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Biomedical subjects

S G Oliver

Publications and source records attributed to S G Oliver.

At least 73 records · Page 4Linked to original sources

Functional evidence that the principal DNA replication origin of the Streptomyces coelicolor chromosome is close to the dnaA-gyrB region.

The degree of overrepresentation of selected chromosomal genes in rapidly growing cultures of Streptomyces coelicolor was assessed by quantitative DNA hybridization analysis. The results are consistent with the hypothesis that the principal origin of replication is close to the dnaA-gyrB region, in the center of the linear chromosome, and that replication proceeds bidirectionally.

Chromosome Mapping↗

Periodic selection in longterm continuous-flow cultures of the filamentous fungus Fusarium graminearum.

By monitoring increases and decreases in the proportion of cycloheximide-resistant macroconidia, periodic selection was observed in populations of the filamentous fungus Fusarium graminearum, grown in glucose-limited chemostat cultures. The results indicated that periodic selection of advantageous mutants of F. graminearum occurred at intervals of about 124 h at both high (D = 0.19 h-1, approximately 34 generations) and low (D = 0.06 h-1, approximately 11 generations) dilution rates. Several 'adaptive' peaks (each indicating the appearance of an advantageous mutation) were observed before morphological (highly branched) mutants appeared in the populations; these mutants have previously been observed to have a selective advantage over the parental strain. At intervals, macroconidia harvested from the chemostat were used to inoculate plates of non-antibiotic-containing agar medium, and it was possible to monitor periodic selection in the original chemostat culture using second generation macroconidia harvested from these cultures. The proportion of cycloheximide-, potassium chlorate-, and p-fluoro-DL-phenylalanine-resistant macroconidia in these second generation macroconidia changed in a pattern similar to that observed when monitoring the proportion of cycloheximide-resistant macroconidia in the first generation population harvested directly from the chemostat. The experiments demonstrated that populations of filamentous fungi are heterogeneous and that much of this heterogeneity may already be present at the end of batch growth, i.e., before the onset of continuous cultivation.

Cycloheximide↗

The complete DNA sequence of yeast chromosome III.

The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

Base Sequence↗

Studies on plasmid stability, cell metabolism and superoxide dismutase production by Pgk- strains of Saccharomyces cerevisiae.

A double mutant sod1/pgk1 strain of Saccharomyces cerevisiae has been constructed in order to investigate the effects of different environmental conditions on yeast physiology, plasmid stability, and superoxide dismutase (SOD) production. Strains were transformed with yeast episomal plasmids (YEp) containing both PGK1 and SOD1 genes and were grown on fermentable carbon sources and under vigorous aeration. Under these conditions, the presence of the PGK1 gene was made essential for growth and both genes were efficiently expressed. However, plasmid-borne PGK1 was found not to increase the stability of YEp vectors in batch cultures of Pgk- cells. Paradoxically, plasmid stability increased during the respiratory phase of growth. An investigation of the metabolism of Pgk- cells demonstrated that these glycolytic pathway mutants do not appreciably metabolize glycerol. Thus Pgk+, plasmid-containing, cells have a selective advantage during the respiratory phase of batch growth since they can utilize both glycerol and ethanol.

Clone Cells↗

Complementation of the Saccharomyces cerevisiae srb1-1 mutation: an autoselection system for stable plasmid maintenance.

The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain. When transformed into a fragile srb1-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used. Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol). However, in the absence of the osmoticum, nearly 100% of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth.

Cell Division↗

Isolation and characterization of temperature-sensitive mutants of Streptomyces coelicolor A3(2) blocked in macromolecular synthesis.

A collection of temperature-sensitive mutants of Streptomyces coelicolor A3(2) was isolated. The majority of the mutants showed an osmotically remedial phenotype. Mutants defective in macromolecular synthesis were identified and characterized further. Four mutants were found in which DNA replication was defective, but which continued to synthesize RNA and protein at the restrictive temperature (39 degrees C). The kinetics of cessation of DNA synthesis allowed a tentative identification of slow (initiation) and fast (elongation) stop dna mutants. The inhibition of DNA replication in the four mutants was found to be reversible on returning to the permissive temperature (30 degrees C), but only after a delay of about 2 h. Three other mutants were identified which showed not only cessation of DNA replication at the restrictive temperature, but also defects in other macromolecular synthesis events.

