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PubMed1997

Rapid detection of multidrug-resistant tuberculosis.

Transmission of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) presents a serious problem for infection control in hospitals, particularly in the context of co-infection with the human immunodeficiency virus (HIV). We report on the use of molecular genetic tools to allow rapid assessment of samples from patients potentially infected with MDR-TB. Sputum and bronchoalveolar lavage samples were obtained from two HIV-positive patients with suspected tuberculosis, who had previous contact with a known MDR-TB index case. Polymerase chain reaction (PCR) was used directly on clinical samples to amplify genetic loci associated with rifampicin resistance (rpoB), and strain-specific polymorphisms (the direct repeat (DR) region). Drug resistance was determined using a commercially available kit for detection of point mutations in the rpoB gene (Inno-Lipa RifTB; Innogenetics, Belgium), and confirmed by nucleotide sequencing. Strain variation was determined using the spoligotyping method, based on the presence or absence of variable linker sequences within the DR region. In one patient, infection with a MDR strain identical to that of a known index case was demonstrated. A second patient, although positive for M. tuberculosis, was found to be infected with a rifampicin-sensitive strain. Results were obtained within 48 h, allowing appropriate treatment to be initiated and infection control measures to be implemented. PCR-based tests for strain-typing and for identification of rifampicin resistance provide important tools for identifying patients with MDR-TB and for rapid monitoring of potential nosocomial spread of MDR-TB. Prompt confirmation or exclusion of possible transmission allows early clinical intervention to prevent future outbreaks of multidrug-resistant M. tuberculosis.

Antibiotics, Antitubercular↗
PubMed1994

Genetic and molecular analysis of hybrids in the genus Saccharomyces involving S. cerevisiae, S. uvarum and a new species, S. douglasii.

We have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems. The yeasts investigated are three Saccharomyces species: S. cerevisiae, S. uvarum and a new species, S. douglasii. The diploid hybrids from crosses between these species sporulate well but are essentially infertile. The rare viable spores, one per 10(4) to 10(5) asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e. they are generally disomic for at least two or three chromosomes. This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes. This apparent lack of pairing of 'homeologous' chromosomes in meiosis I was analysed in most detail with S. cerevisiae/S. douglasii hybrids. As a genetic tool we studied frequencies of recombination, taking advantage of an S. douglasii breeding stock of some 50 identified mutations in non-switching haploids. Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange. Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten-fold. Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two- to 100-fold. DNA hybridization experiments with S. cerevisiae gene probes gave results consistent with low DNA sequence homologies between S. cerevisiae and S. douglasii. Moreover, by change, our experiments disclosed another Saccharomyces strain (CBS2908, originally classified as S. cerevisiae) with hybridization patterns identical to S. douglasii except for the hybridization with the Ty transposon probes. Crosses between CBS2908 and S. douglasii yielded diploid hybrids with 80-90% spore viability, thus establishing a second member of the S. douglasii species.

Chromosomes, Fungal↗
PubMed2005

Promoter analysis of Mucor rouxii delta9-desaturase: its implication for transcriptional regulation in Saccharomyces cerevisiae.

Promoter study was performed to understand the transcriptional control of delta9-desaturase gene of Mucor rouxii. Several putative cis-elements involved in lipid metabolism were mapped by computational analysis. 5' deletion analysis shows the presence of elements with repressing activity, especially in 122 bp located upstream of the transcription start site. Truncation of these repressor domains showed that the promoter of M. rouxii is functional in Saccharomyces cerevisiae without additional components and is insensitive to nutritional depletion. The promoter also drove effectively the expression of a M. rouxii delta12-desaturase gene, and the linoleic acid content increased with the age of the yeast culture in parallel with the promoter activity. This approach provides a genetic tool for programming heterologous protein production in the yeast.

Base Sequence↗
PubMed2025

Parameters that influence bipartite reporter system expression in Caenorhabditis elegans.

The development of bipartite reporter systems in Caenorhabditis elegans has lagged by more than a decade behind its adoption in Drosophila, the other invertebrate model commonly used to dissect biological mechanisms. Here, we characterize many parameters that influence expression in recently developed C. elegans bipartite systems. We examine how DNA binding site number and spacing influence expression and characterize how these expression parameters vary in distinct tissue types. Furthermore, we examine how both basal promoters and 3' UTR influence the specificity and level of expression. These studies provide both a framework for the rational design of driver and reporter transgenes and molecular and genetic tools for the creation, characterization, and optimization of bipartite system components for expression in other cell types.

Animals↗
PubMed2004

Molecular diversity of new Thermococcales isolates from a single area of hydrothermal deep-sea vents as revealed by randomly amplified polymorphic DNA fingerprinting and 16S rRNA gene sequence analysis.

Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13 degrees N 104 degrees W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales: About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.

Base Sequence↗
PubMed2004

Drosophila, an emerging model for cardiac disease.

A variety of studies that are currently underway may validate the fruit fly as an in vivo model for analyzing genes involved in cardiac function. Many mutations in conserved genetic pathways have been found, including those controlling development and physiology. Because homologous genes control early developmental events as well as functional components of the Drosophila and vertebrate hearts, the fly is the simplest existing model system that can be used to assay genes involved in human congenital heart disease (CHD). The wide variety of genetic tools available to Drosophila researchers offers many technical advantages for rapidly screening through large numbers of candidate genes. Thus, an important future and long-term direction is likely to be the use of Drosophila as a vehicle for analyzing polygenic traits as an aid in human genetics. One can anticipate a time in the not too distant future when mutant lines exist for every gene in vertebrate systems, such as mice and zebrafish. However, one of the enduring problems that will not easily be addressed by such resources will be the tracking of complex traits defined by polygenic variants. For this level of genetic analysis, simple genetic model systems including yeast, Caenorhabditis elegans, and Drosophila melanogaster will undoubtedly play a crucial ongoing role. Of them, Drosophila will be critical for examining gene networks involved in organogenesis and is clearly the system of choice for studying cardiac development, function and aging, since among the simple genetic models it is the only one with a fluid pumping heart.

Animals↗
PubMed1995

Patterns of genetic variability at individual minisatellite loci in minke whale Balaenoptera acutorostrata populations from three different oceans.

The genetic variability at six cloned minisatellite loci was analyzed in minke whale populations from the North Atlantic, North Pacific, and Antarctic Oceans. Three loci displayed only a few different alleles in each of the three populations, with heterozygosity ranging from 0.00 to 0.47, and three loci revealed many different alleles in at least two fo the three populations, with heterozygosity ranging up to 0.98. Using small sample sizes, samples from two adjacent Antarctic Management Areas were not found to differ significantly in allele frequencies at any of the six loci. The use of principal coordinate analysis to detect multilocus disequilibria was explored. No significant evidence was found of intrapopulation heterogeneity within the pooled Antarctic sample. Pronounced interoceanic differences were observed at every locus, confirming the existence of genetic isolation found earlier using more conventional marker systems. The populations from the three oceans appear to have diverged to such a degree that the hypervariable loci have had time to evolve independently and arrive at different evolutionary stages in different populations. The frequency of undetected "null" alleles is remarkably high in minke whale populations compared to human populations and is probably a result of the cloning protocol used. Minisatellite loci are shown to provide a powerful population genetic tool, supplying levels of resolution appropriate to different degrees of evolutionary divergence.

Alleles↗
PubMed1998

New genetic concepts in hypertensive cardiovascular disease.

Hypertension is a complex quantitative trait under polygenic control. The identification of genes responsible for high blood pressure is of major importance, because it provides a mechanistic classification of the common phenotype and guide therapy tailored to the underlying primary abnormality. Experimental studies have identified several quantitative trait loci for blood pressure and other cardiovascular phenotypes. Further strategies that include congenic and subcongenic lines should ultimately lead to positional cloning of the causative genes, but this final step remains elusive at present. Human studies have focused on the rare Mendelian forms of human hypertension or candidate gene studies. Moreover, two recent examples show direct translation of a candidate gene and a quantitative trait locus from the experimental setting to human investigation. These strategies, together with new molecular genetic tools, will ultimately result in the identification of major genes contributing to human essential hypertension.

Animals↗
PubMed2006

In vivo Himar1-based transposon mutagenesis of Francisella tularensis.

Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host.

Animals↗
PubMedDate not supplied

MHC and non-MHC genes in lung tumor susceptibility in the mouse: implications for the study of the different lung tumor types and their cell of origin.

Lung tumorigenesis in the mouse is controlled by multiple genes, which until now largely escaped detection because of the limitations of the available genetic tools. Therefore, we have developed the Recombinant Congenic Strains (RCS), which can be used to separate and map individual tumor susceptibility genes. The two strains, B10.O20/Dem and O20/A (used to produce the RCS series O20.c.B10.O20/Dem) differ in number, type, and degree of malignancy of lung tumors. Thus the genetic control of the different aspects of lung tumorigenesis can now be analyzed using RCS. The tests on the role of the Major Histocompatibility Complex (MHC; H-2 in mice) in lung tumorigenesis show that the H-2 influence is different for alveolar tumors and papillary tumors. In addition, only the development of papillary tumors is influenced by glucocorticoid administration concomitant with the transplacental carcinogen treatment. The MHC influence on lung tumor development is probably related to H-2 effects on the regulation of lung differentiation. In the H-2 congenic strains used papillary tumors developed early, whereas alveolar tumors appeared later, if at all. This differential impact of genetic and hormonal factors on the development of alveolar and papillary tumors suggests that they arise either from different cell types or from a common cell type at different stages of differentiation.

