Showing papers on "Gene published in 2003"

PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes

Abstract: DNA microarrays can be used to identify gene expression changes characteristic of human disease. This is challenging, however, when relevant differences are subtle at the level of individual genes. We introduce an analytical strategy, Gene Set Enrichment Analysis, designed to detect modest but coordinate changes in the expression of groups of functionally related genes. Using this approach, we identify a set of genes involved in oxidative phosphorylation whose expression is coordinately decreased in human diabetic muscle. Expression of these genes is high at sites of insulin-mediated glucose disposal, activated by PGC-1α and correlated with total-body aerobic capacity. Our results associate this gene set with clinically important variation in human metabolism and illustrate the value of pathway relationships in the analysis of genomic profiling experiments.

Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana

Abstract: Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.

Global analysis of protein expression in yeast

Abstract: The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

Systematic functional analysis of the Caenorhabditis elegans genome using RNAi

Abstract: A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour. Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of ∼86% of the 19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans.

Gene Silencing in Cancer in Association with Promoter Hypermethylation

Abstract: This article reviews the mechanisms of gene silencing in cancer and clinical applications of this phenomenon. The silencing of genes, especially tumor-suppressor genes, is a key event in the development of cancer. The silencing can be effected by a disabling mutation or by a shutting down of the promoter region, the site at which transcription of the gene begins.

Targeting RAS signalling pathways in cancer therapy

Abstract: The RAS proteins control signalling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. They are aberrant in most human tumours due to activating mutations in the RAS genes themselves or to alterations in upstream or downstream signalling components. Rational therapies that target the RAS pathways might inhibit tumour growth, survival and spread. Several of these new therapeutic agents are showing promise in the clinic and many more are being developed.

MicroRNA targets in Drosophila

Abstract: Background: The recent discoveries of microRNA (miRNA) genes and characterization of the first few target genes regulated by miRNAs in Caenorhabditis elegans and Drosophila melanogaster have set the stage for elucidation of a novel network of regulatory control. We present a computational method for wholegenome prediction of miRNA target genes. The method is validated using known examples. For each miRNA, target genes are selected on the basis of three properties: sequence complementarity using a position-weighted local alignment algorithm, free energies of RNA-RNA duplexes, and conservation of target sites in related genomes. Application to the D. melanogaster, Drosophila pseudoobscura and Anopheles gambiae genomes identifies several hundred target genes potentially regulated by one or more known miRNAs.

ATM and related protein kinases: safeguarding genome integrity

Abstract: Maintenance of genome stability is essential for avoiding the passage to neoplasia. The DNA-damage response--a cornerstone of genome stability--occurs by a swift transduction of the DNA-damage signal to many cellular pathways. A prime example is the cellular response to DNA double-strand breaks, which activate the ATM protein kinase that, in turn, modulates numerous signalling pathways. ATM mutations lead to the cancer-predisposing genetic disorder ataxia-telangiectasia (A-T). Understanding ATM's mode of action provides new insights into the association between defective responses to DNA damage and cancer, and brings us closer to resolving the issue of cancer predisposition in some A-T carriers.

A Protein Interaction Map of Drosophila melanogaster

Abstract: Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid-based protein-interaction map of the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions. Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a starting point for a systems biology modeling of multicellular organisms, including humans.

Genes required for mycobacterial growth defined by high density mutagenesis

Abstract: Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.

A Gene-Coexpression Network for Global Discovery of Conserved Genetic Modules

Abstract: To elucidate gene function on a global scale, we identified pairs of genes that are coexpressed over 3182 DNA microarrays from humans, flies, worms, and yeast. We found 22,163 such coexpression relationships, each of which has been conserved across evolution. This conservation implies that the coexpression of these gene pairs confers a selective advantage and therefore that these genes are functionally related. Manyof these relationships provide strong evidence for the involvement of new genes in core biological functions such as the cell cycle, secretion, and protein expression. We experimentallyconfirmed the predictions implied bysome of these links and identified cell proliferation functions for several genes. By assembling these links into a gene-coexpression network, we found several components that were animal-specific as well as interrelationships between newly evolved and ancient modules.

Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease

Abstract: Genes and mechanisms involved in common complex diseases, such as the autoimmune disorders that affect approximately 5% of the population, remain obscure. Here we identify polymorphisms of the cytotoxic T lymphocyte antigen 4 gene (CTLA4)—which encodes a vital negative regulatory molecule of the immune system—as candidates for primary determinants of risk of the common autoimmune disorders Graves' disease, autoimmune hypothyroidism and type 1 diabetes. In humans, disease susceptibility was mapped to a non-coding 6.1?kb 3′ region of CTLA4, the common allelic variation of which was correlated with lower messenger RNA levels of the soluble alternative splice form of CTLA4. In the mouse model of type 1 diabetes, susceptibility was also associated with variation in CTLA-4 gene splicing with reduced production of a splice form encoding a molecule lacking the CD80/CD86 ligand-binding domain. Genetic mapping of variants conferring a small disease risk can identify pathways in complex disorders, as exemplified by our discovery of inherited, quantitative alterations of CTLA4 contributing to autoimmune tissue destruction.

