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At least 109 records · Page 6Linked to original sources
PubMed1993

A genetic system controlling mitochondrial fusion in the slime mould, Physarum polycephalum.

We have identified two distinct mitochondrial phenotypes, namely, Mif+ (mitochondrial fusion) and Mif- (mitochondrial fusion-deficient), and have studied the genetic system that controls mitochondrial fusion in the slime mould, Physarum polycephalum. A mitochondrial plasmid of approximately 16 kbp was identified in all Mif+ plasmodial strains. This plasmid is apparently responsible for promoting mitochondrial fusion, and it is inserted into the mitochondrial DNA (mtDNA) in successive sexual crossing with Mif- strains. This recombinant mtDNA and the unchanged free plasmid spread through the mitochondrial population via the promotion of mitochondrial fusion. The Mif+ strains with the plasmid were further classified as being two types: high frequency and low frequency mitochondrial fusion. Restriction analysis of the mtDNA suggested that the high frequency mitochondrial fusion type was more often heteroplasmic; within each plasmodium, mtDNAs of both parental types were usually present, in addition to the presence of the plasmid. Genetic analysis with the progeny obtained from crossing myxamoebae derived from three different isolates suggested that these progeny carried different alleles at a nuclear locus that controlled the frequency of mitochondrial fusion. These alleles (mitochondrial mating-type alleles, mitA1, 2 and 3) appear to function like the mating type of the myxamoebae; mitochondrial fusion occurs at high frequency with the combination of unlike alleles, but at low frequency with the combination of like alleles.

Alleles↗
PubMed1997

A yeast genetic system for selecting small molecule inhibitors of protein-protein interactions in nanodroplets.

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein-protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein-protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These "nanodroplets" contain defined media, cells, and one or more beads containing approximately 100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein-protein interactions in living cells or organisms.

Activin Receptors, Type I↗
PubMed1971

Three closely linked genetic systems relevant to transplantation.

The concept that antigens of a major histocompatibility locus (HL-A), the mixed leukocyte reaction, and the rejection of a graft are expressions of the same genetic region has been generally accepted. WE HAVE PRESENTED EXPERIMENTS THAT CHALLENGE THE ABOVE CONCEPT AND SUGGEST THAT THE REJECTION TIME OF SKIN AND ORGAN TRANSPLANTS IS DEPENDENT ON IMMUNIZATION AGAINST THE PRODUCTS OF TWO, AND POSSIBLY THREE, SEPARATE, BUT CLOSELY LINKED, GENETIC SYSTEMS: HL-A, mixed leukocyte reaction (MLR-S), and hypersensitivity delayed reaction (HDR). The findings presented and discussed here suggest that the HL-A antigens are not the primary factors involved in the cell-mediated component of allotransplantation, and that these antigens and phenotypes of the mixed leukocyte reaction can only be used as a guide in predicting the survival of allografts that are obtained from unrelated donors.

Female↗
PubMed1997

Detection of Pneumocystis carinii in tracheal aspirates of intubated patients using calcofluor-white (Fungi-Fluor) and immunofluorescence antibody (Genetic Systems) stains.

OBJECTIVE: To compare the detection rate of Pneumocystis carinii in endotracheal aspirates with that rate in bronchoalveolar lavage fluid, using calcofluor-white (Fungi-Fluor) and immunofluorescence antibody (Genetic Systems) staining methods. DESIGN: Prospective, consecutive cases. SETTING: Medical intensive care unit at Ben Taub General Hospital. PATIENTS: Thirty-one intubated patients admitted with respiratory failure and suspected P. carinii pneumonia. INTERVENTIONS: An endotracheal aspirate specimen was obtained after maximally advancing a closed-system suction catheter, instilling aliquot portions of saline, and suctioning the lavage fluid. This procedure was followed within 30 mins by fiberoptic bronchoscopy and bronchoalveolar lavage. MEASUREMENTS AND MAIN RESULTS: Endotracheal aspirate and bronchoalveolar lavage specimens from each patient were mixed with Saccomano's fixative, blended, and centrifuged. Using a modified method for P. carinii cysts, the sediment was stained with the test calcofluor-white stain Solution A and the test antibody stain. The test antibody stain on the bronchoalveolar lavage specimens was positive for P. carinii for 13 patients and was used as the standard for comparison. In the endotracheal aspirate specimens, the test antibody stain detected 12 (92%) P. carinii-positive patients while the test calcofluor-white stain detected ten (77%) P. carinii-positive patients. CONCLUSIONS: We described a simple method for obtaining, processing, and staining endotracheal aspirate specimens for P. carinii. Obtaining an endotracheal aspirate specimen did not require specially trained personnel or a specialized and more expensive catheter, and was not associated with any complications.

