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American Society of Plant Biologists New Molecular Phenotypes in the dst Mutants of Arabidopsis Revealed by DNA Microarray Analysis
a Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 3 To whom correspondence should be addressed. E-mail green{at}msu.edu; fax 517-353-9168
In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.
Cells must be able to adjust their gene expression patterns quickly to respond to intracellular and extracellular stimuli; therefore, it is necessary for certain transcripts to reach new steady state levels rapidly. The steady state levels of eukaryotic mRNAs are determined by both their rate of synthesis and their rate of degradation. The control of mRNA stability is a major determinant of steady state mRNA levels in the cell and often has a great impact on the level at which a particular gene is expressed. Furthermore, mRNA stability affects the rate at which new steady state RNA levels are achieved after changes in transcription; the more unstable the mRNA, the more rapidly it reaches a new steady state (Abler and Green, 1996  ).
Most recent studies on mRNA stability in eukaryotes have focused on transcripts that are relatively unstable, generally with half-lives of <60 min (Ross, 1995
The majority of mRNAs fall in the stable range of mRNA half-lives for a given organism (Taylor and Green, 1995
The most widely studied instability determinants are the mammalian AU-rich elements (AREs). AREs are found in the 3' untranslated region (UTR) of several of the most unstable mammalian transcripts, such as lymphokine, cytokine, and protooncogene mRNAs (Chen and Shyu, 1995
In plants, in addition to AREs, an instability determinant called DST (downstream) (Newman et al., 1993
To gain insights into the cellular components involved in the DST-mediated mRNA degradation pathway, we isolated Arabidopsis mutants defective in their ability to recognize DST elements. dst1 and dst2 were isolated based on their ability to stabilize specific DST-containing transgene mRNAs (Johnson et al., 2000
Genes regulated differentially in mutant backgrounds have been identified in the past using a variety of techniques, such as differential display and subtractive hybridization (Kehoe et al., 1999
Recently published studies have demonstrated how microarrays containing a large number of Arabidopsis genes can provide a powerful tool for plant gene discovery, functional analysis, and elucidation of genetic regulatory networks (Schenk et al., 2000
In this report, we describe the use of DNA microarrays containing >11,000 Arabidopsis ESTs, representing
Generation of a 600-Element DNA Microarray As a first step toward analyzing the dst1 mutant for additional changes in gene expression, a DNA microarray representing  600 Arabidopsis genes was assembled. The clones included were mainly ESTs from the Michigan State University (MSU) collection (Newman et al., 1994). Table 1 includes a description of these genes, grouped according to function or sequence similarity. Because the dst1 mutant affects RNA metabolism, half of the clones included in the microarray were those predicted to be associated with some aspect of RNA metabolism. The criterion for the selection of many of these EST clones was their sequence similarity to potential RNA metabolism genes described previously in Arabidopsis or other organisms.
To date, the SAUR-AC1 transcript is the only known target for the DST-dependent mRNA degradation pathway in Arabidopsis (Johnson et al., 2000 ). To identify additional direct targets of this pathway, genes with possible DST elements were included. The strategy to identify genes containing potential DST elements was to combine the results of several pattern searches using different degenerate criteria, because our knowledge of what constitutes a functional DST element is still limited (Newman et al., 1993; Sullivan and Green, 1996). Because AREs also function in plants as mRNA instability elements (Ohme-Takagi et al., 1993), genes with AU-rich elements were selected in a similar manner by searching for variations of the element AUUUA in an AU-rich context in the 3' UTR. As a positive control, the HPH and GUS coding sequences also were included so that the mutant phenotype of dst1 could be confirmed. Approximately 300 additional clones were included in the microarray to expand coverage beyond genes associated with RNA metabolism. A complete list of the clones in this array and the raw microarray expression data can be found at http://www.bch.msu.edu/pamgreen/Perez-Amador_etal/600_list.htm.
