High-throughput yeast two-hybrid screening.

Charting the interactions among proteins is essential for understanding biological processes. While a number of complementary technologies for detecting protein interactions are available, the yeast two-hybrid system is one of the few that have been successfully scaled up. Two-hybrid screens have been used to construct extensive protein interaction maps for humans and several model organisms, and these maps have proven invaluable for studies on a variety of biological systems. These maps, however, have not come close to covering all proteins or interactions detectable by yeast two-hybrid. This is due in part to the difficulty of using library screening methods to sample all possible binary combinations of proteins. Ideally, every binary pair of proteins would be tested individually to ensure that every detectable interaction is identified. For organisms with large proteomes, however, this is not economically feasible and instead efficient pooling schemes must be implemented. The high-throughput two-hybrid screening methods presented here are designed to efficiently maximize coverage for selected sets of proteins or entire proteomes. We present two high-throughput screening protocols. Both methods are designed to identify interactors for any number of bait proteins expressed as DNA-binding domain (BD) fusions. The choice of which protocol to use depends largely on the nature of the available library of proteins fused to an activation domain (AD). The first protocol is appropriate for screening a library of AD clones, such as a cDNA library, a domain library, or a large pool of AD clones. By contrast, the second protocol is appropriate for screening a large array of individual sequence-verified AD clones. This protocol screens small pools of AD clones from the array in a two-phase scheme. Although the methods presented were developed using the LexA version of the yeast two-hybrid system, we include notes as appropriate to accommodate users of other versions.

DroID 2011: a comprehensive, integrated resource for protein, transcription factor, RNA and gene interactions for Drosophila.

DroID (http://droidb.org/), the Drosophila Interactions Database, is a comprehensive public resource for Drosophila gene and protein interactions. DroID contains genetic interactions and experimentally detected protein-protein interactions curated from the literature and from external databases, and predicted protein interactions based on experiments in other species. Protein interactions are annotated with experimental details and periodically updated confidence scores. Data in DroID is accessible through user-friendly, intuitive interfaces that allow simple or advanced searches and graphical visualization of interaction networks. DroID has been expanded to include interaction types that enable more complete analyses of the genetic networks that underlie biological processes. In addition to protein-protein and genetic interactions, the database now includes transcription factor-gene and regulatory RNA-gene interactions. In addition, DroID now has more gene expression data that can be used to search and filter interaction networks. Orthologous gene mappings of Drosophila genes to other organisms are also available to facilitate finding interactions based on gene names and identifiers for a number of common model organisms and humans. Improvements have been made to the web and graphical interfaces to help biologists gain a comprehensive view of the interaction networks relevant to the genes and systems that they study.

Rsf1p is required for an efficient metabolic shift from fermentative to glycerol-based respiratory growth in S. cerevisiae.

Previous studies from this laboratory indicated that the product of the RSF1 gene of S. cerevisiae is present in both nucleus and mitochondria, and they suggested that Rsf1p acts as a transcriptional modulator. To investigate this latter question, we performed transcriptome profiling of an rsf1 mutant strain and its wild-type parent during a shift from glucose-based fermentative to glycerol-based respiratory growth to identify genes whose expression is regulated by Rsf1p. Loss of Rsf1p engendered a decrease in transcript levels from many genes encoding components of the electron transport chain and various other mitochondrially-localized products. The earlier studies further showed that rsf1 cells exhibit a growth defect on medium containing glycerol, but not ethanol, as sole carbon source. Importantly, transcriptome profiling of the rsf1 mutant during shift from glucose- to glycerol-based medium revealed that the product of this gene plays a major role in both orchestration of the transition to, and maintenance of, efficient growth on glycerol as sole carbon source. An increase in transcript levels from genes encoding products that function in the stress response, and an imbalance between expression of genes encoding glycerol anabolic and catabolic enzymes, was observed in the rsf1 mutant during steady-state growth on glycerol- but not ethanol-based medium; this suggests the presence of partially separate transcriptional regulatory systems for transition to respiratory growth on each of these two carbon sources. Genes whose expression is affected by loss of Rsf1p, which lacks a known DNA-binding motif, lack a common DNA sequence motif in their upstream regions. These and other data presented here strongly suggest that the transcriptional effects exerted by Rsf1p are mediated via interaction with other transcription factors.