Benzenesulfonates↗
PubMed2003

Genetic biases for showy males: are some genetic systems especially conducive to sexual selection?

Male secondary sexual characters (conspicuous ornaments, signals, colors) are among nature's most striking features. Yet, it is unclear why certain groups of organisms are more likely than others to evolve these traits. One explanation for such taxonomic biases is that some genetic systems may be especially conducive to sexual selection. Here, we present theory and simulation results demonstrating that rare alleles encoding either male ornaments or female preferences for those ornaments are better protected against random loss in species with ZZZW or ZZZO sex chromosome systems (male homogamety) than in species with XXXY or XXXO systems (male heterogamety). Moreover, this protection is much stronger in diploid than haplodiploid species. We also present empirical data showing that male secondary sexual characters are better developed in diploid than haplodiploid species and in diploid species with male homogamety than in those with male heterogamety. Thus, taxonomic biases for showy males may stem from differences in sex chromosome systems.

Alleles↗
PubMed1993

Typogenetics: an artificial genetic system.

Cellular automata are now used to model various natural phenomena. In particular, they provide a logical universe in which artificial cells can be embedded in the form of propagating virtual automata. In this paper, we propose a molecular automaton which is an attempt to implement the "molecular logic of the living state" in an artificial biochemistry. This automaton is an artificial genetic system composed of two classes of interacting artificial molecules. It was shown to be self-replicating and to possess features that are analogous to those of the postulated prebiotic molecular systems.

Amino Acids↗
PubMed1989

A novel genetic system to detect protein-protein interactions.

Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.

DNA Restriction Enzymes↗
PubMed2005

[Segregation analysis of genetic system of quantitative traits in plants].

Based on the traditional polygene inheritance model of quantitative traits, the mixed major gene and polygene inheritance model was raised and considered as the general model, while pure major gene or pure polygene inheritance model being only the specific case of the general model. From the proposed theory, the segregation analysis procedure was established for studying the genetic system of quantitative traits of plants. At present this procedure can be used to evaluate the genetic effects of individual major genes (up to 2-3 major genes) and the collective genetic effects of polygenes as well as their heritability values. The present paper introduces the process of the establishment of the procedure, main achievements and application results. An example was given to illustrate the steps, methods and effectiveness of the procedure.

Crosses, Genetic↗
PubMed2004

Development of a reverse-genetics system for Avian pneumovirus demonstrates that the small hydrophobic (SH) and attachment (G) genes are not essential for virus viability.

Avian pneumovirus (APV) is a member of the genus Metapneumovirus of the subfamily Pneumovirinae. This study describes the development of a reverse-genetics system for APV. A minigenome system was used to optimize the expression of the nucleoprotein, phosphoprotein, M2 and large polymerase proteins when transfected into Vero cells under the control of the bacteriophage T7 promoter. Subsequently, cDNA was transcribed from the virion RNA to make a full-length antigenome, which was also cloned under the control of the T7 promoter. Transfection of the full-length genome plasmid, together with the plasmids expressing the functional proteins in the transcription and replication complex, generated APV in the transfected cells. The recombinant virus was passaged and was identified by cytopathic effect (CPE) that was typical of APV, the presence of a unique restriction-endonuclease site in the cDNA copy of the genome and immunofluorescence staining with anti-APV antibodies. Replacement of the full-length wild-type antigenome with one lacking the small hydrophobic (SH) protein and the attachment (G) genes generated a virus that grew more slowly and produced atypical CPE with syncytia much larger than those seen with wild-type virus.

Animals↗
PubMed1997

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells.

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5' and 3' deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the beta-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9 x 10(-4), whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8 x 10(-4). The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.

Animals↗
PubMed2004

Development of a genetic system in chitinase-producing streptomyces and the application of an allosamidin-insensitive chitinase gene to homologous overexpression.