Expression Levels for Most Genes Are Similar in dst1 and the Parental Plants To determine whether additional molecular differences existed, poly(A)+ RNA from leaves of 40-day-old dst1 and parental plants was extracted and used in a DNA microarray experiment. After hybridization and normalization of data, several genes were identified with changes in mRNA levels >1.5-fold. As expected, HPH-DST and GUS-DST were among the genes with increased mRNA levels in dst1. SAUR-AC1 also was increased in the mutant. In addition, a DEAD box RNA helicase RH15 (At5g11200) and EST 125O9T7 were identified as being increased at the mRNA level in the dst1 mutant. When the sequence from EST 125O9T7 was compared with the TAIR database (http://www.arabidopsis.org/), the translation showed high similarity (63.7%) to the SAUR-AC1 protein and several other SAUR-like proteins, as depicted in Figure 1A . As a result, this gene was named SAUR-like1. Most interestingly, although the 3' UTR of this gene lacks a classic DST element, it contains multiple DST-like subdomains (i.e., two ATAGAT-like and three GTA-like subdomains), as shown in Figure 1B.
RNA gel blot analysis was used to confirm the differential regulation of these genes identified by DNA microarray analysis. In previous experiments, RNA gel blot analysis had proven an effective method to measure subtle differences in mRNA abundance between dst1 and parental plants (Johnson et al., 2000 ). In addition, this approach has other advantages. RNA gel blot analysis traditionally has been relied on for accurate analysis of gene expression at the level of mRNA abundance. Moreover, once differentially abundant mRNAs are identified using one combination of mutant and wild type, these mRNAs can be investigated easily in additional mutants. Finally, RNA gel blot analysis provides the means to test the biological reproducibility of data obtained in microarray experiments, because additional samples can be tested readily. Accordingly, the dst2 mutant was included in this analysis to compare the molecular phenotypes between dst1 and dst2. Like dst1, dst2 has no known phenotype other than increased HPH-DST, GUS-DST, and SAUR-AC1 mRNA levels caused by increased stability.
EST clones to be investigated were radiolabeled and used as probes on the RNA gel blots. The translation elongation factor eIF4A, which was used to normalize RNA gel blot data as described previously (Johnson et al., 2000
11,521-Element Microarray Reveals Additional Genes with Altered Gene Expression in the dst1 Mutant To expand on the 600-element microarray analysis, high-density arrays containing 11,521 clones (prepared at the Arabidopsis Functional Genomics Consortium DNA microarray facility at MSU) were used. Poly(A)+-selected RNA from leaves of 5-week-old dst1 and parental plants was extracted, labeled with Cy3- and Cy5-CTP, and used for the hybridization. Analysis was performed using four independently grown sample sets to control for biological variability. Each sample set consisted of the dst1 mutant and parental plants. For each set, two microarrays were used, each with reverse labeling, for a total of eight microarray slides (raw data are available at http://genome-www4.stanford.edu/cgi-bin/SMD/cluster/QuerySetup.pl, under the experimenter name Green). DNA microarray expression data were normalized as indicated in Methods to calculate the ratios of the fluorescence intensities of the two probes. The number of clones with a ratio >1.5-fold was determined. Although a relatively low cutoff ratio of 1.5 was used, the data were reproducible and were confirmed by RNA gel blot analysis (see below). When the expression data, represented as the median of eight microarray slides, were plotted, 36 clones (31 ESTs and five clones for HPH and GUS) with  1.5-fold increased or decreased mRNA levels could be identified (Figure 3)
. Most of these clones showed a difference of 1.5-fold or greater on at least seven of the eight slides analyzed. Each EST was mapped to the Arabidopsis genome by conducting a BLAST search against the Arabidopsis genome sequence database. Multiple ESTs representing the same gene also were identified in this manner, revealing that the 31 ESTs corresponded to 25 genes. The redundancy among the ESTs present in the array allowed us to verify the microarray data further, because ESTs for the same genes showed similar changes in expression. In some cases, ESTs corresponding to the same gene showed slightly different expression ratios (e.g., the four ESTs corresponding to RAP2.4 ranged from two- to five-fold higher in dst1, with a mean of 3.9-fold); this variability could be attributable to variable probe or target length, genetic redundancy, or a combination thereof. The transgenes HPH and GUS, as well as the DEAD box RNA helicase gene RH15, again were detected with increased mRNA levels in dst1.
