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A broad range of genomics and proteomics technologies are increasingly being integrated into emerging research fields such as pharmacogenomics, pharmacoproteomics, chemogenomics, chemical genetics, and chemical biology. Here we review applications of genomic and proteomic technologies to drug mechanism-of-action studies and how these are beginning to impact the drug discovery process.

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:

Clustering large data sets is a central challenge in gene expression analysis. The hybridization of synthetic oligonucleotides to arrayed cDNAs yields a fingerprint for each cDNA clone. Cluster analysis of these fingerprints can identify clones corresponding to the same gene. We have developed a novel algorithm for cluster analysis that is based on graph theoretic techniques. Unlike other methods, it does not assume that the clusters are hierarchically structured and does not require prior knowledge on the number of clusters. In tests with simulated libraries the algorithm outperformed the Greedy method and demonstrated high speed and robustness to high error rate. Good solution quality was also obtained in a blind test on real cDNA fingerprints., (Copyright 2000 Academic Press.)

The genomics era is providing us with vast amounts of information derived from whole-genome sequencing. This will doubtlessly revolutionise biology and the way novel medicines will be discovered. To leverage this information efficiently, however, technologies in addition to high-throughput sequencing are required. DNA microarray technology is one technology that has already shown great potential for both basic research and drug discovery. With particular emphasis on antibacterial research we will summarise in this review the key technological aspects and most important applications of DNA microarrays demonstrated so far.

We describe the use of oligonucleotide fingerprinting for the generation of a normalized cDNA library from unfertilized sea urchin eggs and report the preliminary analysis of this library, which resulted in the establishment of a partial gene catalogue of the sea urchin egg. In an analysis of 21,925 cDNA clones by hybridization with 217 oligonucleotide probes, we were able to identify 6291 clusters corresponding to different transcripts, ranging in size from 1 to 265 clones. This corresponds to an average 3.5-fold normalization of the starting library. The normalized library represents about one-third of all genes expressed in the sea urchin egg. To generate sequence information for the transcripts represented by the clusters, representative clones selected from 711 clusters were sequenced. The construction and preliminary analysis of the normalized library are the first steps in the assembly of an increasingly complete collection of maternal genes expressed in the sea urchin egg, which will provide a number of insights into the early development of this well-characterized model organism., (Copyright 1999 Academic Press.)

Arabidopsis thaliana is a small flowering plant that serves as the major model system in plant molecular genetics. The efforts of many scientists have produced genetic maps that provide extensive coverage of the genome (http://genome-www. stanford.edu/Arabidopsis/maps.html). Recently, detailed YAC, BAC, P1 and cosmid-based physical maps (that is, representations of genomic regions as sets of overlapping clones of corresponding libraries) have been established that extend over wide genomic areas ranging from several hundreds of kilobases to entire chromosomes. These maps provide an entry to gain deeper insight into the A. thaliana genome structure. A. thaliana has been chosen as the subject of the first large-scale project intended to determine the full genome sequence of a plant. This sequencing project, together with the increasing interest in map-based gene cloning, has highlighted the requirement for a complete and accurate physical map of this plant species. To supply the scientific community with a high-quality resource, we present here a complete physical map of A. thaliana using essentially the IGF BAC library. The map consists of 27 contigs that cover the entire genome, except for the presumptive centromeric regions, nucleolar organization regions (NOR) and telomeric areas. This is the first reported map of a complex organism based entirely on BAC clones and it represents the most homogeneous and complete physical map established to date for any plant genome. Furthermore, the analysis performed here serves as a model for an efficient physical mapping procedure using BAC clones that can be applied to other complex genomes.

In order to generate a physical map of the Arabidopsis thaliana genome based on bacterial artificial chromosome clones (BACs), an iterative high throughput hybridisation strategy was applied and its efficiency was evaluated. Thus, probes generated from both ends of 500 BAC clones selected from the Arabidopsis-IGF-BAC library were hybridised to the entire library gridded on high density filters. The 1000 hybridisation reactions identified 4496 clones (41.8% of the complete library, or 50.3% if organellar, centromeric, and ribosomal DNA carrying clones are excluded) which were assembled into a minimum of 220 contigs. These results demonstrate the viability of the applied 'double-end clone-limited/sampling without replacement' hybridisation strategy for the generation of a high resolution physical map, and provide a highly useful resource for map-based gene cloning approaches and further genome analysis.

CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be involved in other human genetic diseases. To identify new candidate genes, we have undertaken a large-scale screening project for CAG/CTG ([CAG]n) and CGG/GCC ([CGG]n) repeats in human brain reference cDNAs. Here, we present the final classification for 597 cDNAs selected by CAG and CGG hybridization from two libraries (100,128 clones) and the updated characterization of [CAG]n- and [CGG]n-positive cDNAs (repeat polymorphism and cDNA localization). We have selected 124 CAG and 83 CGG hybridization-positive clones representing new genes, from which 49 CAG and 7 CGG repeats could be identified. New [CAG]n and [CGG]n with more than seven to nine units were rare (1/2000), and perfect [CAG]n 9 were more likely polymorphic. Overall, highly polymorphic to monomorphic new [CAG]n > 9 and [CGG]n > 7 were characterized. The comparison of our data with other [CAG]n and [CGG]n resources suggests that the screening of reference cDNAs leads to unique sources of new [CAG]n and [CGG]n and will enhance the study of enlarged triplet repeats in human genetic diseases., (Copyright 1998 Academic Press.)

