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Cloning Procedures. Unless otherwise indicated, all PCR products and their target vectors were digested with restriction endonucleases whose sites were incorporated into the 5' end of primers (Table 1) and were then ligated to each other. Standard molecular techniques were used for subcloning (1).

Constructs for Transactivation and Protein-Protein Interaction Assays. For fusion constructs with the GAL4-DNA-binding domain (DBD), DNA fragments corresponding to full-length growth-regulating factors GRF1 and GRF2 as well as their various segments and the complete C terminus of GRF5 were amplified by PCR with cDNA clones as templates, digested with restriction endonucleases, and ligated in frame to the GAL4-DBD sequence of pGBT8, which was derived from pGBT9 to have additional restriction-enzyme sites in the multicloning site (Clontech). For the fusion construct with GAL4-AD, DNA fragments for full-length GIF1 were amplified by PCR and ligated to the GAL4-AD sequence of pACT2 (Clontech) after restriction digestion.

Constructs for the in Vitro Binding Assay. For the GIF1 proteins tagged with hemagglutinin (HA)-epitope, DNA fragments encoding the full-length GRF1 and its segment proteins, including the QLQ or WRC domain, were amplified by PCR, digested, and ligated in frame to the HA-epitope sequence of the pHB6 bacterial expression vector (Roche Molecular Biochemicals). For the GST fusion of GIF1 proteins, PCR-amplified DNA fragments for full-length GIF1 and its segments containing the SNH or QG domain were ligated in frame to the GST sequence of pGEX4T-1 (Amersham Pharmacia Biosciences).

Construct for Transient Expression. DNA fragments corresponding to full-length GIF1 were amplified by PCR, digested, and ligated in frame upstream of the Enhanced Yellow Fluorescent Protein (EYFP) sequence in the pAVA554 vector, resulting in the GIF1-EYFP fusion gene.

GIF1-RNAi Construct. A GRF1-specific DNA region encoding the QG domain was amplified by PCR by using two sets of primers for sense and antisense inserts. The 3' end of PCR products for sense inserts was cut with KpnI and ligated into the pHANNIBAL vector (2), which was prepared to have one end digested with KpnI and the other end filled in by Klenow fragment after XhoI-digestion. The resulting plasmid was cut again with BamHI/ClaI, and DNA fragments for antisense inserts were ligated to it after corresponding restriction-enzyme digestion. The construct made in pHANNIBAL was subcloned as NotI fragments into the binary vector pART27 (2) to complete the GIF1-RNAi plasmid.

1. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, eds. Sambrook, J., Fritsch, E. F. & Maniatis, T. (Cold Spring Harbor Lab. Press, Plainview, NY).

2. Wesley, S. V., Helliwell, C. A., Smith N. A., Wang, M. B., Rouse, D. T., Liu, Q., Gooding, P. S., Singh, S. P., Abbott, D., Stoutjesdijk, P. A., et al.. (2001) Plant J. 27, 581-590.