Twenty genes, including the transgenes HPH and GUS, showed increased mRNA levels, whereas seven displayed decreased levels of mRNA in the dst1 mutant compared with those of the parental plants, as listed in Tables 2 and 3, respectively. After correction for redundancy in the ESTs and excluding the transgenes HPH and GUS, 25 endogenous genes whose expression levels were altered in dst1 were identified. The probable biological functions of these genes, listed in Tables 2 and 3, were based on annotation by the Munich Information center for Protein Sequences (MIPS) Arabidopsis thaliana database. The 3' UTRs of all of the genes identified as being regulated differentially in dst1 were analyzed for the presence of either a classic DST element or DST-like subdomains. Seven genes were identified that contain possible DST-like sequences in their 3' UTRs (Table 4). Therefore, these genes could be hypothesized to be primary targets of the DST-mediated decay pathway that is deficient in the mutant.
RNA Gel Blot Analysis Confirms DNA Microarray Data To determine whether the transcript changes identified in the dst1 mutant by microarray analysis were reliable, total RNA was obtained from the same plants that were used for each of the four microarray experiments and examined by RNA gel blot analysis. Several genes exhibiting altered mRNA levels in the dst1 mutant as determined from analysis of the 11,521 microarrays were tested. All of the genes exhibited increased or decreased mRNA abundance, as expected. Although mRNA changes were relatively small (1.5- to 3.9-fold), in all cases, differences in gene expression detected by the microarray translated into similar fold differences as determined by RNA gel blot analysis (Figure 4) .
Again, RNA levels in dst2 also were monitored in these experiments. Using this approach, molecular markers specific for each dst mutant were identified. Most of the genes that had increased mRNA abundance in the dst1 mutant were unaffected in the dst2 mutant. As demonstrated in Figure 5A , RAP2.4, RH15, and ESTs 120E6T7, F2H12T7, and 141P19T7 showed mRNA levels similar to those of the parental plants in the dst2 mutant. HPH and GUS mRNA levels were increased in both mutants. The EST for one gene, 168C16T7, which shows high similarity to a cadmium-induced AP2 protein, had mRNA levels that were increased in both mutants, although the increase was greater in dst1 than in dst2 ( 3.5-fold versus two-fold compared with that of the parental type). EST 139I3T7, which encodes an unknown protein, may have marginally reduced mRNA levels in the dst2 mutant.
Of particular interest was the analysis of the seven genes with lower mRNA abundance in dst1 compared with that in the parental plants. Most of these genes either were increased (ESTs 111G9T7, E6E1T7, 170N19T7, and H5A10T7) or unaffected (ESTs 171P10T7 and 201O1T7) in the dst2 mutant, as is evident from the histograms shown in Figure 5B. The only exception was EST G4A11T7; this EST codes for an unknown protein, and the mRNA levels for this gene also were diminished in dst2. SAUR-AC1 and SAUR-like1 also were analyzed by RNA gel blot analysis and found to be increased in both the dst1 and dst2 mutants (Figure 2), although they were beneath our detection limit on the majority of the slides used for the microarray experiments. These signals were below detection because, in most experiments, the signals were too close to background to calculate a valid ratio. However, in the experiments in which we could detect a valid ratio, increase in dst1 was observed, as expected (data not shown).
Multiple Replicates Remove Nonreproducible Changes
To monitor the reproducibility of the microarray hybridizations, for individual pairs of technical replica slides, the number of clones with a ratio >1.5 was calculated. For each, a clone was considered reproducible if it was represented in the final set of 36 clones showing
Cluster Analysis of Genes with Altered Expression Levels in dst1 Clustering allows the grouping of genes with similar expression profiles and provides a holistic view because coexpressed genes involved in similar processes provide clues regarding the biological significance of the pathway being studied. We used cluster analysis to compare gene expression data for the 25 genes identified in this study using data from the 47 Arabidopsis microarray experiments (113 slides) available in the Stanford Microarray Database (SMD) (http://genome-www.stanford.edu/Microarray) at the time of the analysis. A portion of the resulting clusters is shown in Figure 7 . As expected, when EST redundancy was not removed, ESTs representing the same gene clustered next to or in the immediate vicinity of each other, thus validating the observed pattern of gene expression (data not shown).