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.).

Abstract: A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley (Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3′-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5′-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 ± 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.

A gene expression atlas of the central nervous system based on bacterial artificial chromosomes

Abstract: The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.

Identifying biological themes within lists of genes with EASE

Abstract: EASE is a customizable software application for rapid biological interpretation of gene lists that result from the analysis of microarray, proteomics, SAGE and other high-throughput genomic data. The biological themes returned by EASE recapitulate manually determined themes in previously published gene lists and are robust to varying methods of normalization, intensity calculation and statistical selection of genes. EASE is a powerful tool for rapidly converting the results of functional genomics studies from 'genes' to 'themes'.

Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as Transcriptional Activators in Abscisic Acid Signaling

Abstract: In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA). We reported previously that MYC and MYB recognition sites in the rd22 promoter region function as cis-acting elements in the drought- and ABA-induced gene expression of rd22. bHLH- and MYB-related transcription factors, rd22BP1 (renamed AtMYC2) and AtMYB2, interact specifically with the MYC and MYB recognition sites, respectively, in vitro and activate the transcription of the β-glucuronidase reporter gene driven by the MYC and MYB recognition sites in Arabidopsis leaf protoplasts. Here, we show that transgenic plants overexpressing AtMYC2 and/or AtMYB2 cDNAs have higher sensitivity to ABA. The ABA-induced gene expression of rd22 and AtADH1 was enhanced in these transgenic plants. Microarray analysis of the transgenic plants overexpressing both AtMYC2 and AtMYB2 cDNAs revealed that several ABA-inducible genes also are upregulated in the transgenic plants. By contrast, a Ds insertion mutant of the AtMYC2 gene was less sensitive to ABA and showed significantly decreased ABA-induced gene expression of rd22 and AtADH1. These results indicate that both AtMYC2 and AtMYB2 proteins function as transcriptional activators in ABA-inducible gene expression under drought stress in plants.

Global Mapping of the Yeast Genetic Interaction Network: Discovering Gene and Drug Function

Abstract: budding yeast Saccharomyces cerevisiae, ~80% of the ~6000 genes are nonessential, indi- cating that many biological processes are buffered from the phenotypic consequences of genetic per- turbation. To examine these functional relation- ships we developed a method called Synthetic Genetic Array (SGA) analysis, which automates yeast genetics and enables a systematic and high- throughput construction of double mutants from an ordered array of ~4700 viable gene deletion mutants. In particular, double mutants showing reduced fitness (a synthetic sick phenotype) or lethality (a synthetic lethal phenotype) define functional relationships between genes and their corresponding pathways. We have undertaken a project to generate a synthetic genetic interaction network for the yeast cell with the expectation that it will represent a global map of functional relationships amongst most genes. We found that synthetic genetic interactions are more common than anticipated previously, with an average query gene displaying ~30 different interactions. Cluster analysis of a compendium of ~132 SGA screens revealed that genes displaying similar patterns of genetic interactions often encode proteins within the same pathway or complex; therefore, the yeast genetic interaction network predicts precise molec- ular roles of previously uncharacterized genes. Moreover, because a gene deletion mutation pro- vides a model for the effect of a compound that inhibits its corresponding gene product, our com- pendium of synthetic genetic profiles provides a key for determining the cellular targets of small molecules and drugs. Finally, the surprisingly large number of synthetic genetic interactions observed for defined mutations of inbred laboratory yeast strains suggests that digenic interactions of this type may also occur frequently amongst different alleles of genes found within the individuals of an outbred population and thus similar genetic inter- actions may underlie many of the inherited pheno- types in other organisms.

Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes

Abstract: MicroRNAs (miRNAs) are ∼21-nucleotide noncoding RNAs that have been identified in both animals and plants. Although in animals there is direct evidence implicating particular miRNAs in the control of developmental timing, to date it is not known whether plant miRNAs also play a role in regulating temporal transitions. Through an activation-tagging approach, we demonstrate that miRNA 172 (miR172) causes early flowering and disrupts the specification of floral organ identity when overexpressed in Arabidopsis. miR172 normally is expressed in a temporal manner, consistent with its proposed role in flowering time control. The regulatory target of miR172 is a subfamily of APETALA2 (AP2) transcription factor genes. We present evidence that miR172 downregulates these target genes by a translational mechanism rather than by RNA cleavage. Gain-of-function and loss-of-function analyses indicate that two of the AP2-like target genes normally act as floral repressors, supporting the notion that miR172 regulates flowering time by downregulating AP2-like target genes.

