Expresión de la resistencia en plantas de tomate a nematodos formadores de nódulos (Meloidogyne javanica)

La resistencia genética mediada por los genes R es uno de los sistemas de defensa de las plantas frente a patógenos y se activa una vez que los patógenos han superado la defensa basal que otorgan la cutícula y pared celular. Los mecanismos de resistencia genética se inician a su vez, por el reconocimiento de productos derivados de genes de avirulencia de los patógenos (avr) por parte de las proteínas R. Tanto la respuesta de defensa basal como la respuesta de defensa por genes R están influenciadas por patrones de regulación hormonal, que incluye a las principales hormonas vegetales ácido salicílico (SA), ácido jasmónico (JA) y etileno (ET). En tomate (Solanum lycopersicum) uno de los genes R es el gen MiG1, que confiere resistencia a nematodos formadores de nódulos (Meloidogyne javanica, M. incognita y M. arenaria). Uno de los eventos más importantes que caracterizan a la respuesta de resistencia es la reacción hipersensible (HR), que está mediada por la activación temprana de una serie de sistemas enzimáticos, entre los que destaca el de las peroxidasas (PRXs) Clase III. Su función es importante tanto para limitar el establecimiento y expansión del nematodo, al generar ambientes altamente tóxicos por su contribución en la producción masiva de ROS, como por su implicación en la síntesis y depósito de lignina generando barreras estructurales en el sitio de infección. Además de estos mecanismos de defensa asociados a la resistencia constitutiva, las plantas pueden desarrollar resistencia sistémica adquirida (SAR) que en la naturaleza ocurre, en ocasiones, en una fase posterior a que la planta haya sufrido el ataque de un patógeno. Así mismo hay diferentes productos de origen químico como el benzotiadiazol o BTH (ácido S-metil benzol-(1,2,3)-tiadiozole-7-carbónico ester) que pueden generar esta misma respuesta SAR. Como resultado, la planta adquiere resistencia sistémica frente a nuevos ataques de patógenos. En este contexto, el presente trabajo aborda en primer lugar el análisis comparativo, mediante microarrays de oligonucleótidos, de los transcriptomas de los sistemas radicales de plantas de tomate de 8 semanas de edad de dos variedades, una portadora del gen de resistencia MiG1 (Motelle) y otra carente del mismo y, por tanto, susceptible (Moneymaker), antes y después de la infección por M. javanica. Previo a la infección se observó que la expresión de un gran número de transcritos era más acusada en la variedad resistente que en la susceptible, entre ellos el propio gen MiG1 o los genes PrG1 (o P4), LEJA1 y ER24, lo que indica que, en ausencia de infección, las rutas hormonales del SA, JA y ET están más activas en la raíz de la variedad resistente. Por el contrario, un número mucho menor de transcritos presentaban su expresión más reducida en Motelle que en Moneymaker, destacando un gen de señalización para sintetizar la hormona giberelina (GA). La infección por M. javanica causa importantes cambios transcripcionales en todo el sistema radical que modifican sustancialmente las diferencias basales entre plantas Motelle y Moneymaker, incluida la sobreexpresión en la variedad resistente de los transcritos de MiG1, que se reduce parcialmente, mientras que las rutas hormonales del SA y el JA continuan más activas que en la susceptible (evidente por los genes PrG1 y LEJA1). Además, los cambios asociados a la infección del nematodo se evidencian por las grandes diferencias entre los dos tiempos post-infección considerados, de tal forma que en la fase temprana (2 dpi) de la interacción compatible predomina la sobreexpresión de genes de pared celular y en la tardía (12 dpi) los relacionados con el ARN. En el análisis de la interacción incompatible, aunque también hay muchas diferencias entre ambas fases, hay que destacar la expresión diferencial común de los genes loxA y mcpi (sobrexpresados) y del gen loxD (reprimido) por su implicación en defensa en otras interacciones planta-patógeno. Cabe destacar que entre las interacciones compatible e incompatible hubo muy pocos genes en común. En la etapa temprana de la interacción compatible destacó la activación de genes de pared celular y la represión de la señalización; en cambio, en la interacción incompatible hubo proteínas principalmente implicadas en defensa. A los 12 días, en la interacción compatible los genes relacionados con el ARN y la pared celular se sobreexpresaban principalmente, y se reprimían los de proteínas y transporte, mientras que en la incompatible se sobreexpresaron los relacionados con el estrés, el metabolismo secundario