Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.

Dynamics of Gene Expression Revealed by Comparison of Serial Analysis of Gene Expression Transcript Profiles from Yeast Grown on Two Different Carbon SourcesD⃞

We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE). Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source. A major fraction (>20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of ∼6300 yeast genes. Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters). In oleate-grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal β-oxidation enzymes required for degradation of fatty acids. The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes. We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2 and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins. Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment. We describe a statistical method for analysis of data obtained by SAGE.

Fundamental patterns underlying gene expression profiles: Simplicity from complexity

Analysis of previously published sets of DNA microarray gene expression data by singular value decomposition has uncovered underlying patterns or “characteristic modes” in their temporal profiles. These patterns contribute unequally to the structure of the expression profiles. Moreover, the essential features of a given set of expression profiles are captured using just a small number of characteristic modes. This leads to the striking conclusion that the transcriptional response of a genome is orchestrated in a few fundamental patterns of gene expression change. These patterns are both simple and robust, dominating the alterations in expression of genes throughout the genome. Moreover, the characteristic modes of gene expression change in response to environmental perturbations are similar in such distant organisms as yeast and human cells. This analysis reveals simple regularities in the seemingly complex transcriptional transitions of diverse cells to new states, and these provide insights into the operation of the underlying genetic networks.

From hematopoiesis to neuropoiesis: Evidence of overlapping genetic programs

It is reasonable to propose that gene expression profiles of purified stem cells could give clues for the molecular mechanisms of stem cell behavior. We took advantage of cDNA subtraction to identify a set of genes selectively expressed in mouse adult hematopoietic stem cells (HSC) as opposed to bone marrow (BM). Analysis of HSC-enriched genes revealed several key regulatory gene candidates, including two novel seven transmembrane (7TM) receptors. Furthermore, by using cDNA microarray techniques we found a large set of HSC-enriched genes that are expressed in mouse neurospheres (a population greatly enriched for neural progenitor cells), but not present in terminally differentiated neural cells. In situ hybridization demonstrated that many of them, including one HSC-enriched 7TM receptor, were selectively expressed in the germinal zones of fetal and adult brain, the regions harboring mouse neural stem cells. We propose that at least some of the transcripts that are selectively and commonly expressed in two or more types of stem cells define a functionally conserved group of genes evolved to participate in basic stem cell functions, including stem cell self-renewal.

Genome-wide gene expression profiles of the developing mouse hippocampus

We have analyzed the developmental molecular programs of the mouse hippocampus, a cortical structure critical for learning and memory, by means of large-scale DNA microarray techniques. Of 11,000 genes and expressed sequence tags examined, 1,926 showed dynamic changes during hippocampal development from embryonic day 16 to postnatal day 30. Gene-cluster analysis was used to group these genes into 16 distinct clusters with striking patterns that appear to correlate with major developmental hallmarks and cellular events. These include genes involved in neuronal proliferation, differentiation, and synapse formation. A complete list of the transcriptional changes has been compiled into a comprehensive gene profile database (http://BrainGenomics.Princeton.edu), which should prove valuable in advancing our understanding of the molecular and genetic programs underlying both the development and the functions of the mammalian brain.