Although the cluster analysis was performed using the limited number of genes that were identified in this study, certain common expression patterns could be found. The most prominent cluster is composed of three genes that are regulated in a diurnal fashion and share several other expression characteristics. This cluster is characterized by genes whose transcripts are decreased in abundance in the dst1 mutant and include genes encoding a protein similar to a stem-specific protein, senescence-associated protein, and xylosidase. These genes show tissue-preferential expression with a high level of expression in leaves and a low level in flowers and roots, and are increased by light. Two of the three genes in this cluster, senescence-associated protein and xylosidase, have unstable transcripts (see Discussion), contain possible DST-like sequences in their 3' UTRs, and are increased in the dst2 mutant. Apart from the aforementioned cluster, a second cluster of coordinately expressed genes was identified. This cluster includes genes for chalcone synthase and a protein similar to chalcone flavanone isomerase, both of which show increased mRNA levels in the dst1 mutant. These genes are regulated in a diurnal fashion as well but are decreased by light and have higher levels of expression in flowers compared with leaves or roots. In addition, these mRNAs are increased in a mutant defective for protein import into chloroplasts. No other common characteristic gene expression patterns could be seen. However, eight genes (senescence-associated protein, putative patatin protein, protein similar to CCR, putative membrane channel protein, protein similar to APR2, calmodulin-like protein, an unknown protein, and chalcone synthase) are regulated in a circadian manner, which suggests a possible connection between the DST-mediated decay pathway and circadian rhythms. Four of these eight genes also contain DST-like subdomains in their 3' UTRs and could be direct targets of the dst1 mutation.
Microarray technology has great potential for the characterization of mutants, especially those with no visible aberrant phenotype. Here, we report on the dst1 mutant, which fits into this category. To evaluate the molecular phenotypes of this mutant, DNA microarray technology was used, which allowed the identification of primary as well as secondary effects and also revealed clues to the identification of possible relationships among genes.
Transcripts Altered in dst1 Have Predominantly Increased Levels Although Some Decrease in Abundance
Our analysis showed that the expression of several genes decreases in dst1. Decreases in gene expression could be explained by the same defect if the DST recognition component acts as both a repressor and an activator in a context-dependent manner, similar to some transcriptional regulators. For example, Drosophila Drap1 and dCtBP are bifunctional transcription factors with distinct activation and repression functions (Phippen et al., 2000
The genes identified in this study encode proteins involved in a variety of cellular processes. The DEAD box RNA helicase, RH15, is part of a large gene family in Arabidopsis. RNA helicases are primarily RNA-unwinding enzymes and have been implicated in a variety of molecular processes, including mRNA splicing, ribosome assembly, and translation initiation (Aubourg et al., 1999
Most transcripts that are affected in the dst1 mutant relative to the parental type are expected to fall into one of two categories. One category consists of mRNAs that are the direct targets of the DST-mediated decay pathway, the primary transcripts, whereas the second category consists of transcripts, referred to as secondary transcripts, that are increased or diminished as a result of the secondary effect of changes in the primary transcripts. A number of genes showing altered levels of gene expression in dst1 may be caused by secondary effects, because these genes do not appear to contain a consensus DST element or DST-like subdomains in their 3' UTRs. Secondary effects would be expected if altered levels of DST-containing mRNAs influence the abundance of other RNAs. This situation is not unique to dst1; secondary effects would be expected in all microarray experiments comparing mutants with the wild type. However, this study allowed us to predict the primary and secondary effects of the dst1 mutation based on the presence or absence of DST-like sequences, respectively. Although the precise sequences required for a functional DST element have not been elucidated fully, our data provide us with the advantage of identifying at least some of the most likely primary effects. For example, none of the 140 possible DST-containing sequences found by motif searching tools in the 600-element array were affected in dst1. This finding suggests that the DST element is more complex in Arabidopsis than in soybean and that it is difficult to identify this element with simple sequence search tools. It appears that multiple subdomains of the DST element are sufficient for its function as an instability determinant, because seven of the 25 genes identified contain DST-like subdomains. Earlier mutagenesis studies of individual subdomains also are consistent with this hypothesis (Feldbrügge et al., 2001 Additionally, initial microarray experiments conducted to monitor mRNA stability on a global basis have indicated that a number of transcripts identified in this study may be unstable (R.A. Gutiérrez, R.M. Ewing, J.M. Cherry, and P.J. Green, unpublished data). These results are encouraging for future studies, because primary targets of the DST-mediated decay pathway are expected to be stabilized in the dst1 mutant.
