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Cloning of the Arabidopsis Clock Gene TOC1 , an Autoregulatory Response Regulator Homolog
Carl Strayer, Tokitaka Oyama, Thomas F. Schultz, Ramanujam Raman, David E. Somers, Paloma Más, Satchidananda Panda, and Joel A. Kreps

Supplementary Material

Automated bioluminescence assay
Period length phenotype of toc1 mapping populations (cab::luc transgenic seedlings) and ccr2::luc transgenic lines was determined using an automated bioluminescence assay. A fragment containing 1.6 kb of CCR2 upstream sequence was cloned into pPZPluc, which is similar to pPZPluc+ but containing an unmodified luc ORF (Promega). Arabidopsis plants (ecotype Columbia) were transformed by Agrobacterium-mediated floral dip transformation procedure [S. J. Clough and A. F. Bent, Plant J.16, 735 (1998)]. Seeds were surface-sterilized, germinated, and grown on agar media in 12:12 light (60 to 85 μmol m-2 s-1):dark cycles for 6 to 7 days as described [D. E. Somers, P. F. Devlin, S. A. Kay, Science282, 1488 (1998)]. Two days before assay, seedlings in agar plugs were transplanted into 96-well microtiter plates (Dynex Technologies), luciferin solution added to each well, plates covered with clear adhesive film, and the film over each well perforated to allow gas exchange. Seedlings were returned to entraining conditions for 2 days. The plates were then stacked vertically in the plate-handling apparatus of a Packard TopCount Multiplate Scintillation Counter (Packard Instruments) kept in constant conditions (DD or LL, constant temperature) and bioluminescence was automatically recorded for each seedling for 5 to 10 s once every 1 to 2 hours, depending on experiment. For LL assays, modified clear microtiter plates were intercalated between sample plates, and lighting was provided by red LED lamps (Quantum Devices) placed on either side of the stack. Incident fluence rate at the surface of sample plates was 15 to 50 μmol m-2 s-1.

Map-based cloning
Markers EG7F2 and LFY were obtained from Research Genetics. Additional SSLPs and CAPs markers were developed from publicly available genomic sequence and physical clone sequence and mapping information produced through the efforts of the AGI, and our own analysis of unordered AGI BAC clones from this region. cab::luc period phenotype and molecular marker genotypes were scored in F2 individuals and/or F3 families of two mapping populations (Web fig. 1). Genomic DNA was amplified by PCR and products sequenced directly [D. E. Somers, T. F. Schultz, M. Milnamow, S. A. Kay, Cell101, 319 (2000)]. TOC1 cDNAs identified from EST databases (H1G8T7 and AI998263) were obtained (from the Arabidopsis Biological Resource Center and Genome Systems Inc., respectively) and sequenced, and compared to genomic sequence to determine gene structure.

Northern blot analysis of TOC1 transcript
TOC1 probe was prepared from a 2.7-kb Sac I-Dra I restriction fragment of cDNA clone H1G8T7 (this fragment was also used as a control template in the experiment presented in Fig. 3E). Total RNA was extracted from 10- to 15-day-old seedlings and Northern blots prepared and hybridized as described [D. E. Somers, T. F. Schultz, M. Milnamow, S. A. Kay, Cell101, 319 (2000)]. For LD and DD time courses, samples were prepared from ecotype Columbia plants; for LL time courses, ecotype C24 seedlings (toc1-1 and wt parental line) were used. TOC1 and rDNA probes were random primer labeled with [32P]dCTP. Quantification and analysis of hybridized blots was done using a Phosphorimager and ImageQuant Software (Molecular Dynamics).

