Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays

BACKGROUND: Long oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear. RESULTS: Unmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 μm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR. CONCLUSIONS: Microarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied

The TIGR Rice Genome Annotation Resource: improvements and new features

In The Institute for Genomic Research Rice Genome Annotation project (), we have continued to update the rice genome sequence with new data and improve the quality of the annotation. In our current release of annotation (Release 4.0; January 12, 2006), we have identified 42 653 non-transposable element-related genes encoding 49 472 gene models as a result of the detection of alternative splicing. We have refined our identification methods for transposable element-related genes resulting in 13 237 genes that are related to transposable elements. Through incorporation of multiple transcript and proteomic expression data sets, we have been able to annotate 24 799 genes (31 739 gene models), representing ∼50% of the total gene models, as expressed in the rice genome. All structural and functional annotation is viewable through our Rice Genome Browser which currently supports 59 tracks. Enhanced data access is available through web interfaces, FTP downloads and a Data Extractor tool developed in order to support discrete dataset downloads

Nerve growth factor regulation of m4 muscarinic receptor mRNA stability but not gene transcription requires mitogen-activated protein kinase activity

Nerve growth factor (NGF) up-regulated steady-state levels of m4 muscarinic acetylcholine receptor (mAChR) mRNA in PC12 cells. Up-regulation of mRNA levels was associated with a corresponding increase in mAChR binding sites. Two other growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), up-regulated m4 mRNA and mAChR binding sites. Treatment of PC12 cells with NGF and bFGF, but not EGF, has previously been demonstrated to result in sustained activation of mitogen-activated protein kinase (MAPK). Analogously, NGF and bFGF, but not EGF, increased the stability of m4 mRNA in PC12 cells. In HER-PC12 cells, a clonal PC12 cell transfectant overexpressing EGF receptors and displaying sustained MAPK activation upon receptor stimulation, EGF treatment stabilized the m4 transcript. A synthetic inhibitor of MAPK kinase, PD98059, inhibited growth factor-induced stabilization of the m4 transcript in both PC12 and HER-PC12 cells. These findings demonstrate that the MAPK pathway is involved in transcript stabilization. Cycloheximide pretreatment abolished the post- transcriptional effect of NGF, indicating that de novo protein synthesis was required for the observed increase in m4 mRNA stability. By contrast, cycloheximide had no discernible post-transcriptional effect if added after NGF treatment, suggesting that an inducible yet stable protein factor was involved in m4 mRNA decay. An unusually well conserved 137 nucleotides of m4 3\u27-untranslated region has been identified by sequence comparison with other mRNAs that are post-transcriptionally regulated by NGF. In PC12 cells that heterologously overexpress this region, we demonstrate that NGF no longer stabilizes endogenous m4 mRNA. This conserved region probably represents an NGF-responsive element involved in mRNA stability regulation. Finally, transcription of the m4 gene can be induced by all three growth factors but is not dependent on MAPK activity, unlike growth factor-induced m4 mRNA stabilization

Adenosine A(2A) receptor mRNA regulation by nerve growth factor is TrkA- , Src-, and Ras-dependent via extracellular regulated kinase and stress- activated protein kinase/c-Jun NH2-terminal kinase

We have shown previously that nerve growth factor (NGF) down-regulates adenosine A(2A) receptor (A(2A)AR) mRNA in PC12 cells. To define cellular mechanisms that modulate A(2A)AR expression, A(2A)AR mRNA and protein levels were examined in three PC12 sublines: i) PC12nnr5 cells, which lack the high affinity NGF receptor TrkA, ii) srcDN2 cells, which overexpress kinase- defective Src, and iii) 17.26 cells, which overexpress a dominant-inhibitory Ras. In the absence of functional TrkA, Src, or Ras, NGF-induced down- regulation of A(2A)AR mRNA and protein was significantly impaired. However, regulation of A(2A)AR expression was reconstituted in PC12nnr5 cells stably transfected with TrkA. Whereas NGF stimulated the mitogen-activated protein kinases p38, extracellular regulated kinase 1 and 2 (ERK1/ERK2), and stress- activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in PC12 cells, these kinases were activated only partially or not at all in srcDN2 and 17.26 cells. Inhibiting ERK1/ERK2 with PD98059 or inhibiting SAPK/JNK by transfecting cells with a dominant-negative SAPKβ/JNK3 mutant partially blocked NGF-induced down-regulation of A(2A)AR expression in PC12 cells. In contrast, inhibiting p38 with SB203580 had no effect on the regulation of A(2A)AR mRNA and protein levels. Treating SAPKβ/JNK3 mutant-transfected PC12 cells with PD98059 completely abolished the NGF-induced decrease in A(2A)AR mRNA and protein levels. These results reveal a role for ERKI/ERK2 and SAPK/JNK in regulating A(2A)AR expression

A role of p75 in NGF-mediated down-regulation of the A(2A) adenosine receptors in PC12 cells

