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PubMedDate not supplied

Symposium on microbiology update: old friends and new enemies. Listeria monocytogenes.

Listeria monocytogenes, one of the "new enemies" in food microbiology, is a human and animal pathogen that is widespread in nature. The organism is a transient constituent of the intestinal flora excreted by 1-10% of healthy humans. It is an extremely hardy organism and can survive for many years in the cold in naturally infected sources. L. monocytogenes has been isolated from a wide variety of foods, including dairy products, meats, and fish. Although most of the foodborne listeriosis outbreaks have been linked to the consumption of dairy products, recent sporadic cases have been associated with meats, as well as other foods. It is now recognized that listeriolysin 0, a 60-kilodalton protein, is one of the major virulence factors of the organism. All strains of L. monocytogenes are pathogenic by definition although some appear to be more virulent than others. There have been recent reports of hemolytic isolates of L. monocytogenes, which are nonpathogenic for mice. Attachment to and penetration of cells also appear to be prerequisites for human infection. Cultural methodology for isolating the organism from foods has been in a state of flux since 1985. Rapid methods using both ELISA and DNA technology have been developed. Because of the widespread nature of the organism, it is nearly impossible to eliminate it from the food supply. However, by using a hazard analysis-critical control point approach, the health hazard associated with this organism can be reduced to a minimum.

Food Microbiology↗
PubMed1987

The molecular biology of the colony-stimulating factors.

Our understanding of the biochemistry and molecular biology of the colony stimulating factors (CSF) and the regulation of their production has advanced rapidly with the application of recombinant DNA technology. This report reviews several aspects of the regulation of CSF production and the structural features relating to their function. The structural gene for hGM-CSF is present as a single copy, is 2.5 kilobase long region and is located on the long arm of chromosome 5. The genes for human multi-CSF and M-CSF are also on chromosome 5. The gene for hGM-CSF is organized into four exons. The intron-exon boundaries contain a single consensus splice donor and acceptor sequence. CSFs are elaborated by endothelial cells, fibroblasts, and T lymphocytes in response to inflammatory mediators. Many of these same cell types respond to inflammatory stimuli with production of additional immune mediators such as IL-1, IL-2, or the interferons. Insights are now emerging into important structure--function relationships of the hematopoietic growth factors and molecular events in the regulation of CSF gene expression.

Chromosomes, Human, Pair 5↗
PubMed2005

Genetic screening and diagnosis.

PURPOSE OF REVIEW: To review the latest developments in screening and diagnosis of non-chromosomal genetic diseases. RECENT FINDINGS: Major recent advances include the completion of the Human Genome Project, the use of microarray and related technologies for mass screening and diagnosis of thousands of genetic abnormalities, and non-invasive prenatal diagnosis using fetal DNA in maternal plasma. SUMMARY: The rapid development in molecular biological technologies makes it possible to screen and to diagnosis thousands of genetic conditions, mutations and also predispositions to chronic diseases or traits, either prenatally or after birth. Clinical application of non-invasive prenatal diagnosis using fetal DNA in maternal plasma has become a reality. The arrival of the molecular genetic era also leads to many new ethical, social and medico-legal problems and dilemmas that obstetricians will have to face in the near future. There is an urgent need for the development of a new model for provision of genetic screening and diagnosis.

Congenital Abnormalities↗
PubMed1991

Rapid electroblotting of small DNA fragments from polyacrylamide gels.

With the recent advances in PCR technology, the need for a simplified analysis of small double-stranded DNA fragments (less than 1.5 kb) has increased dramatically. An easy and rapid procedure has been developed for the separation, transfer and probe analysis of small double-stranded DNA fragments from polyacrylamide gels.

DNA↗
PubMed2001

DNA microarray analysis of gene expression in endothelial cells in response to 24-h shear stress.

The recently developed DNA microarray technology provides a powerful and efficient tool to rapidly compare the differential expression of a large number of genes. Using the DNA microarray approach, we investigated gene expression profiles in cultured human aortic endothelial cells (HAECs) in response to 24 h of laminar shear stress at 12 dyn/cm(2). This relatively long-term shearing of cultured HAECs led to the modulation of the expression of a number of genes. Several genes related to inflammation and EC proliferation were downregulated, suggesting that 24-h shearing may keep ECs in a relatively noninflammatory and nonproliferative state compared with static cells. Some genes were significantly upregulated by the 24-h shear stress; these includes genes involved in EC survival and angiogenesis (Tie2 and Flk-1) and vascular remodeling (matrix metalloproteinase 1). These results provide information on the profile of gene expression in shear-adapted ECs, which is the case for the native ECs in the straight part of the aorta in vivo.

Adult↗
PubMed2001

Novel use of guanidinium isothiocyanate in the isolation of Mycobacterium tuberculosis DNA from clinical material.

