Exploring the new world of the genome with DNA microarrays.

Thousands of genes are being discovered for the first time by sequencing the genomes of model organisms, an exhilarating reminder that much of the natural world remains to be explored at the molecular level. DNA microarrays provide a natural vehicle for this exploration. The model organisms are the first for which comprehensive genome-wide surveys of gene expression patterns or function are possible. The results can be viewed as maps that reflect the order and logic of the genetic program, rather than the physical order of genes on chromosomes. Exploration of the genome using DNA microarrays and other genome-scale technologies should narrow the gap in our knowledge of gene function and molecular biology between the currently-favoured model organisms and other species.

Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association.

We determined the distribution of repressor-activator protein 1 (Rap1) and the accessory silencing proteins Sir2, Sir3 and Sir4 in vivo on the entire yeast genome, at a resolution of 2 kb. Rap1 is central to the cellular economy during rapid growth, targeting 294 loci, about 5% of yeast genes, and participating in the activation of 37% of all RNA polymerase II initiation events in exponentially growing cells. Although the DNA sequence recognized by Rap1 is found in both coding and intergenic sequences, the binding of Rap1 to the genome was highly specific to intergenic regions with the potential to act as promoters. This global phenomenon, which may be a general characteristic of sequence-specific transcriptional factors, indicates the existence of a genome-wide molecular mechanism for marking promoter regions.

Large-scale identification of secreted and membrane-associated gene products using DNA microarrays.

Membrane-associated and secreted proteins are an important class of proteins and include receptors, transporters, adhesion molecules, hormones and cytokines. Although algorithms have been developed to recognize potential amino-terminal membrane-targeting signals or transmembrane domains in protein sequences, their accuracy is limited and they require knowledge of the entire coding sequence, including the N terminus, which is not currently available for most of the genes in most organisms, including human. Several experimental approaches for identifying secreted and membrane proteins have been described, but none have taken a comprehensive genomic approach. Furthermore, none of these methods allow easy classification of clones from arrayed cDNA libraries, for which large-scale gene-expression data are now becoming available through the use of DNA microarrays. We describe here a rapid and efficient method for identifying genes that encode secreted or membrane proteins. mRNA species bound to membrane-associated polysomes were separated from other mRNAs by sedimentation equilibrium or sedimentation velocity. The distribution of individual transcripts in the 'membrane-bound' and 'cytosolic' fractions was quantitated for thousands of genes by hybridization to DNA microarrays. Transcripts known to encode secreted or membrane proteins were enriched in the membrane-bound fractions, whereas those known to encode cytoplasmic proteins were enriched in the fractions containing mRNAs associated with free and cytoplasmic ribosomes. On this basis, we identified over 275 human genes and 285 yeast genes that are likely to encode previously unrecognized secreted or membrane proteins.

Genomic mismatch scanning: a new approach to genetic linkage mapping.

Genomic mismatch scanning (GMS) is a new method of genetic linkage analysis that does not require conventional polymorphic markers or gel electrophoresis. GMS is ideally suited to affected-relative-pair mapping. DNA fragments from all regions of identity-by-descent between two relatives are isolated based on their ability to form extensive mismatch-free hybrid molecules. The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step. Here we demonstrate the practicality of GMS in a model organism, Saccharomyces cerevisiae. GMS is likely to be applicable to other organisms, including humans, and may be of particular value in mapping complex genetic traits.

Use of a cDNA microarray to analyse gene expression patterns in human cancer.

The development and progression of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. Microarrays of cDNA provide a powerful tool for studying these complex phenomena. The tumorigenic properties of a human melanoma cell line, UACC-903, can be suppressed by introduction of a normal human chromosome 6, resulting in a reduction of growth rate, restoration of contact inhibition, and suppression of both soft agar clonogenicity and tumorigenicity in nude mice. We used a high density microarray of 1,161 DNA elements to search for differences in gene expression associated with tumour suppression in this system. Fluorescent probes for hybridization were derived from two sources of cellular mRNA [UACC-903 and UACC-903(+6)] which were labelled with different fluors to provide a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene. The fluorescence signals representing hybridization to each arrayed gene were analysed to determine the relative abundance in the two samples of mRNAs corresponding to each gene. Previously unrecognized alterations in the expression of specific genes provide leads for further investigation of the genetic basis of the tumorigenic phenotype of these cells.