Hippocampal cellular stress responses after global brain ischemia and reperfusion.

Brain ischemia and reperfusion (I/R) induce neuronal intracellular stress responses, including the heat-shock response (HSR) and the unfolded protein response (UPR), but the roles of each in neuronal survival or death are not well understood. We assessed the relative expression of UPR (ATF4, CHOP, GRP78, XBP-1) and HSR-related (HSP70 and HSC70) mRNAs and proteins after brain I/R. We evaluated these in hippocampal CA1 and CA3 after normothermic, transient global forebrain ischemia and up to 42 h of reperfusion. In CA1, chop and xbp-1 mRNA showed maximal 14- and 12-fold increases, and the only protein increase observed was for 30-kDa XBP-1. CA3 showed induction of only xbp-1. GRP78 protein declined in CA1, but increased twofold and then declined in CA3. Transcription of hsp70 was an order of magnitude greater than that of any UPR-induced transcript in either CA1 or CA3. HSP70 translation in CA1 lagged CA3 by approximately 24 h. We conclude that (a) in terms of functional end products, the ER stress response after brain ischemia and reperfusion more closely resembles the integrated stress response than the UPR; and (b) the HSR leads to quantitatively greater mRNA production in postischemic neurons, suggesting that cytoplasmic stress predominates over ER stress in reperfused neurons.

Transcriptome profiling of Saccharomyces cerevisiae during a transition from fermentative to glycerol-based respiratory growth reveals extensive metabolic and structural remodeling.

Transcriptome analyses using a wild-type strain of Saccharomyces cerevisiae were performed to assess the overall pattern of gene expression during the transition from glucose-based fermentative to glycerol-based respiratory growth. These experiments revealed a complex suite of metabolic and structural changes associated with the adaptation process. Alterations in gene expression leading to remodeling of various membrane transport systems and the cortical actin cytoskeleton were observed. Transition to respiratory growth was accompanied by alterations in transcript patterns demonstrating not only a general stress response, as seen in earlier studies, but also the oxidative and osmotic stress responses. In some contrast to earlier studies, these experiments identified modulation of expression for many genes specifying transcription factors during the transition to glycerol-based growth. Importantly and unexpectedly, an ordered series of changes was seen in transcript levels from genes encoding components of the TFIID, SAGA (Spt-Ada-Gcn5-Acetyltransferase), and SLIK (Saga LIKe) complexes and all three RNA polymerases, suggesting a modulation of structure for the basal transcriptional machinery during adaptation to respiratory growth. In concert with data given in earlier studies, the results presented here highlight important aspects of metabolic and other adaptations to respiratory growth in yeast that are common to utilization of multiple carbon sources. Importantly, they also identify aspects specific to adaptation of this organism to growth on glycerol as sole carbon source.

The YJR127C/ZMS1 gene product is involved in glycerol-based respiratory growth of the yeast Saccharomyces cerevisiae.

A putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website. Real time RT-PCR assays showed that expression of YJR127C/ZMS1 was glucose-repressible, and a deletion mutant for the gene showed a growth defect on glycerol-based but not on glucose- or ethanol-based medium. Real time RT-PCR analyses identified severely attenuated transcript levels from GUT1 and GUT2 to be the source of that growth defect, the products of GUT1 and GUT2 are required for glycerol utilization. mRNA levels from a large group of mitochondria- and respiration-related nuclear genes also were shown to be attenuated in the deletion mutant. Importantly, transcript levels from the mitochondrial OLI1 gene, which has an associated organellar UAS, were attenuated in the DeltaYJR127C mutant during glycerol-based growth, but those from COX3 (OXI2), which lacks an associated mitochondrial UAS, were not. Transcriptome analysis of the glycerol-grown deletion mutant showed that genes in several metabolic and other categories are affected by loss of this gene product, including protein transport, signal transduction, and others. Thus, the product of YJR127C/ZMS1 is involved in transcriptional control for genes in both cellular genetic compartments, many of which specify products required for glycerol-based growth, respiration, and other functions.