Molecular Markers to Expedite the Characterization of and Differentiation between dst1 and dst2
Before this study, it was difficult to distinguish between dst1 and dst2 because the known phenotypes of the two mutants were identical and F2 progeny of a cross between the mutants do not show additive increases in the abundance of DST-containing mRNAs (Johnson et al., 2000
Circadian Association of the dst1 Mutation
Conclusions and Future Prospects
As a result of the stringency of our parameters to avoid false positives, the 25 genes identified in this study are most likely an underrepresentation of the molecular phenotypes of dst1. For example, ratios derived from the microarray results from lowly expressed genes, such as SAUR-AC1 and SAUR-like1, which have low levels of expression in the absence of auxin (McClure and Guilfoyle, 1987 Beyond extending our knowledge of the DST-mediated decay pathway in plants, this study provides additional general insights. Our study shows that subtle changes in gene expression can be measured reliably using multiple microarrays, which should enhance the global investigation of gene expression patterns under several conditions. This analysis demonstrates that new molecular phenotypes for mutants without a visible phenotype can be identified using DNA microarray technology. Also, with full genome microarrays, it should be possible to catalog all of the molecular phenotypes for any mutant. Furthermore, new hypotheses and associations, such as the potential link between the dst1 mutation and circadian rhythms, can be developed using the publicly available databases. Future mutant analyses via DNA microarray analysis should have even greater utility, particularly for the analysis of mutants identified by reverse genetic approaches (e.g., T-DNA insertions) that have no apparent mutant phenotype.
Plant Material All Arabidopsis thaliana plants described in this report are from the accession Columbia; they were grown in growth chambers under 16 hr of light and 60% relative humidity at 20°C. Tissue from the parental line (p1519-31) and dst1 and dst2 homozygous mutants (Johnson et al., 2000 ) were harvested from 35- to 40-day-old plants. All tissue was harvested from plants grown in parallel under the same conditions in different growth chambers. Seed from the dst mutants and the parental line are freely available upon request for basic research from the corresponding author. They cannot be distributed using Arabidopsis Biological Resource Center (Ohio State University, Columbus) accession numbers because they contain proprietary materials.
Generation of a 600-Element DNA Microarray
Selected Arabidopsis EST clones were obtained from the PRL2 EST collection (Newman et al., 1994 Plasmid DNA from the Michigan State University (MSU) collection was diluted to a final concentration of 1 to 3 ng·µL-1 in TE (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). PCR reactions, in a final volume of 100 µL, contained 40 pmol of the corresponding primers, 0.2 mM of each deoxynucleotide triphosphate (dNTP), 2 to 5 ng of plasmid DNA, and 2 units of Taq DNA polymerase in 1 x reaction buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, and 2 mM MgCl2). After PCR, DNA was precipitated in ethanol and resuspended in 20 µL of 3 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). To determine the quality and quantity of the amplified DNA, 1 µL of the final resuspension was analyzed by electrophoresis in 1.0% agarose gels. PCR products with low concentration (<200 ng·µL-1) or showing multiple bands were discarded and replaced with a new PCR amplification product derived from a different EST clone corresponding to the same gene. If no alternative EST was available, a new PCR amplification was performed using a gene-specific primer designed for the 5' end of the EST clone along with the corresponding 3' vector primer. In this way, we ensured that only high-quality PCR products were included on the 600-element DNA microarray. To confirm the identity of the amplified DNAs, we sequenced 12 randomly selected clones. In each case, the sequence obtained matched the EST sequence deposited in the database.
For clones obtained from genomic DNA, DNA was extracted from total aboveground tissue of mature Arabidopsis plants using the method of Dellaporta et al. (1983) DNA from the PCR reactions was transferred to master DNA plates and stored at 4°C until printing. The final concentration of DNA for printing was estimated to be 200 to 400 ng·µL-1. DNAs were arranged as four subgrids of 12 x 13 and printed twice on poly-L-lysine–coated slides. Printing, handling, and use of the 600-element DNA microarray were performed with the same methods used for the 11,521-element MSU DNA microarray, as indicated below.
