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note
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6 plaques screened. One cDNA was made up of 1681 base pairs (bp) and encodes a full-length protein. Amino acid sequences of RS2 and PHAN proteins from Antirrhinum and Arabidopsis were aligned using Pileup (Genetics Computer Group, University of Wisconsin, Madison, WI) with a gap weight of 5 and a length weight of 0.1. An 11.5-kb Bam HI genomic clone was isolated from a size-selected genomic library. Introns were identified by restriction analysis of the genomic and cDNA clones, followed by polymerase chain reaction (PCR) and sequence analysis. An intron of ∼2.4 kb is present in the 5′ UTR.
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note
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1 screens at a frequency of 1 in ∼12,000 plants. The nature of the mutation in the rs2-mum1 and rs2-mum2 alleles was determined by restriction analysis.
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Five micrograms of total RNA from vegetative shoot apices, young floral corollas (<5 mm), young leaves (<5 mm), and fully expanded leaves of either wild type or phan-607 mutants (6) were used in cDNA synthesis with an oligo(dT) primer. One percent of each cDNA pool was used as a template in PCR reactions with AmSTM-specific primers capable of amplifying 419 bp of cDNA surrounding the position of the 405-bp intron (6). Specific PCR products of 419 bp were detected by Southern (DNA blot) hybridization with an AmSTM probe. Control amplification with primers specific for the Antirrhinum UBIQ-UITIN gene (X67957) under the same conditions indicated that similar amounts of cDNA were present in all samples.
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note
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The authors thank M. Tsiantis, R. Schneeberger, M. Freeling, and J. Langdale for discussions of their independent cloning of rs2; our colleagues at Yale University and Cold Spring Harbor Laboratory for useful discussions; and T. Mulligan and J. Truglio for help in the field and in the laboratory. P.W.B. was supported by a grant from NIH to M. Freeling. This research was supported by an award from USDA to T.N.
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