Detection of alternative splicing variants
For RT-PCR characterization of splice variants, first-strand synthesis was performed using an exon 5-specific complementary primer (5´-CAAGACCACCATCACGAGCATGAAC-3´) and total RNA prepared from 14-day-old seedlings harvested at ZT11 in LD conditions. RT reactions were performed using SuperScriptII RT-PCR kit (Gibco BRL) according to manufacturer's recommendations. PCR was performed using a 5´UTR-exon 2 32P-labeled primer pair (5´-GTTCTGATTTGGCCATGGAAGT-3´ and 5´-TCACCTGCCTTGCTGATTTCAC-3´). Serial dilutions of a TOC1 cDNA restriction fragment were used to generate a standard curve. Products were resolved on a 6% sequencing gel and quantified. PCR was also performed using the RT reactions and a primer spanning the correct exon 1-exon 2 splice junction (preserving the TOC1 ORF) (5´-GATTTCACTGCAGTCAT/TTG-3´, where "/" denotes the splice junction) paired with the 5´UTR primer to confirm the presence of this species in toc1-2 RNA extracts. The toc1-2 splice variant sequence was determined by sequencing of RT-PCR products made using primers flanking the exon 1-exon 2 splice junction.

Cloning of TL1 cDNA
The TL1 cDNA was amplified using total RNA derived from wild-type tissue and oligo-dT primer for first strand synthesis, and gene specific primers (5´-TTTGAGGTCTGAGTCTATGGG-3´ and 5´-GCTCTTCATGATTTTGTAGACGC-3´) for PCR using the SuperScriptII kit following manufacturer's recommendations.

toc1::luc+ reporter construct
Using BAC (genomic) DNA as template, a PCR fragment was produced encompassing a Hind III site 2.5 kb upstream of the TOC1 ORF and extending to 7 nucleotides 5´ of the ATG, where the 3´ primer encoded a Hind III site. This was digested with Hind III and cloned into the Hind III site of pPZPluc+, a vector derived from pPZP221 [P. Hajdukiewicz, Z. Svab, P. Maliga, Plant. Mol. Biol.25, 989 (1994)] containing the ORF for a modified version of beetle luciferase (Promega) plus a viral (Omega) translational enhancer. Transgenic Arabidopsis plants were produced as described above and imaged [A. J. Millar, I. A. Carré, C. A. Strayer, N.-H. Chua, S. A. Kay, Science267, 1161 (1995)].

Visualization of YFP::TOC1 fusion protein
The YFP coding region was derived from the vector pEYFP (Clontech) and the TOC1 coding sequence was derived from cDNA clone H1G8T7. This fusion was cloned into the vector pRTL2 [J. C. Carrington, D. D. Freed, A. J. Leinicke, Plant Cell3, 953 (1991)], which contains a CaMV 35S promoter with a duplicated transcriptional enhancer and a TEV translational enhancer. Tobacco BY2 cells were transfected as described [P. Mas and R. N. Beachy, J. Cell Biol.147, 945 (1999)]. The fusion protein was visualized using a standard FITC filter set, Chroma Technology Corp., and Olympus IX70 confocal microscope.

Supplemental Figure 1. Positional cloning of TOC1. Yellow bar at top represents Arabidopsis chromosome 5 [size in centimorgans (cM) is indicated]. The ~1 cM region centromere-proximal to the LFY locus (genetic and physical position shown) and surrounding TOC1 is enlarged below. Genetic markers used to define TOC1 interval are shown, and number of recombinants identified in each interval is indicated. TOC1 was delimited to the interval between markers EG7F2 and MFB13-C. P1 clones used as Arabidopsis Genome Initiative sequencing substrates are shown below. Predicted genes in the TOC1 interval are shown at bottom; arrows indicate direction of transcription.

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Supplemental Figure 2. Distribution of period length in a toc1-2 mapping population. A toc1-2 homozygous M3 plant (C24) was outcrossed to the wild type (Laer). F2 seedlings were assayed as described [D. E. Somers, P. F. Devlin, S. A. Kay, Science282, 1488 (1998)], and period lengths were determined as described in Fig. 1. Note that wild-type period length varies significantly among ecotypes [K. Swarup et al., Plant J. 20, 67 (1999); C. Strayer, D. E. Somers, S. A. Kay, unpublished data], which is reflected in the shorter period length of all classes in this hybrid population as compared to isogenic C24 populations.

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