11,521-Element Arabidopsis Functional Genomics Consortium DNA Microarray
Total RNA Extraction, Poly(A)+ RNA Purification, and RNA Gel Blot Hybridization
Labeling of Poly(A)+ RNA
DNA Microarray Hybridization and Analysis Hybridization was performed in a water bath at 65°C for 12 to 20 hr. After hybridization, the microarray was washed for 5 min in 1 x SSC and 0.2% SDS, 5 min in 0.1 x SSC and 0.2% SDS, and 30 sec in 0.1 x SSC without SDS. Then it was dried by centrifugation at 62g for 5 min. The slide was scanned once in a ScanArray 3000 or 5000 (GSI Lumonics) for both channels 1 and 2 (corresponding to Cy3- and Cy5-labeled probes, respectively) at 10-µm resolution. The image files obtained were analyzed using ScanAlyze software (version 2.32; M. Eisen, Stanford University, http://genome-ww4.stanford.edu/MicroArray/SMD/restech.html). To ensure that only spots of high quality were used in the analysis, quality control measurements produced by the ScanAlyze software were used. For example, the GTB2 value represents the fraction of pixels within each spot that are >1.5-fold of the background measurement. Spots with GTB2 values <0.50 for either channel were removed and not considered for further analysis.
Data from each channel were transformed to the natural logarithm, and a Z score was calculated to normalize the channel values to account for variation in RNA labeling. For the Z score calculation [Z = (
Hierarchical clustering was performed using Cluster and Treeview software (Eisen et al., 1998 Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use in noncommercial research purposes.
Accession Numbers
We thank Dr. Christoph Benning for critical reading of the manuscript and members of the Green laboratory for helpful discussions. We also thank Tom Newman for donating EST clones and the members of the microarray facility of the Arabidopsis Functional Genomics Consortium (AFGC) at MSU for technical advice. Zihua Hi is acknowledged for helping to collect some of the clones for the 600 element array. This work was funded by grants from the United States Department of Agriculture (2000-014191), the Department of Energy (DE-FG02-91ER20021), and the National Science Foundation (DBN987638) to P.J.G. M.A.P.-A. received postdoctoral fellowships from North Atlantic Treaty Organization–Spain and the Ministerio de Educatión y Ciencia, Spain.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010295.
1 Current address: Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investi-gaciones Científicas, Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, 46022 Valencia, Spain.
2 Current address: Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637. Received July 20, 2001; accepted September 24, 2001.
Abler, M.L., and Green, P.J. (1996). Control of mRNA stability in higher plants. Plant Mol. Biol. 32, 63–78.[CrossRef][Web of Science][Medline]
Altschul, S.F., Madden, T.L., Schaffer, A.A, Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.
Aubourg, S., Kreis, M., and Lecharny, A. (1999). The DEAD box RNA helicase family in Arabidopsis thaliana. Nucleic Acids Res. 27, 628–636.
Buzby, J.S., Brewer, G., and Nugent, D.J. (1999). Developmental regulation of RNA transcript destabilization by A + U-rich elements is AUF1-dependent. J. Biol. Chem. 274, 33973–33978. Chen, C.Y.A., and Shyu, A.B. (1995). AU-rich elements: Characterization and importance in mRNA degradation. Trends Biochem. Sci. 20, 465–470.[CrossRef][Web of Science][Medline] Dellaporta, S.L., Wood, J., and Hicks, J.B. (1983). A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 1, 19–21.
DeRisi, J.L., Iyer, V.R., and Brown, P.R. (1997). Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686.
Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998). Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868. Feinberg, A.P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.[CrossRef][Web of Science][Medline] Feldbrügge, M., Arizti, M.L., Zamore, P.D., Belasco, J.G., and Green, P.J. (2001). Comparative analysis of the plant mRNA-destabilizing element DST, in mammalian and tobacco cells. Plant Mol. Biol., in press.
Franco, A.R., Gee, M.A., and Guilfoyle, T.J. (1990). Induction and superinduction of auxin-responsive mRNAs with auxin and protein synthesis inhibitors. J. Biol. Chem. 265, 15845–15849. Gil, P., and Green, P.J. (1996). Multiple regions of the Arabidopsis SAUR-AC1 gene control transcript abundance: The 3' untranslated region functions as an mRNA instability determinant. EMBO J. 15, 1678–1686.[Web of Science][Medline] Greenberg, M.E., and Belasco, J.G. (1993). Control of the decay of labile protooncogene and cytokine mRNAs. In Control of Messenger RNA Stability, J. Belasco and G. Brawerman, eds (San Diego, CA: Academic Press), pp. 199–218.