The imprinted mouse insulin-like growth factor type 2 receptor (Igf2r) maps to the middle of a gene-rich region in band A2 of mouse chromosome 17. The t(Lub2) chromosome 17 variant contains a small deletion that removes at least seven genes including Igf2r. We have constructed a YAC contig spanning the entire t(Lub2) deletion and created a restriction map that covers 700 kb. The position, transcription orientation, and imprinted status of the genes immediately flanking Igf2r have been assessed. We show here that the Mas gene, which lies 65 kb upstream to Igf2r, contains a novel 5' exon and is not imprinted in adult tissues. We further show that the recently identified Lx1 gene lies immediately downstream and is also expressed from both parental alleles in adult tissues. The remaining genes in this region have previously been shown to be biallelically expressed.

Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurodegenerative or neuromuscular diseases and may be involved in several other genetic disorders of the central nervous system. To identify new candidate genes, we have undertaken a large-scale screening project for CAG and CTG repeats in human reference cDNAs. We screened 100 128 brain cDNAs by hybridization. We also scanned GenBank expressed sequence tags for the presence of long CAG/CTG repeats in the extremities of cDNAs from several human tissues. Of the selected clones, 286 were found to represent new genes, and 72 have thus far been shown to contain CAG/CTG repeats. Our data indicate that CAG/CTG repeated 10 or more times are more likely to be polymorphic, and that new 3'-directed cDNAs with such repeats are very rare (1/2862). Nine new cDNAs containing polymorphic (observed heterozygote frequency: 0.05-0.90) CAG/CTG repeats have been currently identified in cDNAs. All of the cDNAs have been assigned to chromosomes, and six of them could be mapped with YACs to 1q32-q41, 3p14, 4q28, 3p21 and 12q13.3, 13q13.1-q13.2, and 19q13.43. Three of these clones are highly polymorphic and represent the most likely candidate genes for inherited neurodegenerative diseases and, perhaps, neuropsychiatric disorders of multifactorial origin.

To test the effectiveness of ordering shotgun DNA-templates prior to sequence analysis, the 450 kb left arm of yeast chromosome XII was randomly subcloned into a phagemid vector. Clones were ordered by hybridisation to an average map density of one new insert every 125 bp and are currently used for sequencing the chromosomal fragment. An 11.5 kb overlap between the template map and a DNA fragment that had been sequenced earlier allowed an independent evaluation of the strategy's effectiveness. To this end, clones were selected from the map and tag-sequenced from either end, thus comparing the map position with the actual location within the 11.5 kb. Of 65 selected clones, taken mostly at random from a total of 423, 58 mapped on average about a quarter of a clone length around their predicted position, with the other seven being between 0.6 and 1.5 clone length off. 75-86 sequencing reactions on clones selected from the map would have been sufficient for completely sequencing both strands of the 11.5 kb fragment. The results demonstrate the efficacy of such template sorting, considerably assisting sequencing at relatively little cost on the mapping level.

Sequence tagged sites (STSs) that were generated via Alu-element-mediated polymerase chain reaction (Alu-PCR) and mapped to human Xq26 were used to isolate and overlap yeast artificial chromosomes (YACs). By collating the results of primary pool screening, the order of STSs and YACs was postulated directly. Subsequent isolation of 11 key YACs from 75 positive pools confirmed the proposed contig. Although only a small subset of the available Alu-PCR fragments was used, the STSs were generated at sufficient density to isolate all the YACs required and to identify all except one overlap directly. The results confirmed physical linkage of HPRT to DXS86 and DXS144E. Long-range continuity was determined purely by analysis of the 11 YAC colonies and required no end-rescue. This strategy is therefore an effective approach for the construction of YAC contigs spanning discrete chromosomal regions contained within somatic cell hybrids, with minimal prior knowledge of the region.

We describe the isolation and characterization of yeast artificial chromosome (YAC) clones spanning the male sex determining region on the short arm of the human Y chromosome. The clones were isolated by hybridizing probes in the interval between the genes MIC2 and ZFY to a Y chromosome-enriched YAC library. The YAC clones were consistent with the order of probes established for this interval and may be useful for functional studies of the region in male sex determination. However, many of the YAC clones from this library carried only one arm of the vector ("half-YACs"), deleted sequences from one end, and contained much smaller inserts (148 kb average) than the size of ligated fragments selected by pulsed-field gel electrophoresis (greater than 440 kb). These problems were overcome by protecting DNA with polyamines during YAC library construction and a second Y-enriched YAC library was constructed with an average insert size of 627 kb.