Copper-responsive transcriptional regulation in candida albicans

The copper-containing protein superoxide dismutase is required for the virulence of C. albicans in a mouse model. Previous work in our laboratory has shown that copper uptake and regulation in C. albicans has some similarities to Saccharomyces cerevisiae, including the activation of the copper transporter gene CaCTR1 in low copper conditions by the transcription factor CaMac1p. However, further analysis in this study has demonstrated that the actual mechanism of regulation by CaMac1p is different from that of S. cerevisiae Mac1p.;This thesis demonstrates for the first time that the CaMAC1 gene is transcriptionally autoregulated in a copper-dependent manner. This is in contrast to the S. cerevisiae MAC1 homologue, which is constitutively transcribed. The presence of one binding site for CaMac1p in the promoters of CaCTR1, CaMAC1 and the ferric/cupric reductase gene CaFRE7 is sufficient for copper-responsive regulation. In contrast, two promoter elements are essential for normal levels of copper-dependent activation by S. cerevisiae Mac1p. CaMac1p is also involved in the regulation of the iron-responsive transcriptional repressor gene SFU1 and the alternative oxidase gene AOX2. This work describes key features of the copper uptake system in the human pathogen C. albicans that distinguishes it from similar processes in the model yeast S. cerevisiae. Transcriptional autoregulation of the CaMAC1 gene could enable C. albicans to respond more precisely to environmental changes, conferring an adaptation to the human host that may be an advantage in the disease process

High Throughput Method for Analysis of Repeat Number for 28 Phase Variable Loci of Campylobacter jejuni Strain NCTC11168

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method—the 28-locus-CJ11168 PV-analysis assay—for rapidly determining ON/OFF states of 28 of these phase-variable loci in a large number of individual colonies from C. jejuni strain NCTC11168. The method combines a series of multiplex PCR assays with a fragment analysis assay and automated extraction of fragment length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. Application of this method to analysis of the 28 polyG/polyC tracts in 90 C. jejuni colonies detected a 2.5-fold increase in slippage products as tracts lengthened from G8 to G11 but no difference between tracts of similar length indicating that flanking sequence does not influence slippage rates. Comparison of this observed slippage to previously measured mutation rates for G8 and G11 tracts in C. jejuni indicates that PCR amplification of a DNA sample will over-estimate phase variation frequencies by 20-35-fold. An important output of the 28-locus-CJ11168 PV-analysis assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species

LangoScholey_Test_Sequence_Files

This folder contains the test sequences used to validate the assa

ReadMe

This file is a description of the contents of the archiv

LangoScholey_Archive _Map

Picture showing a map of the files in this archiv

Comparison of two analysis methods for determining the percentage of cells in a population with a gene in an ON state.

<p>The percentage of ON variants in a population was determined by PCR-based fragment analysis of either the relative proportions of peaks obtained using a total DNA extract of the population (x-axis) or from analysis of up to 30 single colonies obtained from serial dilutions of a population (y-axis). The analysis was performed on 16 populations for 28 phase-variable loci. Each circle represents one of the 448 measurements. Black line, line for a 100% correlation between each method. Blue line, linear regression line with 95% confidence interval indicated by shaded area.</p

Multiplex PCR primer mixes.

<p>Multiplex PCR primer mixes.</p

PCR amplification and A-tail reactions.

<p>PCR amplification and A-tail reactions.</p

Output files for PSAnalyse.

<p>Output files for PSAnalyse.</p

Phase-variable genes and repeat tract specific PCR primers.

<p>Phase-variable genes and repeat tract specific PCR primers.</p