A transformation system for Streptomyces sp. AJ9463 strain (allosamidin producer) was successfully developed using protoplasts and a PEG-mediated method. To prepare protoplasts, the concentration of glycine and sucrose in YEME medium were optimized to 0.5% (w/v) and 34.0% (w/v), respectively. When the protoplasts of Streptomyces sp. AJ9463 were transformed with pUWL-KS, transformants could be obtained at a high efficiency of 7.0 x 10(4) transformants per microg DNA. To ensure that the transformation system worked properly, we then constructed a constitutive expression vector pYK1, in which the ermE* promoter drives transcription of the allosamidin-insensitive chitinase gene, chiIS. Although no transformant could be obtained by the genetic system using pYK1 isolated from Escherichia coli DH5alpha, pYK1 isolated from the methylase-deficient mutant E. coli SCS110, could be introduced into Streptomyces sp. AJ9463. This indicates that Streptomyces sp. AJ9463 has a methylation-specific restriction system, and that the chiIS and/or ermE* promoter region of pYK1 includes a restriction site of its endonuclease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that pYK1 in Streptomyces sp. AJ9463 started to obviously express ChiIS from 14-h. Moreover, the pYK1-introduced strain gave a five-fold higher chitinase activity than the wild-type, suggesting that this system can be widely applied for the overexpression and gene functional analysis.

Acetylglucosamine↗
PubMed2003

A reverse genetics system for Borna disease virus.

Borna disease virus (BDV) is an enveloped virus. Its non-segmented, negative-stranded RNA genome has the coding capability for six main polypeptides and has an organization characteristic of members of the order Mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae. Here, the establishment of a reverse genetics system for BDV is described. Intracellular synthesis of a BDV RNA analogue or minigenome (MG) from a plasmid was driven by RNA polymerase I. Co-transfection with plasmids expressing the BDV polymerase (L), nucleoprotein (N) and phosphoprotein (P) under the control of RNA polymerase II allowed for BDV MG replication and expression. This process depended on a delicate N:P ratio, whereas the L:P ratio was less critical. Two isoforms of N, Np40 and Np38, are present in BDV-infected cells but only Np40 was strictly required for virus polymerase activity. BDV p10 polypeptide encoded by the P gene exhibited a strong inhibitory effect on BDV MG expression.

Animals↗
PubMed2006

Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal.

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that replicates and transcribes in the nucleus of infected cells. Therefore, efficient synthesis of BDV RNA in the nucleus is critical for the development of a reverse genetics system for this virus. Here, we report the development of such a system using the RNA polymerase II (Pol II) promoter. The BDV minigenome cDNA was flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences and inserted downstream of the Pol II promoter. To improve the efficacy of minigenome expression, we estimated the effects of several signal sequences within the minigenome constructs. We found that insertion of the SV40 nuclear import sequence into the Pol II constructs significantly enhances the replication of the minigenome even in cells lacking the SV40 large T antigen. This novel system is theoretically applicable to any mammalian cell line and would be valuable for analyzing host- or cell-type-dependent differences in BDV replication and production. We could demonstrate here the cell-type-dependent inhibitory effect of the viral protein X on BDV polymerase activity. This system may be useful for various research fields not only of BDV but also of other negative-sense RNA viruses.

Active Transport, Cell Nucleus↗
PubMed2001

Development of a genetic system for Geobacter sulfurreducens.

Members of the genus Geobacter are the dominant metal-reducing microorganisms in a variety of anaerobic subsurface environments and have been shown to be involved in the bioremediation of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic system was developed for Geobacter sulfurreducens. The antibiotic sensitivity of this organism was characterized, and optimal conditions for plating it at high efficiency were established. A protocol for the introduction of foreign DNA into G. sulfurreducens by electroporation was also developed. Two classes of broad-host-range vectors, IncQ and pBBR1, were found to be capable of replication in G. sulfurreducens. In particular, the IncQ plasmid pCD342 was found to be a suitable expression vector for this organism. When the information and novel methods described above were utilized, the nifD gene of G. sulfurreducens was disrupted by the single-step gene replacement method. Insertional mutagenesis of this key gene in the nitrogen fixation pathway impaired the ability of G. sulfurreducens to grow in medium lacking a source of fixed nitrogen. Expression of the nifD gene in trans complemented this phenotype. This paper constitutes the first report of genetic manipulation of a member of the Geobacter genus.

Anti-Bacterial Agents↗
PubMed2006

Mouse embryonic stem cells as a model genetic system to dissect and exploit the RNA interference machinery.

Conditional gene targeting is often a useful approach to elucidate the in vivo function of a gene. We use this approach to investigate the biological role of the RNA interference (RNAi) pathway in mammals. In addition, the RNAi machinery in mammalian cells can be exploited for gene knock-down experiments. In this chapter, we discuss the variety of experiments that can be performed using genetically engineered embryonic stem (ES) cells. ES cells provide a mammalian genetic system that is physiological, and tractable for mutagenesis and experimentation. This approach is economical and rapid, because it does not require production and breeding of genetically engineered mice.

Animals↗