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+ plants were repeated a minimum of three times with at least two independent constructs. All plants were maintained in a 20°C glasshouse with ambient lighting.
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The wild-type GFP sequence in plasmid pGFP was cleaved with Age I, treated with Klenow fragment, and cleaved with Spe I. This GFP fragment was inserted into the infectious cDNA clone of RCNMV RNA-2 that had been cleaved with Nco I, treated with Klenow fragment, and cleaved with Xba I to produce clone R2GFP. Clone R2GΔH was produced by cleavage of R2GFP with Hpa I (within the GFP coding sequence) and Hind III (within the multiple cloning site of the plasmid) followed by insertion of the ApaL I-Hind III fragment from wild-type RCNMV RNA-2. Clone R2GΔHX was produced by cleavage of clone R2GΔH with Bgl II-Xba I followed by Klenow treatment and religation to yield a 120-nt deletion.
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3542997120
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note
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The RNA-2 fragment was excised from the infectious cDNA clone with ApaL I-Xba I followed by Klenow treatment and ligation into the SnaB I site of pHST2 to yield clones pHST2-RC2.3 (+) and -RC2.4 (-). The 209-nt RNA-2 insert was produced by digestion of pHST2-RC2.3 with Bgl II-Xba I followed by Klenow treatment and religation to yield clone pHST2-ΔBX.
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RNA secondary structure was predicted with M. Zuker's mfold server
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RNA secondary structure was predicted with M. Zuker's mfold server at http://www.ibc.wustl.edu/ ∼zuker/rna/
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3543038460
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note
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The 5′-terminal 121-nt and 3′-terminal 91-nt regions of the 209-nt element were amplified by PCR. The products were cleaved with SnaB I-Xho I and ligated into similarly cleaved pHST2.
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25
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3543018751
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note
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RCNMV RNA-2 inserts of 50 nt or less in pHST2 were produced by annealing of phosphorylated complementary oligonucleotides followed by ligation of the resulting double-stranded DNAs into pHST2 cleaved with SnaB I-Xho I.
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0030608942
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PCR-based mutagenesis of the RNA-1 sequence was performed as by S. Li and M. F. Wilkinson [Biotechniques 23, 588 (1997)]. After amplification, the PCR product was gel-purified, cleaved with Xho I-Bgl II, and ligated into similarly cleaved R1sGFP to generate clone R1sGFP-M.
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note
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We thank D. Cookmeyer, T. Petty, and M. Conkling for reviewing this manuscript; J. Sheen for the sGFP and a GFP plasmid; G. Payne for a GFP plasmid; T. Okuno for the SCNMV RNA-2 cDNA clone; and H. Scholthof for the TBSV pHST2 vector. Supported in part by NSF grant MCB-9419700 to S.A.L.
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