Browsing by Subject "Saccharomyces cerevisiae"

The cell division cycle is the process in which the entirety of a cell's contents is duplicated completely and then equally segregated into two identical daughter cells. The order of the steps in the cell cycle must be followed with fidelity to guarantee two viable cells. Understanding the regulatory mechanisms that control cell-cycle events remains to be a fundamental question in cell biology. In this dissertation, I explore the mechanisms that coordinate and regulate cell-cycle progression in the budding yeast, Saccharomyces cerevisiae.

Cell-cycle events have been shown to be triggered by oscillations in the activity of cyclin dependent kinases (CDKs) when bound to cyclins. However, several studies have shown that some cell-cycle events, such as periodic transcription, can continue in the absence of CDK activity. How are periodic transcription and other cell-cycle events coupled to each other during a wild-type cell cycle? Currently, two models of cell-cycle regulation have been proposed. One model hypothesizes that oscillations in CDK activity controls the timing of cell-cycle events, including periodic transcription. The second model proposes that a transcription factor (TF) network oscillator controls the timing of cell-cycle events, via proper timing of gene expression, including cyclins. By measuring global gene expression dynamics in cells with persistent CDK activity, I show that periodic transcription continues. This result fits with the second model of cell-cycle regulation. Further, I show that during a wild-type cell cycle, checkpoints are responsible for arresting the bulk of periodic transcription. This finding adds a new layer of regulation to the second model, providing a mechanism that coordinates cell-cycle events with a TF network oscillator. Taken together, these data provide further insight into the regulation of the cell cycle.

Hydrogen peroxide is used by animals and plants to deter the growth of microbial invaders by inflicting DNA lesions, protein oxidation and lipid membrane modifications. Pathogens protect themselves with enzymes and scavenging proteins. This study investigated population genetic, biochemical and genetic aspects of peroxide survival in Saccharomyces cerevisiae to address its importance for yeast biology and fungal pathogenicity.

Population genetic analyses of DNA sequences from five loci from 103 strains encompassing the known ecological spectrum of S. cerevisiae show that it is a recombining species with three divergent subgroups, which are associated with soil, fruit, and vineyards. Clinical isolates cluster with fruit isolates but are significantly more resistant to peroxide. Clinical isolates are genetically diverse, indicating multiple origins of the pathogenic lifestyle and eliminating the possibility that peroxide resistance is due to shared ancestry rather than it's importance for than its importance in colonizing the host.

Biochemical aspects of peroxide survival were studied in a resistant (high-survival) clinical isolate, a sensitive (low-survival) laboratory strain and their hybrid. Catalase activity and expression levels are indistinguishable among strains. Co-culture assays and growth curve records indicate that a secreted factor improves survival of the laboratory strain and that the phenotypic difference is most pronounced during exponential growth, excluding mechanisms of the General Stress Response effective during stationary phase. Semi-quantitative expression profiles of stress response candidate genes do not differ, suggesting a novel resistance mechanism.

To elucidate the genetic basis of peroxide survival, the hybrid was sporulated and 200 F1 segregants phenotyped and genotyped for oxidative stress candidate genes. Peroxide survival is a dominant quantitative trait and not linked to catalase, peroxidase or superoxide dismutase genes. 1,246 backcross segregants were phenotyped and 93 segregants selectively genotyped using microarrays. A 14-gene locus on chromosome XVI displayed marker-trait association. One gene, RDS2, encodes a zinc cluster protein acting as a regulator of drug sensitivity and contains a non-synonymous polymorphism whose exchange between the parental strains results a 15% decrease in survival in the clinical strain.

This work establishes a novel function for RDS2 in oxidative stress response and demonstrates the effect a quantitative trait nucleotide has on a clinically relevant phenotype.

Biological systems are highly dynamic, yet our temporal resolution of such

dynamical processes is often limited or difficult to test in the laboratory. The 24-hour

circadian rhythm and the approximately 75-minute cell cycle of a budding yeast cell are

both examples of dynamical processes that contain precisely ordered events, repeating

over each cycle. Organisms utilize such biological clock processes to time a particular

function. Dynamic cellular events are ordered, in part, by coordinated programs of

periodic gene expression. Up to 40% of all mouse genes are periodically expressed with

respect to the circadian cycle, and almost 20% of all yeast genes are periodic during the

cell cycle. Furthermore, more than half of the most frequently prescribed drugs in human

patients target an effector whose expression is under circadian control. Given the large

proportion of genes that are periodically expressed across different biological processes,

it is critically important to understand mechanisms that regulate dynamics in biology.

In this dissertation, I focus on two biological processes that are dynamic and are

not yet fully understood: the eukaryotic cell cycle and malaria parasite development.

Large programs of periodic genes emerge when these biological clock processes are

synchronized and profiled over time. Gene regulatory networks composed of

transcription factors, kinases, and other transcriptional regulators play a critical role in

generating periodicity in gene expression programs, ordering clock events, and

maintaining oscillations in subsequent cycles.

Many previous studies have profiled gene expression during the cell cycle in the

budding yeast Saccharomyces cerevisiae. I have added to this detailed body of work by

demonstrating that regulatory motifs involving negative feedback are required to

maintain normal gene expression levels. Additionally, I showed that many periodic

mRNAs are also periodically abundant at the protein level during the cell cycle. Both

projects provide evidence for the hypothesis that cell-cycle dynamics are driven by a

network of transcription factors with complex protein dynamics and with negative

feedback motifs. Using this ground truth cell-cycle network in S. cerevisiae, I next

performed a comparative transcriptomics study on cell-cycle genes in the less studied,

but more human health relevant fungal pathogen, Cryptococcus neoformans. This work

not only begins to identify a cell-cycle network in C. neoformans but also has

implications for future antifungal drug development, as some genes that are important

for fungal virulence were found to be expressed periodically during the cell cycle.

During infection, the human malaria parasite Plasmodium falciparum cyclically

develops and re-infects red blood cells. Many groups have shown that a very large

program of gene expression occurs during this red blood cell developmental cycle. In

this dissertation, I deploy the experimental and analysis tools that I used to characterize

the fungal cell cycle to ask if a network of transcription factors can explain

developmental gene expression dynamics and cycle period control in malaria.

Biological systems are highly dynamic to respond to environmental signals, grow,

and survive. As the application of genetics and genomics has moved toward

characterizing complex diseases, host-pathogen interactions, or even the cell cycle of a

single yeast cell, it has become increasingly clear that networks of interacting genes are

required to explain biological mechanisms. Results from this dissertation where I

investigate dynamic gene regulatory networks are broadly applicable to our

understanding of both basic molecular biology and of human infectious diseases.