20. G418-resistant Ba/F3 transfectants expressing the indicated proteins and control cells were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2% WEHI conditioned medium as a source of IL-3. Cells were washed twice in the same IL-3free medium and then seeded in IL-3-free medium in 96-well plates at a concentration of 5 ϫ 10 4 cells/ml (10 4 cells per well). The number of wells showing proliferating cells in either the absence or presence of IL-3 was scored after 1 week in culture.

Plants respond to day/night cycling in a number of physiological ways. At the mRNA level, the expression of some genes changes during the 24-hr period. To identify novel genes regulated in this way, we used microarrays containing 11,521 Arabidopsis expressed sequence tags, representing an estimated 7800 unique genes, to determine gene expression levels at 6-hr intervals throughout the day. Eleven percent of the genes, encompassing genes expressed at both high and low levels, showed a diurnal expression pattern. Approximately 2% cycled with a circadian rhythm. By clustering microarray data from 47 additional nonrelated experiments, we identified groups of genes regulated only by the circadian clock. These groups contained the already characterized clock-associated genes LHY , CCA1 , and GI , suggesting that other key circadian clock genes might be found within these clusters. INTRODUCTIONPlants have adapted their growth and development to use the diurnal cycling of light and dark. This is manifested at both the physiological level, with leaf movement, growth, and stomatal opening, and the molecular level, with expression of some genes occurring only at certain times of the day. The day/night cycling of gene expression is called a diurnal rhythm and is achieved primarily by two mechanisms: first, by light, and second, by a free-running internal circadian clock. Circadian clocks have been well characterized in animals, fungi, and bacteria, and in all cases they have a central oscillator that measures time with a molecular feedback loop that cycles over a 24-hr period (Dunlap, 1999). Although a growing number of genes either regulated by the clock or affecting clock function have been identified in plants, a full picture has yet to emerge.The ability of plants to respond to light is achieved through photoreceptors. In Arabidopsis, two classes of photoreceptors are known: the red/far-red receptors, phytochrome A to E (Sharrock and Quail, 1989;Clack et al., 1994), and the blue light receptors, CRY1 (Ahmad and Cashmore, 1993), CRY2 (Guo et al., 1998), and NPH1 (Liscum and Briggs, 1995). Using these photoreceptors, a plant can detect a range of light intensities and wavelengths, with which it senses not only whether light is present but also from which direction the light is coming and whether there is competing vegetation (reviewed in Ballare, 1999). The best characterized of the photoreceptors are the phytochromes, for which the events that convert the light signal into transcriptional regulation have been described. Phytochrome is transported into the nucleus in a light-dependent manner (Sakamoto and Nagatani, 1996;Kircher et al., 1999). In the nucleus, it interacts with a basic helix-loop-helix transcription factor, PIF3 (Ni et al., 1998(Ni et al., , 1999, which has been shown to bind to the G box element found in the promoters of many light-activated genes (Giuliano et al., 1988;Martinez-Garcia et al., 2000). This chain of events allows the plants to respond to light after germination, by stopping hypocotyl elongat...

Small RNAs from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPEN-DENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of RDR2. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop1-1 loss-offunction mutant by using Illumina's sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic Ϸ22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.miRNA ͉ mop1 ͉ rdr2 ͉ small RNA T he small RNAs found in a typical plant cell include a small number of highly abundant, mainly 21-nt microRNAs (miRNAs) and a large number of small interfering RNAs (mainly 24 nt heterochromatic siRNAs, or simply siRNAs) recognizing many diverse sequences. In addition, several additional subclasses of varying and in some cases overlapping functional importance have been described, including the transacting siRNAs (ta-siRNAs) (1-3), natural antisense siRNAs, a type thus far observed only under stress conditions (4, 5), and natural antisense miRNAs (6).MicroRNAs have a variety of regulatory roles in development and stress responses (for review, see ref. 7). In addition, work in Arabidopsis has led to the hypothesis that transcription of repeats is performed by the plant-specific RNA polymerase IV (pol IV) followed by reverse transcription and cleavage by RDR2 and DCL3, respectively. These repeated sequences include transposons and retrotransposons in plants (8), and this series of events produces a complex set of heterochromatic siRNAs (for review, see ref. 9). The genome size of plants varies substantially among species mainly caused by variation in content of repeated DNA. The complexity of siRNAs is correspondingly greater in rice than in Arabidopsis (10), consistent with larger numbers of repeated sequences in rice.The maize b1 locus is an excellent model for paramutation, a phenomenon in which alleles communicate in trans, resulting in meiotically heritable gene expression changes (11). Molecular work, combined with fine-structure recombination mapping, has demonstrated that this activity is mediated by tandem repeats at b1 that are required to e...