y el de hormonas y se reprimieron los de ARN, señalización, metabolismo de hormonas y proteínas. Por otra parte, la técnica de silenciamiento génico VIGS reveló que el gen TGA 1a está implicado en la resistencia mediada por el gen MiG1a M. javanica. Así mismo se evaluó el transcriptoma de todo el sistema radical de la variedad susceptible tras la aplicación del inductor BTH, y se comparó con el transcriptoma de la resistente. Los resultados obtenidos revelan que el tratamiento con BTH en hojas de Moneymaker ejerce notables cambios transcripcionales en la raíz; entre otros, la activación de factores de transcripción Myb (THM16 y THM 27) y del gen ACC oxidasa. Las respuestas inducidas por el BTH parecen ser de corta duración ya que no hubo transcritos diferenciales comunes a las dos fases temporales de la infección comparadas (2 y 12 dpi). El transcriptoma de Moneymaker tratada con BTH resultó ser muy diferente al de la variedad resistente Motelle, ambas sin infectar, destacando la mayor expresión en el primero del gen LeEXP2, una expansina relacionada con defensa frente a nematodos. Las respuestas inducidas por los nematodos en Moneymaker-BTH también fueron muy distintas a las observadas previamente en la interacción incompatible mediada por MiG1, pues sólo se detectaron 2 genes sobreexpresados comunes a ambos eventos. Finalmente, se abordó el estudio de la expresión diferencial de genes que codifican PRXs y su relación con la resistencia en la interacción tomate/M. javanica. Para ello, se realizó en primer lugar el estudio del análisis del transcriptoma de tomate de la interacción compatible, obtenido en un estudio previo a partir de tejido radical infectado en distintos tiempos de infección. Se han identificado 16 unigenes de PRXs con expresión diferencial de los cuales 15 se relacionan por primera vez con la respuesta a la infección de nematodos. La mayoría de los genes de PRXs identificados, 11, aparecen fuertemente reprimidos en el sitio de alimentación, en las células gigantes (CG). Dada la implicación directa de las PRXs en la activación del mecanismo de producción de ROS, la supresión de la expresión génica local de genes de PRXs en el sitio de establecimiento y alimentación pone de manifiesto la capacidad del nematodo para modular y superar la respuesta de defensa de la planta de tomate en la interacción compatible. Posteriormente, de estos genes identificados se han elegido 4: SGN-U143455, SGN-U143841 y SGN-U144042 reprimidos en el sitio de infección y SGN-U144671 inducido, cuyos cambios de expresión se han determinado mediante análisis por qRT-PCR y de hibridación in situ en dos tiempos de infección (2 dpi y 4 dpi) y en distintos tejidos radicales de tomate resistente y susceptible. Los patrones de expresión obtenidos demuestran que en la interacción incompatible la transcripción global de los 4 genes estudiados se dispara en la etapa más temprana en el sitio de infección, detectándose la localización in situ de transcritos en el citoplasma de las células corticales de la zona meristemática afectadas por el nematodo. A 4 dpi se observó que los niveles de expresión en el sitio de infección cambian de tendencia y los genes SGN-U144671 y SGN-U144042 se reprimen significativamente. Los diferentes perfiles de expresión de los genes PRXs en los dos tiempos de infección sugieren que su inducción en las primeras 48 horas es crucial para la respuesta de defensa relacionada con la resistencia frente a la invasión del nematodo. Por último, al analizar el tejido radical sistémico, se detectó una inducción significativa de la expresión en la fase más tardía de la infección del gen SGN-U144042 en el genotipo susceptible y del SGN-U143841 en ambos genotipos. En este estudio se describe por primera vez la inducción de la expresión sistémica de genes de PRXs en tomate durante la interacción compatible e incompatible con M. javanica lo que sugiere su posible implicación funcional en la respuesta de defensa SAR activada por la infección previa del nematodo. ABSTRACT Plants defend themselves from pathogens by constitutive and/or induced defenses. A common type of induced defense involves plant resistance genes (R), which are normally activated in response to attack by specific pathogen species. Typically, a specific plant R protein recognizes a specific pathogen avirulence (avr) compound. This initiates a complex biochemical cascade inside the plant that results in synthesis of antipathogen compounds. This response can involve chemical signaling, transcription, translation, enzymes and metabolism, and numerous plant hormones such as salicylic acid (SA), jasmonates (JA) and ethylene (ET). Induced