Animals↗
PubMed2006

Live bacterial vaccines--a review and identification of potential hazards.

The use of live bacteria to induce an immune response to itself or to a carried vaccine component is an attractive vaccine strategy. Advantages of live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. Derivatives of pathogenic and non-pathogenic food related bacteria are currently being evaluated as live vaccines. However, pathogenic bacteria demands for attenuation to weaken its virulence. The use of bacteria as vaccine delivery vehicles implies construction of recombinant strains that contain the gene cassette encoding the antigen. With the increased knowledge of mucosal immunity and the availability of genetic tools for heterologous gene expression the concept of live vaccine vehicles gains renewed interest. However, administration of live bacterial vaccines poses some risks. In addition, vaccination using recombinant bacteria results in the release of live recombinant organisms into nature. This places these vaccines in the debate on application of genetically modified organisms. In this review we give an overview of live bacterial vaccines on the market and describe the development of new live vaccines with a focus on attenuated bacteria and food-related lactic acid bacteria. Furthermore, we outline the safety concerns and identify the hazards associated with live bacterial vaccines and try to give some suggestions of what to consider during their development.

Journal Article↗
PubMed2004

RNA interference and its current application in mammals.

OBJECTIVE: The aim of this review was to assess RNA interference (RNAi) and its possibility as a potential and powerful tool to develop highly specific double-stranded RNA (dsRNA) or small interfering RNA (siRNA) based gene-silencing therapeutics. DATA SOURCES: The data used in this review were obtained from the current RNAi-related research reports. STUDY SELECTION: dsRNA-mediated RNAi has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. The discovery that synthetic duplexes of 21 nucleotides siRNAs trigger gene-specific silencing in mammalian cells has further expanded the utility of RNAi in to the mammalian system. DATA EXTRACTION: The currently published papers reporting the discovery and mechanism of RNAi phenomena and application of RNAi on gene function in mammalian cells were included. DATA SYNTHESIS: Since the recent development of RNAi technology in the mammalian system, investigators have used RNAi to elucidate gene function, and to develop gene-based therapeutics by delivery exogenous siRNA or siRNA expressing vector. The general and sequence-specific inhibitory effects of RNAi that will be selective, long-term, and systemic to modulate gene targets mentioned in similar reports have caused much concern about its effectiveness in mammals and its eventual use as a therapeutic mordality. CONCLUSIONS: It is certain that the ability of RNAi in mammals to silence specific genes, either when transfected directly as siRNAs or when generated from DNA vectors, will undoubtedly accelerate the study of gene function and might also be used as a potentially useful method to develop highly gene-specific therapeutic methods. It is also expected that RNAi might one day be used to treat human diseases.

Animals↗
PubMed2006

Transgenic labeling of taste receptor cells in model fish under the control of the 5'-upstream region of medaka phospholipase C-beta 2 gene.

Vertebrate taste receptor cells express signaling molecules such as taste receptors and effectors to convert taste stimuli to inner cellular signals. Phospholipase C-beta2 (PLC-beta2) is an effector enzyme that is necessary to transduce taste signals in the mouse. It was shown that a subset of the plc-beta2 expressing cells also express taste receptor molecules, T1Rs or T2Rs, in mammals and fish. To label plc-beta2 expressing cells in the model fish species, we constructed a transgene by linking the 5'-upstream region of the medaka plc-beta2 gene to a green fluorescent protein (GFP) gene. The resulting transgenic medaka exhibited GFP signals in taste buds of the lips and the pharyngeal region. Detailed observation revealed that the GFP signals were in a subpopulation of taste bud cells, and co-localized with the transcript of endogenous plc-beta2 gene. Zebrafish introduced with the same transgene showed GFP signals in a subpopulation of taste bud cells of the lips and the pharyngeal region as in the case of medaka. This is the first report of successful labeling of taste receptor cells in two model fish species under the control of the plc-beta2 promoter. This promoter will be a useful genetic tool to study the vertebrate taste system in general.

Animals↗
PubMed1992

Beauty and the yeast: compartmental organization of the secretory pathway.