Cell Division↗

An integrated approach to studying regulation of production of the antibiotic methylenomycin by Streptomyces coelicolor A3(2).

A physiological and molecular biological study was made of the control of methylenomycin biosynthesis by Streptomyces coelicolor A3(2). A simple and reliable assay for this antibiotic was developed. Conditions that permit the synthesis of methylenomycin by S. coelicolor cultures grown in defined medium were elucidated: a readily assimilated carbon and nitrogen source is required. Under these conditions methylenomycin is produced late in the growth phase, at the time of transition from exponential to linear growth. Provided that the phosphate concentration in the medium is kept high, there is synthesis of methylenomycin but not of the other secondary metabolites that this strain can produce. These conditions were used to study the transcription of the methylenomycin gene cluster during the transition from primary to secondary metabolism. The biosynthetic genes of at least one of the mmy transcription units appear to be transcribed before the mmr resistance determinant. The possibility that methylenomycin induces the transcription of mmr is discussed.

Acetates↗

Heritable damage to yeast caused by transformation.

The introduction of plasmid DNA into yeast by transformation or electroporation, but not by cytoduction, results in the induction of a slow growth phenotype. This phenotype is inherited as a dominant Mendelian trait, which is only exhibited in the absence of the native 2 mu nuclear DNA plasmid of yeast. The use of recombinant DNA technology in yeast, therefore, does not necessarily manipulate the genome in a precise and completely defined way.

DNA↗

Genetic analysis of a meiotic recombination hotspot on chromosome III of Saccharomyces cerevisiae.

In a previous study, we analyzed meiotic recombination events that occurred in the 22-kb region (LEU2 to CEN3) of chromosome III of Saccharomyces cerevisiae. We found one region with an enhanced level of crossovers (a hotspot) and one region with a depressed level of crossovers. In this study, we show that about one-third of the crossovers that occur between LEU2 and CEN3 are initiated in a 1.3-kb region located approximately 6 kb from the centromere. Both crossovers and gene conversion events are initiated at this site. Events initiated at this position can be resolved as crossovers in regions located either centromere-distally or centromere-proximally from the initiation site.

Base Sequence↗

The trpC gene of Phanerochaete chrysosporium is unique in containing an intron but nevertheless maintains the order of functional domains seen in other fungi.

The Phanerochaete chryososporium trpC gene has been isolated by complementation of an Escherichia coli trpC mutant. The full extent of the fungal gene, determined by sequence analysis, was found to be 2414bp. This includes a single intron of 50bp, the presence of which was confirmed by RNA-primed polymerase chain reaction analysis. This features makes the P. chrysosporium gene unique when compared to equivalent genes from other filamentous fungi. The P. chrysosporium trpC gene encodes a single protein containing three enzyme activities involved in tryptophan biosynthesis arranged in the order: NH2-GAT-IGPS-PRAI-COOH. This order is conserved in all filamentous fungi so far examined and, indeed, is the gene order within the E. coli trp operon.

Amino Acid Sequence↗

Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae.

The srb1-1 mutation of Saccharomyces cerevisiae is an ochre allele which renders the yeast dependent on an osmotic stabilizer for growth and gives the cells the ability to lyse on transfer to hypotonic conditions. A DNA fragment which complements both of these phenotypic effects has been cloned. This clone contains a functional gene which is transcribed into a 2.3-kb polyadenylated mRNA molecule. Transformation of yeast strains carrying defined suppressible alleles demonstrated that the cloned fragment does not contain a nonsense suppressor. Integrative transformation and gene disruption experiments, when combined with classical genetic analysis, confirmed that the cloned fragment contained the wild-type SRB1 gene. The integrated marker was used to map SRB1 to chromosome XV by Southern hybridization and pulsed-field gel electrophoresis. A disruption mutant created by the insertion of a TRP1 marker into SRB1 displayed only the lysis ability phenotype and was not dependent on an osmotic stabilizer for growth. Lysis ability was acquired by growth in (or transfer to) an osmotically stabilized environment, but only under conditions which permitted budding. It is inferred that budding cells lyse with a higher probability and that weak points in the wall at the site of budding are involved in the process. The biotechnological potential of the cloned gene and the disruption mutant is discussed.