Nucleic acid amplification technologies offer great promise for the rapid, sensitive and specific diagnosis of tuberculosis. However, the isolation of inhibitor-free DNA from biological specimens is a bottleneck of the PCR assay. Here we describe a simple method for the isolation of PCR-amplifiable DNA of Mycobacterium tuberculosis from all types of samples of pulmonary and extrapulmonary origin tested. Briefly, it involves concentration of the bacilli by high-speed centrifugation, removal of PCR inhibitors by a wash solution containing guanidinium isothiocyanate and the release of bacterial DNA by heating in the presence of detergents and Chelex-100 resin. The entire process is accomplished within approximately 3 h. The method has been validated on 780 samples of human, bovine and guinea pig origin including sputum, cerebrospinal fluid, pulmonary fluids, pus, fine needle aspirate, tissue, blood and milk.

Animals↗
PubMed1998

The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes.

BACKGROUND: . Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects. RESULTS: . We describe a series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre-lox site-specific recombination to catalyze plasmid fusion between the univector - a plasmid containing the gene of interest - and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3' end gene fusions using homologous recombination in Escherichia coli. CONCLUSIONS: . Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis.

Amino Acid Sequence↗
PubMedDate not supplied

Recombinant DNA technology and clinical dentistry.

The introduction of recombinant DNA technology in the late 1970s led to rapid advancements in our knowledge of genes and genomic structure in the 1980s. Such technology, applied to the human genome, provides new information concerning the nature and possible treatment of inherited disorders throughout the human lifespan. Methods to clone structural genes of interest to clinical dentistry now provide strategies to produce genetically engineered biomaterials for prosthetic dentistry. This methodology is also being used to clone microbial DNAs for periodontal disease diagnosis, therapy, and prognostic applications.

DNA, Recombinant↗
PubMed1997

Use of first nucleotide change technology to determine the frequency of factor V Leiden in a population of Australian blood donors.

Activated protein C resistance (APCR), the most common risk factor for venous thrombosis, is the result of a G to A base substitution at nucleotide 1691 (R506Q) in the factor V gene. Current techniques to detect the factor V Leiden mutation, such as determination of restriction length polymorphisms, do not have the capacity to screen large numbers of samples in a rapid, cost-effective test. The aim of this study was to apply the first nucleotide change (FNC) technology, to the detection of the factor V Leiden mutation. After preliminary amplification of genomic DNA by polymerase chain reaction (PCR), an allele-specific primer was hybridised to the PCR product and extended using fluorescent terminating dideoxynucleotides which were detected by colorimetric assay. Using this ELISA-based assay, the prevalence of the factor V Leiden mutation was determined in an Australian blood donor population (n = 500). A total of 18 heterozygotes were identified (3.6%) and all of these were confirmed with conventional MnlI restriction digest. No homozygotes for the variant allele were detected. We conclude from this study that the frequency of 3.6% is compatible with others published for Caucasian populations. In addition, the FNC technology shows promise as the basis for a rapid, automated DNA based test for factor V Leiden.

Adenine↗
PubMedDate not supplied

Opinion--genome testing and quality assurance.

Recent discoveries and innovations in DNA technology have provided scientists with the ability to rapidly identify complex organisms, or samples therefrom, by detailed study of their genes and genome. In various disciplines, most notably in human forensic identifications, parentage tests and disease diagnosis, these activities have moved to commercial practice. However the rigour of quality assurance in these and other areas has not, in general, kept pace with technological innovation in molecular diagnostics. Insufficient resources are given to objective and ongoing evaluation of the quality of molecular diagnostic services, yet sustained quality is the key to long term credibility of the industry. The rapidly expanding commercial prospects for this technology must be accompanied by a planned commitment of resources to cost efficient quality assurance programs which include both declared and undeclared trials.

Biotechnology↗
PubMed2004

Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk.

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg(2+) concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 x 10(6) to 3 x 10(1) copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.

Animals↗
PubMed2001

Production of transgenic rodents by the microinjection of cloned DNA into fertilized one-cell eggs.

Transgenic technologies that enable rapid movement between genotype and phenotype through specific loss-of-function, overexpression, or misexpression phenotypes will be crucial in the elucidation of gene sequences emerging from genome projects. This article describes detailed procedures for the generation of transgenic mice and rats by the injection of cloned DNA into the pronuclei of fertilized one-cell eggs.

Animal Husbandry↗
PubMed2000

Genetic changes in solid tumors.

Although most solid tumors are treated surgically, determining the genetic changes present in the tumor of an individual patient is becoming increasingly important for managing the oncology patient. Our knowledge of the genetic alterations that characterize and predispose to solid tumors continues to expand. Concurrently, the advent of newer technologies such as DNA chips has the potential to enable a more rapid and comprehensive assessment of these changes. The ultimate goal of this new information and technology is to provide sensitive and specific tests that reduce unnecessary procedures and optimize therapy. This review addresses the utility of molecular testing in evaluating cancer. A review of the current technology and hereditary cancer syndromes is also presented.

