Search PubMed⌕ Search

Biomedical subjects

W Xiao

Publications and source records attributed to W Xiao.

155 records · Page 9Linked to original sources
PubMed1990

Cloning of industrial Saccharomyces 2-microns plasmid variants by in vivo site-specific recombination.

Southern analyses defined several industrial Saccharomyces yeast strains with extensive 2-microns DNA polymorphism. Variants included insertions and deletions up to several hundred base pairs. To facilitate the investigation of yeast plasmid evolution we developed a novel method of cloning 2-microns plasmids by taking advantage of 2-microns circle in vivo site-specific recombination and an SMRI gene as a dominant selectable marker. This method can be applied to other organisms for the isolation of plasmid variants and provides a new approach to in vivo plasmid construction.

Cloning, Molecular↗
PubMed1990

Branched chain amino acid regulation of the ILV2 locus in Saccharomyces cerevisiae.

Mutant regulatory loci of the branched pathway for the biosynthesis of isoleucine-valine and leucine were identified with the unusual phenotype of an amino acid dependent auxotrophy. Two mutant loci, bcs1 and bcs2, conferred branched chain amino acid sensitivity and showed independent segregation. Linkage studies defined bcs1 as a cis-acting regulatory site of ILV2 (SMR1). ILV2 upstream deletion analyses and high-copy transformation of the positive regulatory locus LEU3 ruled out the possibility of LEU3 protein binding palindromes mediating the branched chain amino acid dependent auxotrophy. In the presence of leucine and valine, the general amino acid control system (GCN4) was epistatic to bcs1 and bcs2, and under nonstarvation conditions GCN4 strains showed an increased acetolactate synthase activity over gcn4 strains. Thus in addition to general regulation of ILV2, GCN4 functions in basal level expression when the locus is subject to specific repression by pathway end product.

Gene Expression Regulation, Enzymologic↗
PubMed1990

[Dynamic changes in abnormal afferent activities following peripheral nerve injury].

Peripheral nerve injury by chronic compression caused a decrease in conductive velocity or a delay in the transmission of the action potential, later blocking it completely following demyelination of the damaged nerve region. Abnormal firing activities were recorded from the injured peripheral nerve fibers after the 6-7th postoperative day. There were different patterns of abnormal firing from fibers. Regular tonic ectopic firings with high frequency were always recorded from A beta fibers. A delta/C fibers, however, had burst or irregular spikes. A single antidromic shock to the damaged region sometimes induced ectopic spikes following the expected one. The demyelinated region of the damaged nerve was very sensitive to both tetraethylammonium, a K+ channel blocker, and noradrenaline. The authors suggest that new ion channels or receptors, absent in normal, form in the damaged region.

Action Potentials↗
PubMed1989

The construction of recombinant industrial yeasts free of bacterial sequences by directed gene replacement into a nonessential region of the genome.

The yeast SMR1 gene was used as a dominant resistance-selectable marker for industrial yeast transformation and for targeting integration of an economically important gene at the homologous ILV2 locus. A MEL1 gene, which codes for alpha-galactosidase, was inserted into a dispensable upstream region of SMR1 in vitro; different treatments of the plasmid (pWX813) prior to transformation resulted in 3' end, 5' end and replacement integrations that exhibited distinct integrant structures. One-step replacement within a nonessential region of the host genome generated a stable integration of MEL1 devoid of bacterial plasmid DNA. Using this method, we have constructed several alpha-galactosidase positive industrial Saccharomyces strains. Our study provides a general method for stable gene transfer in most industrial Saccharomyces yeasts, including those used in the baking, brewing (ale and lager), distilling, wine and sake industries, with solely nucleotide sequences of interest. The absence of bacterial DNA in the integrant structure facilitates the commercial application of recombinant DNA technology in the food and beverage industry.

Acetolactate Synthase↗
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↗
PubMed1988

Generation of an ilv bradytrophic phenocopy in yeast by antisense RNA.

We report for the first time on the regulation of gene expression in yeast by antisense RNA. Chimaeric genes were constructed containing the 5' upstream and partial coding sequence of SMR1--a sulfometuron methyl resistant allele of the ILV2 locus. Such fragments were placed 5' to 3' and 3' to 5' under control of the GAL10 promoter and CYC1 terminator in a high copy YEp plasmid. Following galactose induction only transformants containing antisense RNA genes showed biological activity against SMR1 gene expression. Antisense RNA inhibited synthesis of the SMR1 gene product acetolactate synthase and thus repressed cellular growth which resulted in a bradytrophic auxotroph revertable by addition of isoleucine and valine. Antisense RNA inhibition was enhanced in galactose medium containing sulfometuron methyl and in gcn4 cells deficient for positive regulation of the ILV2 locus. This system can be used to study factors that interfere with antisense RNA function and to assign biological function to randomly cloned DNA fragments.

Acetolactate Synthase↗
PubMed1988

The yeast ILV2 gene is under general amino acid control.

The yeast ILV2 gene encodes acetolactate synthase, the first enzyme in the biosynthesis of isoleucine and valine. Its multiple regulation has precluded the clear demonstration of whether ILV2 is under general amino acid control. Nonderepressible gcn4 strains were used as recipients for transformation with a YCp plasmid carrying GCN4. Parental gcn4 cells and their isogenic GCN4 transformants were evaluated for ALS derepression following induced amino acid starvation. GCN4 cells showed 1.5- to 1.7-fold derepression but no derepression was observed in isogenic control gcn4 strains. A similar depression of ILV2 mRNA was also observed. Genetic evidence for general amino acid control was the gcn4 suppression of high level resistance to sulfometuron methyl by the SMRI-410 allele of ILV2.

Acetolactate Synthase↗
PubMedDate not supplied

Methods for the characterization of phosphatase-stabilized 2-5A-antisense chimeras.

We have developed chromatographic and spectrophotometric assays for determining the degree of thiolation in phosphatase-resistant 5'-monothiophosphate-capped 2-5A-antisense chimeras. Concomitantly, we have explored the reactivity of this 5'-monophosphorothioate moiety with reporter reagents such as 5-iodoacetomidofluorescein and 5,5'-dithiobis(2-nitrobenzoic acid). On the basis of these reactions, analyses for 5'-monothiophosphate-functionalized 2-5A-antisense chimeras were made possible. Kinetic experiments demonstrated that oligonucleotide backbone negative charge could retard mixed disulfide formation in the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with 5'-monothiophosphorylated 2-5A-antisense chimeras.

Adenine Nucleotides↗