Sequencing and comparison of yeast species to identify genes and regulatory elements

Abstract: Identifying the functional elements encoded in a genome is one of the principal challenges in modern biology. Comparative genomics should offer a powerful, general approach. Here, we present a comparative analysis of the yeast Saccharomyces cerevisiae based on high-quality draft sequences of three related species (S. paradoxus, S. mikatae and S. bayanus). We first aligned the genomes and characterized their evolution, defining the regions and mechanisms of change. We then developed methods for direct identification of genes and regulatory motifs. The gene analysis yielded a major revision to the yeast gene catalogue, affecting approximately 15% of all genes and reducing the total count by about 500 genes. The motif analysis automatically identified 72 genome-wide elements, including most known regulatory motifs and numerous new motifs. We inferred a putative function for most of these motifs, and provided insights into their combinatorial interactions. The results have implications for genome analysis of diverse organisms, including the human.

Role of MicroRNAs in Plant and Animal Development

Abstract: Small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), are key components of an evolutionarily conserved system of RNA-based gene regulation in eukaryotes. They are involved in many molecular interactions, including defense against viruses and regulation of gene expression during development. miRNAs interfere with expression of messenger RNAs encoding factors that control developmental timing, stem cell maintenance, and other developmental and physiological processes in plants and animals. miRNAs are negative regulators that function as specificity determinants, or guides, within complexes that inhibit protein synthesis (animals) or promote degradation (plants) of mRNA targets.

Control of leaf morphogenesis by microRNAs

Abstract: Plants with altered microRNA metabolism have pleiotropic developmental defects, but direct evidence for microRNAs regulating specific aspects of plant morphogenesis has been lacking In a genetic screen, we identified the JAW locus, which produces a microRNA that can guide messenger RNA cleavage of several TCP genes controlling leaf development MicroRNA-guided cleavage of TCP4 mRNA is necessary to prevent aberrant activity of the TCP4 gene expressed from its native promoter In addition, overexpression of wild-type and microRNA-resistant TCP variants demonstrates that mRNA cleavage is largely sufficient to restrict TCP function to its normal domain of activity TCP genes with microRNA target sequences are found in a wide range of species, indicating that microRNA-mediated control of leaf morphogenesis is conserved in plants with very different leaf forms

A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference

Abstract: RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.

Noise in eukaryotic gene expression

Abstract: Transcription in eukaryotic cells has been described as quantal, with pulses of messenger RNA produced in a probabilistic manner. This description reflects the inherently stochastic nature of gene expression, known to be a major factor in the heterogeneous response of individual cells within a clonal population to an inducing stimulus. Here we show in Saccharomyces cerevisiae that stochasticity (noise) arising from transcription contributes significantly to the level of heterogeneity within a eukaryotic clonal population, in contrast to observations in prokaryotes, and that such noise can be modulated at the translational level. We use a stochastic model of transcription initiation specific to eukaryotes to show that pulsatile mRNA production, through reinitiation, is crucial for the dependence of noise on transcriptional efficiency, highlighting a key difference between eukaryotic and prokaryotic sources of noise. Furthermore, we explore the propagation of noise in a gene cascade network and demonstrate experimentally that increased noise in the transcription of a regulatory protein leads to increased cell-cell variability in the target gene output, resulting in prolonged bistable expression states. This result has implications for the role of noise in phenotypic variation and cellular differentiation.

Human Gene Mutation Database (HGMD): 2003 update.

Abstract: The Human Gene Mutation Database (HGMD) constitutes a comprehensive core collection of data on germ-line mutations in nuclear genes underlying or associated with human inherited disease (www.hgmd.org). Data catalogued includes: single base-pair substitutions in coding, regulatory and splicing-relevant regions; micro-deletions and micro-insertions; indels; triplet repeat expansions as well as gross deletions; insertions; duplications; and complex rearrangements. Each mutation is entered into HGMD only once in order to avoid confusion between recurrent and identical-by-descent lesions. By March 2003, the database contained in excess of 39,415 different lesions detected in 1,516 different nuclear genes, with new entries currently accumulating at a rate exceeding 5,000 per annum. Since its inception, HGMD has been expanded to include cDNA reference sequences for more than 87% of listed genes, splice junction sequences, disease-associated and functional polymorphisms, as well as links to data present in publicly available online locus-specific mutation databases. Although HGMD has recently entered into a licensing agreement with Celera Genomics (Rockville, MD), mutation data will continue to be made freely available via the Internet.