Harmer, S.L., Hogenesch, J.B., Straum, M., Chang, H.S., Han, B., Zhu, T., Wang, X., Kreps, J.A., and Kay, S.A. (2000). Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290, 2110–2113. Higgs, D.C., Barnes, L.J., and Colbert, J.T. (1995). Abundance and half-life of the distinct oat phytochrome A3 and A4 mRNAs. Plant Mol. Biol. 29, 367–377.[Medline] Johnson, M.A., Baker, E.J., Colbert, J.T., and Green, P.J. (1998). Determinants of mRNA stability in plants. A look beyond transcription. In Mechanisms Determining mRNA Stability and Translation in Plants, J. Bailey-Serres and D.R. Gallie, eds (Rockville, MD: American Society of Plant Physiologists), pp. 40–53.
Johnson, M.A., Perez-Amador, M.A., Lidder, P., and Green, P.J. (2000). Mutants of Arabidopsis defective in a sequence-specific mRNA degradation pathway. Proc. Natl. Acad. Sci. USA 97, 13991–13996. Kehoe, D.M., Villand, P., and Somerville, S. (1999). DNA microarrays for studies of higher plants and other photosynthetic organisms. Trends Plant Sci. 4, 38–41.[CrossRef][Web of Science][Medline]
Kiledjian, M., DeMaria, C.T., Brewer, G., and Novick, K. (1997). Identification of AUF1 (heterogeneous nuclear ribonucleoprotein D) as a component of the
Loflin, P., Chen, C.Y.A., and Shyu, A.B. (1999). Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element. Genes Dev. 13, 1884–1897. McClure, B.A., and Guilfoyle, T. (1987). Characterization of a class of small auxin-inducible soybean polyadenylated RNAs. Plant Mol. Biol. 9, 611–623.[CrossRef][Web of Science]
McClure, B.A., and Guilfoyle, T.J. (1989). Rapid redistribution of auxin-regulated RNAs during gravitropism. Science 243, 91–93.
McClure, B.A., Hagen, G., Brown, C.S., Gee, M.A., and Guilfoyle, T.J. (1989). Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean. Plant Cell 1, 229–239.
Moser, M.J., Holley, W.R., Chatterjee, A., and Mian, I.S. (1997). The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains. Nucleic Acids Res. 25, 5110–5118.
Newman, T.C., Ohme-Takagi, M., Taylor, C.B., and Green, P.J. (1993). DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobacco. Plant Cell 5, 701–714. Newman, T.C., de Bruijn, F., Green, P.J., Keegstra, K., Kende, H., McIntosh, L., Ohlrogge, J., Raikhel, N.V., Somerville, S.C., Tomashow, M., Retzel, E., and Somerville, C.R. (1994). Genes galore: A summary of methods for accessing results from partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol. 106, 1241–1255.[Abstract]
Ohme-Takagi, M., Taylor, C.B., Newman, T.C., and Green, P.J. (1993). The effect of sequences with high AU content on mRNA stability in tobacco. Proc. Natl. Acad. Sci. USA 90, 11811–11815.
Okamuro, J., Caster, B., Villarroel, R., Van Montagu, M., and Jofuku, K.D. (1997). The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc. Natl. Acad. Sci. USA 94, 7076–7081. Peltz, S.W., and Jacobson, A. (1992). mRNA stability: In trans-it. Curr. Opin. Cell Biol. 4, 979–983.[CrossRef][Medline]
Phippen, T.M., Sweigart, A.L., Moniwa, M., Krumm, A., Davie, J.R., and Parkhurst, S.M. (2000). Drosophila C-terminal binding protein functions as a context-dependent transcriptional co-factor and interferes with both Mad and Groucho transcriptional repression. J. Biol. Chem. 275, 37628–37637.
Reymond, P., Weber, H., Dammond, M., and Farmer, E.E. (2000). Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 12, 707–720.
Ross, J. (1995). mRNA stability in mammalian cells. Microbiol. Rev. 59, 423–450. Ross, J. (1996). Control of mRNA stability in higher eukaryotes. Trends Genet. 12, 171–175.[CrossRef][Web of Science][Medline]
Schaffer, R., Landgraf, J., Accerbi, M., Simon, V.V., Larson, M., and Wisman, E. (2001). Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis. Plant Cell 13, 113–123.
Schena, M., Shalon, D., Davis, R.W., and Brown, P.O. (1995). Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.