plant defense can also activate Class III peroxidases (PRXs), which produce reactive oxygen species (ROS), regulate extracellular H2O2, and play additional roles in plant defense. R-gene activation and the resulting induced defense often remain localized in the specific tissues invaded by the plant pathogen. In other cases, the plant responds by signaling the entire plant to produce defense compounds (systemic induction). Plant defense can also be induced by the exogenous application of natural or synthetic elicitors, such as benzol-(1,2,3)-thiadiazole-7-carbothionic acid. There is much current scientific interest in R-genes and elicitors, because they might be manipulated to increase agricultural yield. Scientists also are interested in systemic induction, because this allows the entire plant to be defended. In this context, one of the aims of this investigation was the transcriptoma analysis of the root systems of two varieties of tomato, the resistant variety (Motelle) that carrier MiG1 and the susceptible (Moneymaker) without MiG1, before and after infection with M. javanica. The overexpression was more pronounced in the transcriptoma of the resistant variety compared with susceptible, before infection, including the MiG1 gene, PrG1 (or P4) genes, LEJA1 and ER24, indicating that hormone SA, JA and ET are active in the resistant variety. Moreover, GA hormone presents an opposite behavior. M. javanica infection causes significant transcriptional changes in both compatible (Moneymaker-M. javanica) and incompatible (Motelle-M. javanica) interaction. In the incompatible transcriptome root system, was notably reduced the expression of the MiG1 gene, and a continuity in the expression of the hormonal pathways of SA and JA. In other hand, transcriptional profile changes during compatible interaction were associated with nematode infection. The large differences between the two times point infection considered (2 dpi and 12 dpi) indicates an overexpression of cell wall related genes in the first phase, and conversely an overexpression of RNA genes in the late phase. Transcriptoma analysis of incompatible interaction, although there were differences between the two phases, should be highlighted the common differential gene expression: loxA and mcpi (overexpressed) and loxD gene (suppressed), as they are involved in defenses in other plant-pathogen interactions. The VIGS tool has provided evidence that TGA 1a is involved in MiG1 mediated resistance to M. javanica. Likewise, the systemic application of BTH was assessed and compared with susceptible and resistant variety. Root system transcriptoma of BTH treatment on leaves showed the activation of Myb transcription factors (THM16 and THM27), the ACC oxidase gene. and the LeEXP2 gene, encoding for an expansin enzyme, related with defense against nematodes. The activation appears to be reduced by subsequent infection and establishment of nematodes. To assist in elucidate the role of tomato PRXs in plant defence against M. javanica, the transcriptome obtained previously from isolated giant cells (GC) and galls at 3 and 7 dpi from the compatible interaction was analysed. A total of 18 different probes corresponding to 16 PRX encoding genes were differentially expressed in infection site compared to the control uninfected root tissues. Most part of them (11) was down-regulated. These results yielded a first insight on 15 of the PRX genes responding to tomato–Meloidogyne interaction and confirm that repression of PRX genes might be crucial for feeding site formation at the initial stages of infection. To study the involvement of PRX genes in resistance response, four genes have been selected: SGN-U143455, SGN-U143841 and SGN-U144042 consistently down-regulated and SGN-U144671 consistently up-regulated at infection site in compatible interaction. The expression changes were determined by qRT-PCR and in situ location at 2 dpi and 4 dpi, and in different root tissues of resistant and susceptible plants. Early upon infection (2 dpi), the transcripts levels of the four genes were strongly increased in infected tissue of resistant genotype. In situ hybridization showed transcript accumulation of them in meristem cortical cells, where the nematode made injury. The results obtained provide strong evidence that early induction of PRX genes is important for defence response of the resistance against nematode invasion. Moreover, the induction patterns of SGN-U144042 gene observed at 4 dpi in distal noninfected root tissue into the susceptible genotype and of SGN-U143841 gene in both genotypes suggest a potential involvement of PRX in the systemic defence response.

Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells

Inhibitors of DNA methyltransferase, typified by 5-aza-2′-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-α. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT’s limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-α2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray HybridizationD⃞

We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: α factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle–regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu

In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs

We describe a method for cloning nucleic acid molecules onto the surfaces of 5-μm microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry ≈106 strands complementary to one of the tags. About 105 copies of each molecule are collected on each microbead. Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or disease-directed, low-density planar microarrays.

Importance of replication in microarray gene expression studies: Statistical methods and evidence from repetitive cDNA hybridizations

We present statistical methods for analyzing replicated cDNA microarray expression data and report the results of a controlled experiment. The study was conducted to investigate inherent variability in gene expression data and the extent to which replication in an experiment produces more consistent and reliable findings. We introduce a statistical model to describe the probability that mRNA is contained in the target sample tissue, converted to probe, and ultimately detected on the slide. We also introduce a method to analyze the combined data from all replicates. Of the 288 genes considered in this controlled experiment, 32 would be expected to produce strong hybridization signals because of the known presence of repetitive sequences within them. Results based on individual replicates, however, show that there are 55, 36, and 58 highly expressed genes in replicates 1, 2, and 3, respectively. On the other hand, an analysis by using the combined data from all 3 replicates reveals that only 2 of the 288 genes are incorrectly classified as expressed. Our experiment shows that any single microarray output is subject to substantial variability. By pooling data from replicates, we can provide a more reliable analysis of gene expression data. Therefore, we conclude that designing experiments with replications will greatly reduce misclassification rates. We recommend that at least three replicates be used in designing experiments by using cDNA microarrays, particularly when gene expression data from single specimens are being analyzed.

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (VH) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Hypoxia-induced gene expression profiling in the euryoxic fish Gillichthys mirabilis

Hypoxia is important in both biomedical and environmental contexts and necessitates rapid adaptive changes in metabolic organization. Mammals, as air breathers, have a limited capacity to withstand sustained exposure to hypoxia. By contrast, some aquatic animals, such as certain fishes, are routinely exposed and resistant to severe environmental hypoxia. Understanding the changes in gene expression in fishes exposed to hypoxic stress could reveal novel mechanisms of tolerance that may shed new light on hypoxia and ischemia in higher vertebrates. Using cDNA microarrays, we have studied gene expression in a hypoxia-tolerant burrow-dwelling goby fish, Gillichthys mirabilis. We show that a coherent picture of a complex transcriptional response can be generated for a nonmodel organism for which sequence data were unavailable. We demonstrate that: (i) although certain shifts in gene expression mirror changes in mammals, novel genes are differentially expressed in fish; and (ii) tissue-specific patterns of expression reflect the different metabolic roles of tissues during hypoxia.

The transcriptional program of a human B cell line in response to Myc

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

Genome-wide Responses to Mitochondrial DysfunctionV⃞

Mitochondrial dysfunction can lead to diverse cellular and organismal responses. We used DNA microarrays to characterize the transcriptional responses to different mitochondrial perturbations in Saccharomyces cerevisiae. We examined respiratory-deficient petite cells and respiratory-competent wild-type cells treated with the inhibitors of oxidative phosphorylation antimycin, carbonyl cyanide m-chlorophenylhydrazone, or oligomycin. We show that respiratory deficiency, but not inhibition of mitochondrial ATP synthesis per se, induces a suite of genes associated with both peroxisomal activities and metabolite-restoration (anaplerotic) pathways that would mitigate the loss of a complete tricarboxylic acid cycle. The array data suggested, and direct microscopic observation of cells expressing a derivative of green fluorescent protein with a peroxisomal matrix-targeting signal confirmed, that respiratory deficiency dramatically induces peroxisome biogenesis. Transcript profiling of cells harboring null alleles of RTG1, RTG2, or RTG3, genes known to control signaling from mitochondria to the nucleus, suggests that there are multiple pathways of cross-talk between these organelles in yeast.