Breast Neoplasms↗
PubMed1989

Prenatal diagnosis and prevention of inherited abnormalities of collagen.

There is now strong evidence for the implication of collagen alpha 1(I), alpha 2(I) and alpha 1(III) mutations in many forms of osteogenesis imperfecta and inherited arterial aneurysms (Ehlers Danlos syndrome type IV). A sizeable proportion of these disorders have detectable abnormalities by conventional protein chemistry, immunofluorescence, or more sophisticated DNA analysis. Everyone of them with specific defects or with linkage to appropriate gene markers is therefore amenable to prevention using conventional prenatal diagnosis by chorionic villus biopsy (with fibroblast culture), fetoscopic biopsy (with fibroblast culture), ultrasound diagnosis of the severely deformed fetus, or gene linkage studies by chorionic villus biopsy or amniocentesis. Already many collagen alpha 1(I), alpha 2(I) and alpha 1(III) mutations have been characterized including point mutations, small and large deletions and regulatory mutations. Many others are likely to be rapidly studied by exploiting recent advances in DNA technology, and other strong candidate genes include collagen II (some chondrodystrophies), collagen VI (certain arterial and cardiovascular diseases) and collagen VII (dystrophic epidermolysis bullosa). Other important common diseases are likely to include osteoporosis, osteoarthritis and cerebral aneurysms. A detailed review is provided of collagen interstitial genes and proteins, together with a description of the various forms of osteogenesis imperfecta and Ehlers Danlos syndrome in which either collagen alpha 1(I), alpha 2(I) or alpha 1(III) mutations have been identified. Appropriate restriction length polymorphisms (RFLPs) useful in identifying carriers of these mutant genes are also described.

Collagen↗
PubMed1990

Clinical application of somatic gene therapy in inborn errors of metabolism.

Rapid advances in recombinant DNA and gene transfer technologies provide the potential for somatic gene therapy of inborn errors of metabolism in which the genetically defective function will be restored by transfer of a normal gene into somatic cells. The therapeutic potential and safety of gene therapy has been explored in cultured cells and experimental animals, but therapeutic clinical trials have not yet been proposed or performed. The technologies which may make somatic gene replacement therapy feasible need to be considered and criticised from a clinical perspective. Clinical trials will be necessary to determine the efficacy of somatic gene therapy and address concerns about safety.

Animals↗
PubMed1994

Nucleic acid probes for the food industry.

Recombinant DNA technology has revolutionised microbiological analysis by providing rapid and sensitive methods for the direct detection and identification of organisms. These advances have been most successfully applied to basic research and to clinical microbiology. Continued improvements in the techniques and in assay formats have now brought them to the stage where they could be useful on an industrial scale. The use of nucleic acid techniques for the identification of bacteria of importance to the food industry is discussed with emphasis on developments in practical applications.

Journal Article↗
PubMed1987

Application of DNA-based diagnosis to patient care: the example of hemophilia A.

DNA-based carrier testing and prenatal diagnosis are rapidly expanding medical applications of recombinant-DNA technology. The ultimate goal of DNA-based diagnosis is the determination of the causative mutation, but, in general, this is possible only for large deletions, insertions, or certain nonsense mutation that, in most diseases, involve only a few percent of affected families. If direct diagnosis of the carrier state or fetal disease state is not feasible, indirect diagnosis can be performed by following the segregation of linked polymorphisms through the family pedigree. For such indirect diagnosis, DNA from multiple family members must be analyzed. Although this procedure is highly accurate in many families, errors can potentially occur because of meiotic recombination, genetic heterogeneity, new mutations, and nonpaternity. In this review, a general introduction to DNA-based diagnosis of mendelian diseases is presented and the methods and strategy are outlined. The use of these techniques for the diagnosis of hemophilia A is then described to illustrate the principles of diagnosis and to highlight some of the complexities encountered. DNA-based diagnosis is in its infancy and has the potential to revolutionize preventive medicine.

DNA, Recombinant↗
PubMed1999

Molecular pathogenesis of human cytomegalovirus infection.

Despite progress in diagnosis and treatment of human cytomegalovirus (CMV) infection, we do not understand why, in hosts with comparable levels of immunosuppression, some CMV infections result in symptomatic CMV disease while others are limited to asymptomatic virus shedding with no discernible clinical consequences. CMV viral detection and quantification are useful for identifying those at highest risk, but do not consistently predict clinical outcome. Factors such as host genotype and immune response are active areas of research. However, the importance of CMV strain variability, recognized since 1976, is now receiving attention. Advances in technology that allow the rapid sequencing of viral DNA for purposes of strain characterization have fueled the renewed interest. The focus of this review will be to summarize our evolving knowledge of CMV strain variability and to document where possible a potential relationship to strain virulence. Studies with the UL55 (gB) envelope glycoprotein will be emphasized because of the ability to clearly identify naturally occurring variants, as well as the increasing number of reports that there are differences in biological activities that may contribute to virulence.

Cytomegalovirus↗