Nerve growth factor (NGF) induces differentiation of the rat pheochromocytoma clone (PC12) by activating the high affinity receptor, p140(trkA), linked to mitogen-activated protein kinase. While the physiological role of the low affinity NGF receptor (p75) has not been clearly defined, this receptor promotes activation of nuclear factor (NF) κB in Schwann cells. PC12 cells express the A(2A) adenosine receptor (AR), whose expression is significantly decreased by NGF treatment. In this study, we determined whether TrkA or p75 is involved in NGF-mediated regulation of A(2A)AR expression. NGF treatment decreased A(2A)AR in a time-dependent manner, with maximal effects observed by 1 day, and continued down-regulation of the receptor for up to 3 days in the presence of NGF. The decrease in A(2A)AR was associated with a more delayed decrease in the steady-state levels of the A(2A)AR mRNA. Down-regulation of the A(2A)AR at 1 day was mimicked by activators of NFκB such as H2O2, and ceramide and was attenuated by the inhibitor pyrrolidine dithiocarbamate or following transient transfection of PC12 cells with a dominant negative IκBα mutant. Moreover, NGF stimulated nuclear accumulation of p65 subunits of NFκB (but not p50 subunits) in PC12 cells, as determined by electrophoretic mobility shift assays and by Western blotting. In contrast, inhibition of TrkA by AG879 or of TrkA-dependent mitogen-activated protein kinase mitogen-activated protein kinase kinase with PD98059 blocked PC12 cell differentiation without affecting A(2A)AR down-regulation, suggesting dissociation be- tween these two phenomena. Taken together, these data provide strong support for the involvement of the p75/NFκB pathway in NGF-mediated down-regulation of A(2A)AR in PC12 cells

Gene expression profile of the aging process in rat liver: Normalizing effects of growth hormone replacement

The mechanisms that control life span and age-related phenotypes are not well understood. It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. To test this hypothesis, and to improve our understanding of the cellular and molecular mechanisms behind the aging process, we have analyzed age-induced changes in gene expression patterns through the application of DNA chip technology. In the present study, the aging process was analyzed in rat liver in the presence or absence of GH replacement. Out of 3,000 genes printed on the microarrays, approximately 1,000 were detected in the rat liver. Among these, 47 unique transcripts were affected by the aging process in male rat livers. The largest groups of age-regulated transcripts encoded proteins involved in intermediary metabolism, mitochondrial respiration, and drug metabolism. Approximately 40% of the differentially expressed gene products were normalized after GH treatment. The majority of those transcripts have previously not been shown to be under GH control. The list of gene products that showed normalized expression levels in GH-treated old rats may shed further insight on the action and mechanism behind the positive effects of GH on, for example, fuel metabolism and body composition observed in different animal and human studies

Molecular cloning, tissue-specific expression, and chromosomal localization of a novel nerve growth factor-regulated G-protein-coupled receptor, nrg-1

A novel and differentially expressed gene, named nrg-1, was identified by EST expression profiling and subsequently isolated as a 2.2-kb full-length clone from a rat PC12 cell cDNA library. Sequence analysis reveals that nrg-1 encodes a putative seven transmembrane spanning domain protein with structural features characteristic of receptors belonging to the G-protein- coupled receptor gene superfamily. The 400-amino-acid protein encoded by nrg- 1 exhibits a high degree of sequence identity (40-44%) to the Edg receptor family; members include Edg-1, Edg-2, Edg-3, Edg-4, and H218. Both Northern analysis and EST expression profiling revealed that whole-tissue distribution of nrg-1 mRNA is restricted, found almost exclusively in brain. Transcripts of nrg-1 could be ubiquitously detected in different brain regions, with very prominent expression in lower brain regions such as the midbrain, pons, medulla, and spinal cord. In PC12 cells, nerve growth factor induces neuronal differentiation and repressed expression of nrg-1. Two other agents that differentiate PC12 cells, fibroblast growth factor and dibutyryl cAMP, down- regulated nrg-1 mRNA levels. Epidermal growth factor, an agent that does not induce differentiation, did not repress nrg-1 mRNA levels. In a PC12 cell mutant that is deficient in protein kinase A activity (AB.11), all three differentiating agents were unable to downregulate nrg-1 mRNA. Hence, protein kinase A appears to be an obligatory cellular component in nrg-1 mRNA regulation. Chromosomal mapping employing a rat somatic cell radiation hybrid panel demonstrated that nrg-1 is linked to marker D8Rat54 and tightly associated with H218 on chromosome 8

Identification of H-Ras, RhoA, Rac1 and Cdc42 responsive genes

The superfamily of small GTP-binding proteins has expanded dramatically in recent years. The Ras family has long been associated with signaling pathways contributing to normal and aberrant cell growth, while Rho-related protein function is to integrate extracellular signals with specific targets regulating cell morphology, cell aggregation, tissue polarity, cell motility and cytokinesis. Recent findings suggest that certain Rho proteins, including RhoA, Racl and Cdc42, can also play a role in signal transduction to the nucleus and cell growth control. However, the nature of the genes regulated by Ras and Rho GTPases, as well as their contribution to their numerous biological effects is still largely unknown. To approach these questions, we investigated the global gene expression pattern induced by activated forms of H-Ras, RhoA, Rac1 and Cdc42 using cDNA microarrays comprising 19 117 unique elements. Using this approach, we identified 1184 genes that were up- or downregulated by at least twofold. Hierarchical cluster analysis revealed the existence of patterns of gene regulation both unique and common to H-Ras V12, RhoA QL, Rac1 QL and Cdc42 QL activation. For example, H-Ras V12 upregulated osteopontin and Akt 1, and H-Ras and RhoA stimulated cyclin G1, cyclin-dependent kinase 8, cyclin A2 and HMGI-C, while Rac1 QL and Cdc42 QL upregulated extracellular matrix and cell adhesion proteins such as alpha-actinin 4, procollagen type I and V and neuropilin. Furthermore, H-Ras V12 downregulated by \u3e eightfold 52 genes compared to only three genes by RhoA QL, Rac1 QL and Cdc42 QL. These results provide key information to begin unraveling the complexity of the molecular mechanisms underlying the transforming potential of Ras and Rho proteins, as well as the numerous morphological and cell cycle effects induced by these small GTPases