Biomedical Engineering Reference
In-Depth Information
deletion) perturbations can be compared to identify the primary target of the stimu-
lus.
S. cerevisiae
has become a model organism to study how eukaryotic cells respond
to stress and define the specific role of stress-induced proteins [25-27]. Moreover,
the high degree of evolutionary conservation of the stress pathways between yeast
and higher eukaryotes indicates that yeasts are a valuable model system for char-
acterization of the stress response in more complex organisms. When challenged
with different stressors, such as heat, ethanol, metal ions, high osmolarity, and oxi-
dants, yeast cells display common molecular mechanisms of defense. The common
events have been identified as the general stress response. Different stressors can
induce reactive oxygen species production that triggers the oxidative stress response,
leading to the acquisition of resistance to subsequent oxidative stress. The connec-
tion between the response elicited by different stress agents and the oxidative stress
response is given primarily by common induction of the transcription of
CTT1
gene
encoding the cytosolic catalase. Indeed, many stress-related genes harbor on their
promoter a STRE (stress response element) sequence. By means of DNA microar-
rays, Gasch et al. [25] explored the genomic expression patterns in
S. cerevisiae
responding to diverse environmental changes. They studied the processes by which
yeast cells respond to temperature, hyper- and hypo-osmotic and hydrogen peroxide
shocks, superoxide-generating drugs (i.e., menadione), sulfhydryl-oxidizing agents
(i.e., diamide), disulfide-reducing agents (i.e., dithiothreitol), amino acid starvation,
nitrogen source depletion, and progression into a stationary phase. This approach
makes it possible to identify a large set of genes sharing similar drastic responses
to almost all these perturbations and to recognize later confirmed involvement of
the transcription factors Yap1p [28], as well as Msn2p [29] and Msn4p [30], in the
response induction. The use of DNAmicroarrays, combined with yeast mutants resis-
tant to a certain drug, or yeasts carrying deletions in genes important for the response
to a given drug, allows not only for an understanding of the mechanisms of drug
action, but also for elucidating the mechanisms of resistance and adaptation to the
toxic agent. Assays of the expression response in yeast cells to treatment with the
immunosuppressant rapamycin [31] showed significant similarity in the metabolic
response profile to the diauxic shift [32].
14.2 CHEMICAL GENETICS AND
S. cerevisiae
Since its beginning concurrent with Mendal seminal studies, classical genetics
has played a central role in the dissection of gene and protein functions. Genetic
approaches can be divided into two main branches: forward and reverse genetics
(Figure 14.3). In forward genetics, random mutations are induced in the genome of
a model organism; then a desirable phenotype is selected (i.e., growth rate decrease,
morphology or phenotype alterations), and the mutation inducing such a phenotype
is characterized (Figure 14.3a) [33]. As suggested by its name, reverse genetics acts
in the opposite direction: the role of a gene in the cell is investigated by studying
the phenotypic effects of its specific mutation (Figure 14.3b) [34]. The principal
problem arising from the use of genetic approaches is that the mutations in essential
