Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

Modern medicine faces the challenge of developing safer and more effective therapies to treat human diseases. Many drugs currently in use were discovered without knowledge of their underlying molecular mechanisms. Understanding their biological targets and modes of action will be essential to design improved second-generation compounds. Here, we describe the use of a genome-wide pool of tagged heterozygotes to assess the cellular effects of 78 compounds in Saccharomyces cerevisiae. Specifically, lanosterol synthase in the sterol biosynthetic pathway was identified as a target of the antianginal drug molsidomine, which may explain its cholesterol-lowering effects. Further, the rRNA processing exosome was identified as a potential target of the cell growth inhibitor 5-fluorouracil. This genome-wide screen validated previously characterized targets or helped identify potentially new modes of action for over half of the compounds tested, providing proof of this principle for analyzing the modes of action of clinically relevant compounds.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

AMP-activated protein kinase (AMPK) is an enzyme that works as a fuel gauge which becomes activated in situations of energy consumption. AMPK functions to restore cellular ATP levels by modifying diverse metabolic and cellular pathways. In the skeletal muscle, AMPK is activated during exercise and is involved in contraction-stimulated glucose transport and fatty acid oxidation. In the heart, AMPK activity increases during ischaemia and functions to sustain ATP, cardiac function and myocardial viability. In the liver, AMPK inhibits the production of glucose, cholesterol and triglycerides and stimulates fatty acid oxidation. Recent studies have shown that AMPK is involved in the mechanism of action of metformin and thiazolidinediones, and the adipocytokines leptin and adiponectin. These data, along with evidence that pharmacological activation of AMPK in vivo improves blood glucose homeostasis, cholesterol concentrations and blood pressure in insulin-resistant rodents, make this enzyme an attractive pharmacological target for the treatment of type 2 diabetes, ischaemic heart disease and other metabolic diseases.

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIBrelated factor (BRF), and B؆. Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B؆ surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Yeast RNA polymerase III (Pol III) is recruited to promoters by the transcription factor TFIIIB (13,14,46). TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF; also termed TFIIIB70), and a third subunit termed BЉ. Together, these three factors form a highly stable protein-DNA complex upstream of the transcription start site at Pol III promoters. This TFIIIB-DNA complex can promote multiple rounds of initiation by polymerase (20). In vivo, TFIIIB is recruited to promoters by the factor TFIIIC (14). In purified systems in vitro, TFIIIB can be positioned at a promoter independent of TFIIIC, provided the promoter contains a functional TATA element (17, 31); an example of such a promoter is the yeast U6 promoter.The preinitiation complexes for RNA Pol II and III and Archaea polymerase are related in several respects. All three complexes contain TBP as an essential component (41,44). In addition, both complexes contain subunits related to the TFIIB family of proteins. The Pol II factor TFIIB contains a Zn binding site at its N terminus and a C-terminal core domain (TFIIBc) containing the TBP and DNA binding activities (1, 32). The Zn binding site of TFIIB is essential for recruitment of Pol II enzyme (3,8,16). The Archaea factor TFB is 32% identical and 56% similar to human TFIIB (34, 36). TFB contains a Zn binding site at its N terminus (48), and the Cterminal core domain of TFB binds TBP-DNA similarly to TFIIB (25). The Pol III factor BRF is homologous to TFIIB and TFB over the Zn binding site and core domain and is about 25% identical and 35% similar to TFIIB and TFB (9,11,30). However, BRF also contains a 30-kDa domain at its C terminus (the C-BRF) which is conserved only among other BRFs and appears to play a major role in interaction with TBP. In two-hybrid and affinity chromatography assays, the C-terminal domain but not the N-terminal domain interacts strongly with TBP (10, 22). In addition, it has been recently shown that the C-BRF alone can form a complex with TBP, BЉ, and DNA (19). The role of the BRF N-terminal domain is not yet clear, although it does...