PubMed Health⌕ Search

Biomedical subjects

G M Arndt

Publications and source records attributed to G M Arndt.

10 recordsLinked to original sources
PubMed2000

A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells.

We describe a rapid cell-based genetic screen using fission yeast for identifying efficient gene suppression constructs (GSCs) from large libraries (10(5)) for any target sequence for use in human cells. In this system, target sequences are fused to the 5' end of the lacZ reporter gene and expressed in yeast. Random fragment expression libraries derived from the target sequence are screened in the fusion gene-expressing strain using the lacZ gene-encoded colony color phenotype. We demonstrate the utility of this screening assay by identifying a range of different GSCs for the fission yeast ura4 gene and human c-myc and Chk1 sequences, including rare efficient suppressors. GSCs specific for c-myc were shown to regulate expression of both a c-myc-lacZ fusion gene and the endogenous c-myc gene in human cells.

Cells, Cultured↗
PubMed2000

The influence of antisense gene location on target gene suppression in the fission yeast Schizosaccharomyces pombe.

A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression. Fission yeast transformants were generated that contained the target lacZ gene at a fixed position and a single copy antisense lacZ gene integrated into various genomic locations, including the same locus as the target gene. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expression in vivo. Instead, increased lacZ downregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number.

Down-Regulation↗
PubMed1997

Colocalization of antisense RNAs and ribozymes with their target mRNAs.

The use of complementary RNA sequences such as antisense RNAs and ribozymes to regulate the expression of specific genes in eukaryotic cells has been well-documented, particularly with their application to both human gene therapy and plant biotechnology. Despite the simplicity of this approach, this technique usually results in only partial suppression of gene expression and, in some instances, even fails to regulate the gene of interest. The variation observed with antisense RNA and ribozyme-mediated regulation is further complicated by the many factors with the potential to impact on the effectiveness of these RNAs. Recent advances in the understanding of the global architecture of the nucleus, chromatin structure, and RNA metabolism provide useful and necessary information for designing novel approaches to improving antisense RNA and ribozyme regulation. These studies predict that the position of genes within the nucleus is not random and that transcripts produced from these genes follow specific tracks in migrating to the cell cytoplasm. These observations have the potential to impact significantly on the ways in which RNA-mediated forms of gene regulation are applied. The purpose of this review is to discuss the concept of colocalizing antisense RNAs and ribozymes with their target mRNAs and to introduce a variety of approaches aimed at achieving this goal.

Cytoplasm↗
PubMed1996

pH sensitivity of Schizosaccharomyces pombe: effect on the cellular phenotype associated with lacZ gene expression.

We report on a series of experiments in Schizosaccharomyces pombe to detect the blue-colour colony phenotype associated with expression of the Escherichia coli lacZ gene. Increasing the pH in solid minimal medium to optimize blue colony colour revealed a pH-sensitive phenotype in auxotrophic strains requiring uracil and leucine as external supplements. This phenotype was observed among common S. pombe stock strains, 5-fluoroorotic acid (5-FOA)selected strains, and random genetic segregants. Growth of prototrophic S. pombe strains 972 and 975 or the adenine auxotrophic strain NCYC 1860 were unaffected by an increase in external pH. Analysis of genetic segregants from three independent crosses indicated that a single auxotrophic marker (ura4- or leu1-32) was sufficient for yeast cell-growth inhibition when the medium pH was increased above 6.6. In contrast, growth of a Saccharomyces cerevisiae strain isogenic to AH22, requiring uracil, leucine and histidine, was unaffected by changes in the pH of the medium. These observations suggest that uptake of uracil and leucine into S. pombe cells is compromised by alterations in external pH. Our results have implications for detection of the lacZ gene-encoded bluecolour colony phenotype in S. pombe, which is optimized by growth in the presence of 5-bromo-4-chloro-3-indolyl- "beta"-D-galactoside (Xgal) at pH 7.0. We discuss the conditions under which this blue-colour phenotype can be routinely observed in S. pombe.

Galactosides↗
PubMed1995

Gene regulation by antisense RNA in the fission yeast Schizosaccharomyces pombe.

This report describes experiments designed to demonstrate the suitability of the fission yeast Schizosaccharomyces pombe as a host for antisense RNA regulation. A lacZ gene-expressing yeast strain was constructed and used as a host for the expression of a series of antisense RNAs complementary to various regions of the target lacZ mRNA. All lacZ antisense genes were placed under control of the thiamine-repressible nmt1 promoter of S. pombe and expressed from episomal plasmids. For each antisense plasmid a corresponding sense control plasmid was constructed. All lacZ antisense genes were shown to express antisense RNAs of the expected size at equivalent steady-state levels. beta-Galactosidase activity in transformed cells expressing the long, short 5' or short 3' lacZ antisense RNAs was shown to be reduced by 45%, 20%, and 10%, respectively, relative to control transformants. Further experiments indicated that antisense RNA regulation in this system was conditional and reversible, with the observed reduction of beta-galactosidase activity being dependent on the transcription of lacZ antisense RNA. Our results represent the first successful example of antisense RNA regulation of gene expression in yeast and establish S. pombe as an experimental model for the biochemical analysis of antisense RNA regulation.