The Transcriptome of the Intraerythrocytic Developmental Cycle of Plasmodium falciparum

Abstract: Plasmodium falciparum is the causative agent of the most burdensome form of human malaria, affecting 200–300 million individuals per year worldwide. The recently sequenced genome of P. falciparum revealed over 5,400 genes, of which 60% encode proteins of unknown function. Insights into the biochemical function and regulation of these genes will provide the foundation for future drug and vaccine development efforts toward eradication of this disease. By analyzing the complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of the HB3 strain of P. falciparum, we demonstrate that at least 60% of the genome is transcriptionally active during this stage. Our data demonstrate that this parasite has evolved an extremely specialized mode of transcriptional regulation that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium-specific functionalities, such as genes involved in erythrocyte invasion. The data reveal that genes contiguous along the chromosomes are rarely coregulated, while transcription from the plastid genome is highly coregulated and likely polycistronic. Comparative genomic hybridization between HB3 and the reference genome strain (3D7) was used to distinguish between genes not expressed during the IDC and genes not detected because of possible sequence variations. Genomic differences between these strains were found almost exclusively in the highly antigenic subtelomeric regions of chromosomes. The simple cascade of gene regulation that directs the asexual development of P. falciparum is unprecedented in eukaryotic biology. The transcriptome of the IDC resembles a “just-in-time” manufacturing process whereby induction of any given gene occurs once per cycle and only at a time when it is required. These data provide to our knowledge the first comprehensive view of the timing of transcription throughout the intraerythrocytic development of P. falciparum and provide a resource for the identification of new chemotherapeutic and vaccine candidates.

Genetics of gene expression surveyed in maize, mouse and man

Abstract: Treating messenger RNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways. Transcript abundances often serve as a surrogate for classical quantitative traits in that the levels of expression are significantly correlated with the classical traits across members of a segregating population. The correlation structure between transcript abundances and classical traits has been used to identify susceptibility loci for complex diseases such as diabetes and allergic asthma. One study recently completed the first comprehensive dissection of transcriptional regulation in budding yeast, giving a detailed glimpse of a genome-wide survey of the genetics of gene expression. Unlike classical quantitative traits, which often represent gross clinical measurements that may be far removed from the biological processes giving rise to them, the genetic linkages associated with transcript abundance affords a closer look at cellular biochemical processes. Here we describe comprehensive genetic screens of mouse, plant and human transcriptomes by considering gene expression values as quantitative traits. We identify a gene expression pattern strongly associated with obesity in a murine cross, and observe two distinct obesity subtypes. Furthermore, we find that these obesity subtypes are under the control of different loci.

The origins of genome complexity.

Abstract: Complete genomic sequences from diverse phylogenetic lineages reveal notable increases in genome complexity from prokaryotes to multicellular eukaryotes. The changes include gradual increases in gene number, resulting from the retention of duplicate genes, and more abrupt increases in the abundance of spliceosomal introns and mobile genetic elements. We argue that many of these modifications emerged passively in response to the long-term population-size reductions that accompanied increases in organism size. According to this model, much of the restructuring of eukaryotic genomes was initiated by nonadaptive processes, and this in turn provided novel substrates for the secondary evolution of phenotypic complexity by natural selection. The enormous long-term effective population sizes of prokaryotes may impose a substantial barrier to the evolution of complex genomes and morphologies.

OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought‐, high‐salt‐ and cold‐responsive gene expression

Abstract: Summary The transcription factors DREBs/CBFs specifically interact with the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element (core motif: G/ACCGAC) and control the expression of many stress-inducible genes in Arabidopsis. In rice, we isolated five cDNAs for DREB homologs: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by cold, whereas expression of OsDREB2A was induced by dehydration and high-salt stresses. The OsDREB1A and OsDREB2A proteins specifically bound to DRE and activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Over-expression of OsDREB1A in transgenic Arabidopsis induced over-expression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought, high-salt, and freezing stresses. This indicated that OsDREB1A has functional similarity to DREB1A. However, in microarray and RNA blot analyses, some stress-inducible target genes of the DREB1A proteins that have only ACCGAC as DRE were not over-expressed in the OsDREB1A transgenic Arabidopsis. The OsDREB1A protein bound to GCCGAC more preferentially than to ACCGAC whereas the DREB1A proteins bound to both GCCGAC and ACCGAC efficiently. The structures of DREB1-type ERF/AP2 domains in monocots are closely related to each other as compared with that in the dicots. OsDREB1A is potentially useful for producing transgenic monocots that are tolerant to drought, high-salt, and/or cold stresses.

Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.

Abstract: Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.