ANX7, a candidate tumor suppressor gene for prostate cancer

The ANX7 gene is located on human chromosome 10q21, a site long hypothesized to harbor a tumor suppressor gene(s) (TSG) associated with prostate and other cancers. To test whether ANX7 might be a candidate TSG, we examined the ANX7-dependent suppression of human tumor cell growth, stage-specific ANX7 expression in 301 prostate specimens on a prostate tissue microarray, and loss of heterozygosity (LOH) of microsatellite markers at or near the ANX7 locus. Here we report that human tumor cell proliferation and colony formation are markedly reduced when the wild-type ANX7 gene is transfected into two prostate tumor cell lines, LNCaP and DU145. Consistently, analysis of ANX7 protein expression in human prostate tumor microarrays reveals a significantly higher rate of loss of ANX7 expression in metastatic and local recurrences of hormone refractory prostate cancer as compared with primary tumors (P = 0.0001). Using four microsatellite markers at or near the ANX7 locus, and laser capture microdissected tumor cells, 35% of the 20 primary prostate tumors show LOH. The microsatellite marker closest to the ANX7 locus showed the highest rate of LOH, including one homozygous deletion. We conclude that the ANX7 gene exhibits many biological and genetic properties expected of a TSG and may play a role in prostate cancer progression.

Amygdala-enriched genes identified by microarray technology are restricted to specific amygdaloid subnuclei

Microarray technology represents a potentially powerful method for identifying cell type- and regionally restricted genes expressed in the brain. Here we have combined a microarray analysis of differential gene expression among five selected brain regions, including the amygdala, cerebellum, hippocampus, olfactory bulb, and periaqueductal gray, with in situ hybridization. On average, 0.3% of the 34,000 genes interrogated were highly enriched in each of the five regions, relative to the others. In situ hybridization performed on a subset of amygdala-enriched genes confirmed in most cases the overall region-specificity predicted by the microarray data and identified additional sites of brain expression not examined on the microarrays. Strikingly, the majority of these genes exhibited boundaries of expression within the amygdala corresponding to cytoarchitectonically defined subnuclei. These results define a unique set of molecular markers for amygdaloid subnuclei and provide tools to genetically dissect their functional roles in different emotional behaviors.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba

Extracts of Ginkgo biloba leaves are consumed as dietary supplements to counteract chronic, age-related neurological disorders. We have applied high-density oligonucleotide microarrays to define the transcriptional effects in the cortex and hippocampus of mice whose diets were supplemented with the herbal extract. Gene expression analysis focused on the mRNAs that showed a more than 3-fold change in their expression. In the cortex, mRNAs for neuronal tyrosine/threonine phosphatase 1, and microtubule-associated τ were significantly enhanced. Hyperphosphorylated τ is the major constituent of the neurofibrillary tangles in the brains of Alzheimer's disease patients. The expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-2, calcium and chloride channels, prolactin, and growth hormone (GH), all of which are associated with brain function, were also up-regulated. In the hippocampus, only transthyretin mRNA was upregulated. Transthyretin plays a role in hormone transport in the brain and possibly a neuroprotective role by amyloid-β sequestration. This study reveals that diets supplemented with Ginkgo biloba extract have notable neuromodulatory effects in vivo and illustrates the utility of genome-wide expression monitoring to investigate the biological actions of complex extracts.

Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B

Human cytomegalovirus (HCMV) infection alters the expression of many cellular genes, including IFN-stimulated genes (ISGs) [Zhu, H., Cong, J.-P., Mamtora, G., Gingeras, T. & Shenk, T. (1998) Proc. Natl. Acad. Sci. USA 95, 14470–14475]. By using high-density cDNA microarrays, we show that the HCMV-regulated gene expression profile in fibroblasts does not differ substantially from the response generated by IFN. Furthermore, we identified the specific viral component triggering this response as the envelope glycoprotein B (gB). Cells treated with gB, but not other herpesviral glycoproteins, exhibited the same transcriptional profile as HCMV-infected cells. Thus, the interaction of gB with its as yet unidentified cellular receptor is the principal mechanism by which HCMV alters cellular gene expression early during infection. These findings highlight a pioneering paradigm for the consequences of virus–receptor interactions.