Biomedical subjects

P R Butler

Publications and source records attributed to P R Butler.

13 recordsLinked to original sources

PubMed2001

Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations.

The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

Blotting, Northern↗

PubMed1999

Analysis of a continuous-culture technique for the selection of mutants tolerant to extreme environmental stress.

An analysis is presented of a continuous-culture technique named "Brown and Oliver Interactive-Continuous Selection" (BOICS; Brown and Oliver, 1982), that was devised for the selection of microbial mutants tolerant to extreme environmental stress. The case in which the stress is due to a growth inhibitor is considered. The possible steady outcomes of competition between mutants in BOICS are analyzed under the assumption that the specific growth rates of the competing mutants depend only on the inhibitor concentration. The analysis indicates that BOICS selects mutants that sustain a given specific growth rate (equal to the dilution rate in the selection experiment) at an increased concentration of the inhibitor. Simulation results support the steady-state analysis. Further simulation results show that BOICS may fail if the specific growth rate of a mutant has to be considered to depend on a factor beside the inhibitor concentration. The choice of experimental conditions that promote the success of BOICS is discussed. An interpretation of BOICS emerges from the analysis. Namely, that BOICS implements (at least approximately) a strategy for the selection of tolerant mutants in which (1) all factors in the growth environment, beside the stress of interest (inhibitor concentration) are maintained constant, and (2) the stress is adjusted as the culture evolves so that it is always maximized subject to a specified minimum value of the mean specific-growth rate being maintained. This interpretation suggests new (possibly improved) ways of implementing essentially the same selection technique. It is noted that a chemostat implements the same strategy in the case that the stress is due to the lack of a growth-rate-limiting nutrient. The outcome of selection for inhibitor tolerant mutants in chemostats, turbidostats, and in BOICS is compared. Only BOICS selects specifically for mutants tolerant to extreme concentrations of the inhibitor.

Adaptation, Physiological↗

PubMed1998

Quantitative analysis of yeast gene function using competition experiments in continuous culture.

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10-90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments is continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype.

Culture Media↗

PubMed1997

Environmental signals triggering methylenomycin production by Streptomyces coelicolor A3(2).

Methylenomycin production by Streptomyces coelicolor A3(2) may be triggered by either of two environmental signals: alanine growth-rate-limiting conditions and/or an acidic pH shock. The production of this SCP1-encoded antibiotic was studied by using batch and chemostat cultures. Batch cultures indicated a role for both nutritional status and culture pH in its regulation. Steady-state methylenomycin production and transcription of an mmy gene under alanine but not glucose growth-rate-limiting conditions was demonstrated in chemostat culture. Transient mmy expression and methylenomycin production occurred following an acidic pH shock. This stimulation of methylenomycin production occurred independently of the nutritional status of the growth environment. Antibiotic production was partially suppressed under alanine compared with glucose growth-rate-limiting conditions following the acidic pH shock. A low specific growth rate was a prerequisite for both steady-state and transient production of methylenomycin.

Acids↗

PubMed1996

Growth rate control of protein and nucleic acid content in Streptomyces coelicolor A3(2) and Escherichia coli B/r.

Escherichia coli possesses regulatory mechanisms that coordinate cell growth with the synthesis of essential macromolecules (protein, RNA and DNA). While fundamental differences have been identified in the growth habit and chromosome structure of E. coli and Streptomyces, little is known about these regulatory mechanisms in filamentous bacteria. This paper reports on the relationship between the macromolecule content of S. coelicolor A3(2) and its specific growth rate. The protein, RNA and DNA contents (g per 100 g biomass) of S. coelicolor A3(2) grown in steady-state continuous culture over a range of specific growth rates (0.025-0.3 h-1) were 31-45, 10-22 and 3.5-4.5% (w/w), respectively. This composition is qualitatively similar to that of other microorganisms. Changes in the macromolecular content of S. coelicolor A3(2) and E. coli B/r with specific growth rate appear to be essentially similar. However, the data indicate that the RNA content of S. coelicolor A3(2), grown under the conditions used, exceeds that of E. coli grown at the same specific growth rate. The data also suggest that overlapping rounds of replication are not a feature of DNA synthesis in S. coelicolor A3(2). This may be a function of the organism's low maximum specific growth rate. Alternatively, it may be a consequence of regulatory mechanisms which act to inhibit the initiation of DNA synthesis in a linear chromosome which is already undergoing replication.

Bacterial Proteins↗

PubMed1985

Use of the flexible film isolator as a single circuit hypoxic chamber for small animals.

PVC isolators are now widely used for housing animals and provide a readily available pretested air-tight chamber (Pendry, 1984; Trexler, 1984). We have adapted a flexible film isolator for use as a hypoxic chamber for small animals. The environment within the chamber can be easily and continuously monitored with indwelling probes, obviating the need for a separate circuit for gas analysis. This design has been used for long-term studies of chronic hypoxia.

Animals↗

PubMed1983

Human platelet phenol sulfotransferase: partial purification and detection of two forms of the enzyme.

To begin to study the usefulness of platelet phenol sulfotransferase (PST) as a possible measure of the enzyme activity in other organs such as the brain, we purified human platelet PST 36-120-fold. Activity toward 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine, 5-hydroxytryptamine (5-HT), and phenol eluted in the same Sephadex G-100 and Affi-Gel Blue column fractions. Specific activities of the enzyme with MHPG, dopamine, 5-HT, and phenol as substrates were 1198, 1068, 401, and 408 units/mg protein, respectively. Optimal assay conditions were established for each substrate. Apparent Km values were 598 microM, 21 microM, 19 microM, and 500 microM for MHPG, dopamine, phenol, and 5-HT, respectively. Apparent Km values for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) with the same four substrates ranged from 0.11 to 0.25 microM. The pH optima were 6.3 for phenol, 6.8 for dopamine, and 7.0 for MHPG and 5-HT. An additional pH optimum at 8.6 was present for 5-HT. A thermolabile form of the enzyme measured with dopamine and 5-HT, as well as a thermostable form measured with phenol, were present. Dichloronitrophenol (10(-5) M) noncompetitively inhibited the thermostable enzyme activity by 96% but decreased the thermolabile activity by only 36%. These studies provide the basis for a more accurate comparison of human platelet PST with the enzyme in the human brain and in other tissues.

Arylsulfotransferase↗

PubMed1974