Replication dynamics of the yeast genome.

Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

Regional and strain-specific gene expression mapping in the adult mouse brain.

To determine the genetic causes and molecular mechanisms responsible for neurobehavioral differences in mice, we used highly parallel gene expression profiling to detect genes that are differentially expressed between the 129SvEv and C57BL/6 mouse strains at baseline and in response to seizure. In addition, we identified genes that are differentially expressed in specific brain regions. We found that approximately 1% of expressed genes are differentially expressed between strains in at least one region of the brain and that the gene expression response to seizure is significantly different between the two inbred strains. The results lead to the identification of differences in gene expression that may account for distinct phenotypes in inbred strains and the unique functions of specific brain regions.

Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation.

The unfolded protein response (UPR) regulates gene expression in response to stress in the endoplasmic reticulum (ER). We determined the transcriptional scope of the UPR using DNA microarrays. Rather than regulating only ER-resident chaperones and phospholipid biosynthesis, as anticipated from earlier work, the UPR affects multiple ER and secretory pathway functions. Studies of UPR targets engaged in ER-associated protein degradation (ERAD) reveal an intimate coordination between these responses: efficient ERAD requires an intact UPR, and UPR induction increases ERAD capacity. Conversely, loss of ERAD leads to constitutive UPR induction. Finally, simultaneous loss of ERAD and the UPR greatly decreases cell viability. Thus, the UPR and ERAD are dynamic responses required for the coordinated disposal of misfolded proteins even in the absence of acute stress.

A genome-wide transcriptional analysis of the mitotic cell cycle.

Progression through the eukaryotic cell cycle is known to be both regulated and accompanied by periodic fluctuation in the expression levels of numerous genes. We report here the genome-wide characterization of mRNA transcript levels during the cell cycle of the budding yeast S. cerevisiae. Cell cycle-dependent periodicity was found for 416 of the 6220 monitored transcripts. More than 25% of the 416 genes were found directly adjacent to other genes in the genome that displayed induction in the same cell cycle phase, suggesting a mechanism for local chromosomal organization in global mRNA regulation. More than 60% of the characterized genes that displayed mRNA fluctuation have already been implicated in cell cycle period-specific biological roles. Because more than 20% of human proteins display significant homology to yeast proteins, these results also link a range of human genes to cell cycle period-specific biological functions.

Direct allelic variation scanning of the yeast genome.

As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).

Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors.

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity and selectivity was determined by analysis of a three-dimensional crystal structure of a CDK2-inhibitor complex. The cellular effects of these compounds were characterized in mammalian cells and yeast. In the latter case the effects were characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays. Purine libraries could provide useful tools for analyzing a variety of signaling and regulatory pathways and may lead to the development of new therapeutics.

Parallel analysis of genetic selections using whole genome oligonucleotide arrays.

Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein-protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.

Genome-wide expression monitoring in Saccharomyces cerevisiae.

The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes. This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry. The measurements are quantitative, sensitive, specific, and reproducible. Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media. Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell. Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.

cDNA Sequences Reveal mRNAs for Two Gα Signal Transducing Proteins from Larval Cilia.

In planktonic larvae of the gastropod mollusk, Haliotis rufescens (red abalone), settlement behavior and subsequent metamorphosis are controlled by two convergent chemosensory pathways that report unique peptide and amino acid signals from the environment. The integration of signals from these two sensory pathways provides for variable amplification, or fine-tuning, of larval responsiveness to the inducers of settlement and metamorphosis. These pathways may be analogous to the neuronal and molecular mechanisms of facilitation and longterm potentiation characterized in other (adult) molluscan systems. Recently, the chemosensory receptors and signal transducers apparently belonging to the regulatory pathway (including a G protein and protein kinase C) have been identified in cilia purified from H. rufescens larvae. These elements retain their sequential receptor-dependent regulation in the isolated cilia in vitro. As a first step toward the molecular genetic dissection of the receptors, transducers, and the mechanisms of their control of settlement behavior and metamorphosis, we present evidence that the cilia purified from these larvae contain polyadenylated mRNA corresponding to unique signal transducers. Purification of this mRNA, enzymatic synthesis of the corresponding cDNAs, amplification by the polymerase chain reaction, cloning, and sequence analysis reveal that the ciliary mRNA includes sequences that apparently code for two G{alpha} signal transducing proteins. One of these is highly homologous to members of the Gq family, recently shown in other systems to control the activity of phospholipase C; the other is more closely related to Gi and Go. These results extend the tractability of the Haliotis system to analyses of cDNA and protein sequences of chemosensory elements from isolated cilia. This is the first time that mRNA has been purified from isolated cilia, and the corresponding cDNA synthesized and characterized.