Our perception of intracellular organelles and cellular architecture was initially based on striking light and electron micrographs of animal and plant cells. The high degree of compartmental organization within specialized mammalian secretory cells aided early efforts to track the movement of proteins through the organelles of the secretory pathway. In contrast, the morphological detail of the yeast Saccharomyces cerevisiae appeared superficially simple, even primitive, by comparison with the higher eukaryotic cells. However, the combination of genetic tools and the development of assays reconstituting vesicular traffic in yeast have facilitated the identification and characterization of individual proteins that function in the secretory pathway. Analogies between the function of yeast and mammalian proteins in vesicular traffic are being drawn with increasing frequency. In this review, the combination of genetic, biochemical, molecular and cell biological approaches used to study compartmental organization in the yeast secretory pathway will be discussed. The rapid progress in our understanding of yeast membrane traffic has revealed the beauty of working with this organism.

Animals↗
PubMed2003

Zebra fish: an uncharted behavior genetic model.

The zebra fish has been a preferred subject of genetic analysis. It produces a large number of offspring that can be kept in small aquaria, it can be easily mutagenized using chemical mutagens (e.g., ethyl nitrosourea [ENU]), and high-resolution genetic maps exist that aid identification of novel genes. Libraries containing large numbers of mutant fish have been generated, and the genetic mechanisms of the development of zebra fish, whose embryo is transparent, have been extensively studied. Given the extensive homology of its genome with that of other vertebrate species including our own and given the available genetic tools, zebra fish has become a popular model organism. Despite this popularity, however, surprisingly little is known about its behavior. It is argued that behavioral analysis is a powerful tool with which the function of the brain may be studied, and the zebra fish will represent an excellent subject of such analysis. The present paper is a proof of concept study that uses pharmacological manipulation (exposure to alcohol) to show that the zebra fish is amenable to the behavioral genetic analysis of aggression and thus may allow us to reveal molecular mechanisms of this behavioral phenomenon relevant to vertebrates.

Aggression↗
PubMed1987

HRAS1-selected, chromosome mediated gene transfer; in situ hybridization with combined biotin and tritium label localizes the oncogene and reveals duplications of the human transgenome.

Chromosomes isolated from a human bladder carcinoma cell line which contains the actively transforming oncogene HRAS1 on chromosome 11 can be used to transform mouse cells. We have analyzed these chromosome mediated gene transformants by in situ hybridization techniques using biotinylated human DNA and a double antibody detection system to visualize the whole of the transgenome in a number of cell lines. In some transformants, where the amount of the transgenome was below the level of detection by the simple biotin system, we used a gold-silver enhancement technique. We have developed a combined in situ hybridization procedure using biotinylated human DNA plus antibodies and 3H-labeled HRAS1 DNA plus autoradiography to locate the actively transforming oncogene within the human transgenome in a selection of these transformants. In each of these there were complex insertions of the transgenome, either at multiple sites or with duplicated inserts at a single site. Each insertion contained a copy of HRAS1. The double in situ hybridization analysis helps define the types of arrangement and rearrangement which can accompany the chromosome mediated gene transfer process and, consequently, the potentials and limitations of the technique as a somatic cell and molecular genetic tool. Our analysis also suggests that multiple copies of the HRAS1 gene may be needed for stable transformation.

Animals↗
PubMed2007

Cre/loxP-mediated chromosome engineering of the mouse genome.

Together with numerous other genome modifications, chromosome engineering offers a very powerful tool to accelerate the functional analysis of the mammalian genome. The technology, based on the Cre/loxP system, is used more and more in the scientific community in order to generate new chromosomes carrying deletions, duplications, inversions and translocations in targeted regions of interest. In this review, we will present the basic principle of the technique either in vivo or in vitro and we will briefly describe some applications to provide highly valuable genetic tools, to decipher the mammalian genome organisation and to analyze human diseases in the mouse.

Animals↗
PubMed1993

Mus spretus-specific LINE-1 DNA probes applied to the cloning of the murine pearl locus.

LINE-1 is the major family of long, interspersed, repetitive DNA sequences found in mammalian genomes. The mouse species Mus spretus contains large LINE-1 subfamilies that are distinguishable from the LINE-1 elements of laboratory Mus domesticus strains by their content of particular nucleotide differences. Oligonucleotides containing these differences act as M. spretus-specific LINE-1 hybridization probes. We have used these probes as a novel genetic tool in conjunction with an interspecific hybrid congenic mouse, in which the M. spretus allele of the pearl gene has been transferred onto a M. domesticus background. From a lambda library prepared from this congenic mouse, four clones were isolated by hybridization to the M. spretus-specific probes. After derivation of genetic markers from these clones, two of them were found to be linked to the pearl gene. These markers are the first two of up to 75 that could be isolated to support cloning the pearl gene. Considering the interspersed nature of LINE-1, we propose that species-specific LINE-1 probes could also be used to isolate markers for many other target genes.

Animals↗