Genome-wide analysis of DNA copy-number changes using cDNA microarrays.

Gene amplifications and deletions frequently contribute to tumorigenesis. Characterization of these DNA copy-number changes is important for both the basic understanding of cancer and its diagnosis. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome. With CGH, differentially labelled test and reference genomic DNAs are co-hybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of DNA copy-number variation. CGH, however, has a limited ( approximately 20 Mb) mapping resolution, and higher-resolution techniques, such as fluorescence in situ hybridization (FISH), are prohibitively labour-intensive on a genomic scale. Array-based CGH, in which fluorescence ratios at arrayed DNA elements provide a locus-by-locus measure of DNA copy-number variation, represents another means of achieving increased mapping resolution. Published array CGH methods have relied on large genomic clone (for example BAC) array targets and have covered only a small fraction of the human genome. cDNAs representing over 30,000 radiation-hybrid (RH)-mapped human genes provide an alternative and readily available genomic resource for mapping DNA copy-number changes. Although cDNA microarrays have been used extensively to characterize variation in human gene expression, human genomic DNA is a far more complex mixture than the mRNA representation of human cells. Therefore, analysis of DNA copy-number variation using cDNA microarrays would require a sensitivity of detection an order of magnitude greater than has been routinely reported. We describe here a cDNA microarray-based CGH method, and its application to DNA copy-number variation analysis in breast cancer cell lines and tumours. Using this assay, we were able to identify gene amplifications and deletions genome-wide and with high resolution, and compare alterations in DNA copy number and gene expression.

Systematic variation in gene expression patterns in human cancer cell lines.

We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

A gene expression database for the molecular pharmacology of cancer.

We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression "signature," irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

A sign inversion mechanism for enzymatic supercoiling of DNA.

Both the introduction and the removal of supertwists by DNA gyrase change the linking number of DNA in steps of two. This surprising finding provides strong evidence that gyrase acts by a mechanism, called sign inversion, whereby a positive supercoil is directly inverted into a negative one via a transient double-strand break.

Exploring the metabolic and genetic control of gene expression on a genomic scale.

DNA microarrays containing virtually every gene of Saccharomyces cerevisiae were used to carry out a comprehensive investigation of the temporal program of gene expression accompanying the metabolic shift from fermentation to respiration. The expression profiles observed for genes with known metabolic functions pointed to features of the metabolic reprogramming that occur during the diauxic shift, and the expression patterns of many previously uncharacterized genes provided clues to their possible functions. The same DNA microarrays were also used to identify genes whose expression was affected by deletion of the transcriptional co-repressor TUP1 or overexpression of the transcriptional activator YAP1. These results demonstrate the feasibility and utility of this approach to genomewide exploration of gene expression patterns.

Quantitative monitoring of gene expression patterns with a complementary DNA microarray.

A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.

Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus.

In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.

Functional analysis of the genes of yeast chromosome V by genetic footprinting.

Genetic footprinting was used to assess the phenotypic effects of Ty1 transposon insertions in 268 predicted genes of chromosome V of Saccharomyces cerevisiae. When seven selection protocols were used, Ty1 insertions in more than half the genes tested (157 of 268) were found to result in a detectable reduction in fitness. Results could not be obtained for fewer than 3 percent of the genes tested (7 of 268). Previously known mutant phenotypes were confirmed, and, for about 30 percent of the genes, new mutant phenotypes were identified.

The transcriptional program of sporulation in budding yeast.

Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

The transcriptional program in the response of human fibroblasts to serum.

The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.

Genome scanning methods.

Recently introduced methods can allow an entire genome to be scanned at one time for sequences linked to trait loci. Representational difference analysis and genomic mismatch scanning are designed, respectively, to isolate and map sequences that differ between genomes discordant for a trait, or that are identical between genomes concordant for a trait. Other methods allow the whole genome to be scanned for regions of increased or decreased copy number, or for differentially methylated sequences.

Observing the living genome.

Dynamic pictures of living genomes are now beginning to emerge from systematic studies of gene expression patterns using DNA microarrays. The rich information represented in the variation in each gene's expression provides the basis for a new kind of genomic map.