Chromosomes, Fungal↗

Functional expression of the yeast Mn-superoxide dismutase gene in Escherichia coli requires deletion of the signal peptide sequence.

Oligodeoxyribonucleotide-directed mutagenesis has been used to delete the leader sequence-coding region from the yeast gene specifying Mn-superoxide dismutase and thus enable its expression in Escherichia coli. The deletion of this leader was demonstrated to be an absolute requirement for the synthesis of an active yeast enzyme in the bacterium. Complementation analysis in E. coli has confirmed that the product of the truncated yeast gene is active in vivo as well as in vitro.

Amino Acid Sequence↗

Replication and recombination in gene establishment in non-Saccharomyces yeasts.

A brief review is given on the establishment of recombinant DNA technology for non-conventional yeasts. The availability of DNA delivery systems, selectable markers for identification of transformants, and the means of replicating and amplifying the recombinant DNA are discussed. Some of the existing transformation systems among non-conventional yeasts are explained.

DNA Replication↗

A downstream activator sequence regulates the expression of the yeast transposon.

The regulation of transcription of the class II yeast transposon Ty 1-17 has been examined using a Ty::LEU2 fusion gene. Deletion analysis has defined a 251 bp region within the coding sequence of the TY A gene which is essential for Ty transcription and is also required for diploid control of Ty expression. This downstream activator sequence (DAS) contains two sequence blocks which show homology to the SV40 enhancer core and three with homology to the diploid-regulation-site from the yeast MAT gene.

Amino Acid Sequence↗

Polymorphisms on the right arm of yeast chromosome III associated with Ty transposition and recombination events.

The region of Saccharomyces cerevisiae chromosome III centromere-distal to the PGK gene is the site of frequent chromosome polymorphisms. We have sequenced this region from fragments of chromosome III isolated from three different yeast strains, GRF88, CN31C and CF4-16B. The sequence analysis demonstrates that these polymorphisms are associated with the presence of Ty and delta elements and defines a region of the chromosome which is a hot-spot for transposition events (the RAHS). The three strains can be arranged into a logical evolutionary series in which successive transposition and recombination events insert Ty elements and fuse them with consequent deletions of chromosome and of transposon sequences. The influence of such events on yeast genome evolution is discussed.

Base Sequence↗

DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence.

Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced. Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence. A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus. Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity.

Base Sequence↗

A 'hot-spot' for Ty transposition on the left arm of yeast chromosome III.

The small ring derivative of Saccharomyces cerevisiae chromosome III, which was formed by a cross-over between HML on the left arm and HMR on the right arm, contains three Ty elements. The class II element Ty 1-17 lies immediately centromere-distal to LEU2 on the left arm while two class I elements are tandemly arranged distal to PGK on the right arm. We have sequenced the regions of chromosome III surrounding Ty 1-17 and have defined a region where a number of transposition events have occurred. This region is flanked by the 5' ends of two tRNA genes, tRNA3Glu on the centromere distal side and tRNA3Leu immediately in front of LEU2. Close to the tRNA3Glu gene there is a region containing degenerate delta sequences organised in opposite orientations. Immediately distal to Ty 1-17 there are two complete solo delta elements, one inserted into the other. The sequence indicates that these two delta sequences were inserted into chromosome II by separate transposition events. A model is presented to explain how this structure arose and the role of solo delta elements in transposon propagation and maintenance is discussed.

Base Sequence↗