Base Sequence↗
PubMed1994

Evidence for Darwinian selection of the 2-micron plasmid STB locus in Saccharomyces cerevisiae.

The 2-microns plasmid of industrial and laboratory strains of Saccharomyces cerevisiae exists as two main polymorphic forms designated type I and type II. Polymorphism is restricted to the 3200-bp right unique region where types I and II show approximately 10% nucleotide divergence in trans-acting REP1 and RAF loci and 30% divergence in the cis-acting STB locus. In addition, the cis-acting STB plasmid partition locus of type II plasmids varies in sequence and copy number of a 125-bp repeat. We devised chimeric and 2-microns plasmid stability experiments to evaluate the effect of STB polymorphism on plasmid fitness in amphiploid industrial and haploid laboratory strains. Reciprocal experiments of type-II STB chimeric plasmids in type-I bakers' yeast or a type-I chimeric plasmid in type-II distillers', wine, or haploid strains showed similar partition efficiencies. However, chimeric and 2-microns plasmids carrying a 250-bp STB from a type-II haploid strain had reduced fitness in a type-II industrial wine strain. These results in conjunction with molecular analyses of 2-microns-like and 2-microns plasmids indicates the coevolution of STB with trans-acting plasmid and host-cell factors.

Biological Evolution↗
PubMed1994

Antisense gene expression in yeast.

The use of antisense and ribozyme RNA to modulate gene expression is emerging as an effective genetic technique. A compilation of successful antisense gene suppression experiments reveals the absence of reports on the use of the yeast Saccharomyces cerevisiae as a host. We examine the field of antisense and ribozyme use in S. cerevisiae and discuss that this result is not due to any lack of attempts and may reflect unique features of S. cerevisiae biology. In an attempt to learn from cellular RNA physiology we review evidence for naturally occurring antisense RNA regulation. Although there are many examples of well characterised overlapping RNA transcripts there is, as yet, no clear evidence suggesting complementary RNA-dependent gene regulation in S. cerevisiae. The application of artificial antisense and ribozyme genes is then discussed with an emphasis on the role of yeast as a model system for the systematic and genetic analysis of antisense and ribozyme RNA function. In addition, potential reasons for the lack of attempts to use antisense or ribozyme genes to create pseudogenetic mutants are considered. We conclude that the application of successful antisense and ribozyme strategies in yeast may have to address features of S. cerevisiae RNA biology and offer experimental approaches that may identify some of these features.

Cell Cycle↗
PubMed1989

FLP-FRT mediated intrachromosomal recombination on a tandemly duplicated YEp integrant at the ILV2 locus of chromosome XIII in Saccharomyces cerevisiae.

A YEp chimaeric plasmid carrying SMR1 and URA3 genetic markers was integrated into chromosome XIII at the ilv2-delta 1 locus in a [cir (o)] background. The 1.5 kb BglII deletion of ilv2-delta 1 allowed the clear identification of an integrant structure which consisted of a direct tandem duplication (TD) of the chimaeric plasmid. Within the integrant structure, a single copy of the plasmid sequence was flanked by a direct duplication of the 2 microns site-specific recombinase (FLP) recognition target (FRT). Isogenic [cir (o)] and [cir+] diploids formed by crossing the [cir (o)] TD strain to complementary haploids were analyzed for plasmid marker loss and chromosomal DNA alterations in the presence and absence of selection pressure for the URA3 and SMR1 plasmid borne markers. [cir (o)] diploids showed no plasmid marker loss and maintained the TD structure. In the absence of selection pressure, the [cir+] diploid underwent FLP-FRT mediated unequal interchromatid recombination, resulting in the breakage-fusion-bridge cycle and homozygotization of chromosome XIII (Rank et al. 1988). Maintenance of selection pressure for the centromere distal plasmid URA3 marker selected against FLP-FRT interchromatid recombinants so that the effects of site specific recombinase on intrachromatid recombination could be evaluated. Intrachromatid recombination at the directly duplicated FRT sites of the TD structure resulted in the loss of a diagnostic internal fragment. These results show that in the presence of FLP, FRT sites separated by up to 13.3 kb of chromosomal DNA function as substrates for intra and interchromatid recombination.

Blotting, Southern↗