lambda DNA injection. Foreign genetic material could be introduced into cells of this strain using a cosmid vector.

Journal Article↗
PubMed2001

Bacterial replication initiator DnaA. Rules for DnaA binding and roles of DnaA in origin unwinding and helicase loading.

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

Bacterial Proteins↗
PubMed2001

SeqA, the Escherichia coli origin sequestration protein, is also a specific transcription factor.

The SeqA protein is a negative regulator of initiation of DNA replication in the Escherichia coli chromosome. Here, we demonstrate that SeqA stimulates transcription from the bacteriophage lambda pR promoter both in vivo and in vitro. The activity of the lambda pL promoter was found not to be affected by this protein. SeqA-mediated stimulation of pR was dependent on the state of template methylation: transcription was activated on fully methylated and hemimethylated templates but not on an unmethylated template. Using electrophoretic mobility shift assay and electron microscopy, we demonstrated that SeqA interacts specifically with a pR promoter region located on both fully methylated and hemimethylated DNA molecules, but not on unmethylated DNA. The activity of SeqA was found to affect the initiation of lambda plasmid replication positively in vivo, probably via pR-dependent expression of lambda replication genes and transcriptional activation of ori lambda. We conclude that, apart from its function in the control of DNA replication, SeqA is also a specific transcription factor.

Bacterial Outer Membrane Proteins↗
PubMed2001

A Vibrio harveyi insertional mutant in the cgtA (obg, yhbZ) gene, whose homologues are present in diverse organisms ranging from bacteria to humans and are essential genes in many bacterial species.

The cgtA gene product is a member of the subfamily of small GTP-binding proteins that have been identified in diverse organisms ranging from bacteria to humans. In bacteria that sporulate or display another special developmental programme, this gene (referred to as cgtA, obg or yhbZ) appears to be involved in the regulation of these processes. However, this gene has also been found to be essential in all bacterial species investigated to date, although its role in bacteria that do not sporulate and do not undergo a specific development remains unknown. Here the authors characterize a Vibrio harveyi mutant bearing a transposon insertion into the cgtA gene. This mutant reveals a multiple phenotype: it grows more slowly than the wild-type strain in a rich medium; its growth is completely inhibited in minimal media; its survival in 3% NaCl is dramatically reduced; it is very sensitive to UV irradiation; it is more susceptible to mutation upon treatment with different mutagens; its luminescence is decreased; its quorum-sensing regulation is less effective than in the wild-type strain; and the elongated shape of the mutant cells may suggest problems with the regulation of cell division and/or DNA replication. These defects in diverse cellular processes found in the insertional cgtA mutant of V. harveyi indicate that in a bacterium that does not sporulate and does not display other special development programmes, the CgtA protein is involved in the regulation of many crucial biochemical reactions, possibly at the stage of signal transduction.

Amino Acid Sequence↗
PubMed2001

Chloramphenicol-sensitive Escherichia coli strain expressing the chloramphenicol acetyltransferase (cat) gene.

An Escherichia coli strain (strain CM2555) bearing the chloramphenicol acetyltransferase (cat) gene was found to be sensitive to chloramphenicol. We demonstrate that the cat gene is efficiently expressed in strain CM2555. Our results suggest that decreased levels of acetyl coenzyme A in cat-expressing CM2555 cells in the presence of chloramphenicol may cause the bacterium to be sensitive to this antibiotic.

Acetyl Coenzyme A↗
PubMed2001

A plasmid cloning vector with precisely regulatable copy number in Escherichia coli.

We have developed a genetic system allowing for precise regulation of plasmid copy number in Escherichia coli cells. A cloning vector based on this system is described in this article. The pTC lambda 3 plasmid is a lambda replicon, but transcription controlling initiation of plasmid DNA replication starts from the PtetA promoter instead of phage lambda PR promoter. Additionally, activity of PtetA promoter is negatively controlled by the TetR repressor whose gene is located on the same plasmid vector and is induced by an analog of tetracycline, autoclaved chlortetracycline (aCT). Using different concentrations of the inducer it is possible to strictly regulate the copy number of pTC lambda 3 and thus the copy number of a cloned gene. The usefulness of the system for the regulatable production of a protein encoded by a gene inserted into pTC lambda 3 plasmid is demonstrated by dependence of beta-galactosidase activity on the lacZ gene dosage.

Chlortetracycline↗
PubMed2001

An introduction to DNA chips: principles, technology, applications and analysis.

This review describes the recently developed GeneChip technology that provides efficient access to genetic information using miniaturised, high-density arrays of DNA or oligonucleotide probes. Such microarrays are powerful tools to study the molecular basis of interactions on a scale that would be impossible using conventional analysis. The recent development of the microarray technology has greatly accelerated the investigation of gene regulation. Arrays are mostly used to identify which genes are turned on or off in a cell or tissue, and also to evaluate the extent of a gene's expression under various conditions. Indeed, this technology has been successfully applied to investigate simultaneous expression of many thousands of genes and to the detection of mutations or polymorphisms, as well as for their mapping and sequencing.

Gene Expression Profiling↗
PubMed2001

The minimal genome paradox.

The concept of a 'minimal genome' has appeared as an attempt to answer the question what the minimum number of genes or minimum amount of DNA to support life is. Since bacteria are cells bearing the smallest genomes, it has been generally accepted that the minimal genome must belong to a bacterial species. Currently the most popular chromosome in studies on a minimal genome belongs to Mycoplasma genitalium, a parasite bacterium whose total genetic material is as small as approximately 580 kb. However, the problem is how we define life, and thus also a minimal genome. M. genitalium is a parasite and requires substances provided by its host. Therefore, if a genome of a parasite can be considered as a minimal genome, why not to consider genomes of bacteriophages? Going further, bacterial plasmids could be considered as minimal genomes. The smallest known DNA region playing the function of the origin of replication, which is sufficient for plasmid survival in natural habitats, is as short as 32 base pairs. However, such a small DNA molecule could not form a circular form and be replicated by cellular enzymes. These facts may lead to an ostensibly paradoxical conclusion that the size of a minimal genome is restricted by the physical size of a DNA molecule able to replicate rather, than by the amount of genetic information.

Journal Article↗