Schenk, P.M., Kazan, K., Wilson, I., Anderson, J.P., Richmond, T., Somerville, S.C., and Manners, J.M. (2000). Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proc. Natl. Acad. Sci. USA 97, 11655–11660.
Seki, M., Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Carninci, P., Hayashizaki, Y., and Shinozaki, K. (2001). Monitoring the expression patterns of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. Plant Cell 13, 61–72.
Shyu, A.-B., Belasco, J.G., and Greenberg, M.E. (1991). Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes Dev. 5, 221–231. Sullivan, M.L., and Green, P.J. (1996). Mutational analysis of the DST element in tobacco cells and transgenic plants: Identification of residues critical for mRNA instability. RNA 2, 308–315.[Abstract]
Taylor, C.B., and Green, P.J. (1991). Genes with homology to fungal and S-gene RNases are expressed in Arabidopsis thaliana. Plant Physiol. 96, 980–984. Taylor, C.B., and Green, P.J. (1995). Identification and characterization of genes with unstable transcripts (GUTs) in tobacco. Plant Mol. Biol. 28, 27–38.[CrossRef][Medline]
Taylor, C.B., Bariola, P.A., DelCardayré, S.B., Raines, R.T., and Green, P.J. (1993). RNS2: A senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc. Natl. Acad. Sci. USA 90, 5118–5122. Tucker, M., Valencia-Sanchez, M.A., Staples, R.R., Chen, J., Denis, C.L., and Parker, R. (2001). The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104, 377–386.[CrossRef][Web of Science][Medline]
Vakalopoulou, E., Schaack, J., and Shenk, T. (1991). A 32-kilodalton protein binds to AU-rich domains in the 3' untranslated regions of rapidly degraded mRNAs. Mol. Cell Biol. 11, 3355–3364. Vasudevan, S., and Peltz, S.W. (2001). Regulated ARE-mediated mRNA decay in Saccharomyces cerevisiae. Mol. Cell 7, 1191–2000.[CrossRef][Web of Science][Medline]
Wildsmith, S.E., and Elcock, F.J. (2001). Microarrays under the microscope. Mol. Pathol. 54, 8–16.
Willy, P.J., Kobayashi, R., and Kadonaga, J.T. (2000). A basal transcription factor that activates or represses transcription. Science 290, 982–985. Yue, H., Eastman, P.S., Wang, B.B., Minor, J., Doctolero, M.H., Nuttall, R.L., Stack, R., Becker, J.W., Montgomery, J.R., Vainer, M., and Johnston, R. (2001). An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression. Nucleic Acids Res. 29, E41. This article has been cited by other articles:
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Abstract
INTRODUCTION
-glucuronidase (GUS), both containing a tetramer of the consensus DST element in the 3' UTR, were introduced into Arabidopsis as selectable and screenable markers, respectively. The presence of the DST elements decreased the mRNA levels for both transgenes, HPH-DST and GUS-DST, resulting in decreased resistance to hygromycin and low GUS activity. The dst mutants were isolated as hygromycin-resistant plants with increased GUS activity because of stabilization of the corresponding mRNAs. In addition, dst mutants have increased levels of SAUR-AC1 mRNA, the only endogenous DST-containing unstable transcript characterized to date (Gil and Green, 1996
-globin mRNA (Kiledjian et al., 1997
Zip-Lox (pZL1 clones; Gibco BRL, Rockville, MD) or pBluescript SK- vector (pBSK clones; Stratagene, La Jolla, CA). To amplify the ESTs by polymerase chain reaction (PCR), we designed universal primers corresponding to the 3' and 5' ends flanking the cloning site of each vector backbone. pZL1 clones were amplified using the 5' primer 5'-CGACTCACTATAGGGAAAGCTGG-3' and the 3' primer 5'-ATTGAATTTAGGTGACACTATAGAAGAGC-3'. pBSK clones were amplified using the 5' primer 5'-CGACTCACTATAGGGCGAATTGG-3' and the 3' primer 5'-GGAAACAGCTATGACCATGATTACG-3'. cDNA clones isolated in our laboratory were cloned in pBluescript SK- (primers as above) or pGEM-T (Promega, Madison, WI) vectors. To amplify clones in pGEM-T, we designed the 5' primer 5'-CGACTC-ACTATAGGGCGAATTGG-3' and the 3' primer 5'-ATTTAGGTGACA-CTATAGAATACTCAAGC-3'. 
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