Biomedical Engineering Reference
In-Depth Information
the treatment. On the contrary, in the homozygote background the overrepresented
bar-coded strains are deleted in genes able to recover, when functional, the toxic effect
of the compound. Furthermore, this assay discloses fundamental information on the
mode of action of compounds lacking a direct protein target, such as in the case of
DNA-damaging agents. A great advancement in disclosing the genetic requirements
for the resistance to DNA-damaging agents was achieved thanks to this approach [69].
Treatment with 12 known DNA-damaging compounds of the competitively grown
homozygous bar-coded collection allowed for the confirmation of the involvement
of genes known to have a role in DNA metabolism, but also the discovery of new
genes responsible for that response. Moreover, comparison of the profile of deletant
strains affected by treatment with the DNA-damaging agent disclosed a fingerprint
(“signature”) of the resistance to every given compound. Compounds with similar
biological effects are supposed to lead to similar chemical genetics profiles [70].
A great deal of effort in that direction is shown in the work of Parsons et al. [71],
endowing the scientific community with a compendium of parallel fitness profiles
obtained by treatment of the yeast haploid deletion collection with 82 chemical
perturbations. In addition to the outstanding data set, Parsons et al. also proposed a
method for the dissection of pathways and proteins affected by the chemicals tested,
based on clustering analysis and probabilistic sparse matrix factorization.
14.3.3 Comparative Expression Profiling
The transcriptional profile of a cell is the most representative portrait of cell status.
The definition of gene expression levels gives a detailed snapshot of the arrangement
of all the structures, molecular components, and reactions present in the cell at a given
moment of its life or in a given condition. When the condition studied is treatment with
a new or knownmolecule, the molecular effects affecting the complete cell system can
be observed by transcriptional analysis. The action of the molecule on the cell can be
observed both if the compound affects a specific target (observing the domino effect
induced by the perturbation of this entity on the cell organization) and in the presence
of a nonselective disturbance. Several efforts in recent years have made possible
the development of a method able to capture, analyze, and demonstrate the entire
expression profile of a cell. To that end, DNA microarray technology represented
a pivotal step forward [72]. The laser scanning of labeled mRNA hybridized to
spotted cDNA gives a quantitative measurement of the expression level of every
annotated ORF of genomically sequenced organisms. In
S. cerevisiae
, microarray
approaches have been used widely to measure gene expression upon cell exposure to
a variety of conditions and stresses [73-76]. A number of studies analyzed the yeast
expression patterns of several different physiological conditions using differently
designed arrays based on probes composed by either DNA fragments [72,77] or
oligonucleotides [78,79].
cDNA microarrays have been used to study yeast gene expression during fun-
damental events such as growth [80], sporulation [81], and in the cell cycle [82].
A brilliant example of the application of transcriptional analysis to the dissection
of the drug mode of action is represented by a study of Hardwick et al. [31].
