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. 2012 Sep 11;7(9):e44361. doi: 10.1371/journal.pone.0044361

Figure 7. Binding of MA-MADS5 to CArG-box elements in the promoters of different ripening genes in banana. a.

Figure 7

Analysis of promoter fragments of different ripening specific genes (MA-SPS, MA-ACS1, MA-ACO1, MA-EXP, MA-LEC) from banana showing the presence of CArG-box elements in different regions of promoter. CArG-box motifs identified in the promoters of different ripening genes have been serially numbered as 1–9 respectively. b ChIP PCR assay for the cis-elements of MA-SPS, MA-ACS1, MA-ACO1, MA-EXP, MA-Lec. Chromatin was prepared from climacteric banana fruit, and was immunoprecipitated using anti-MA-MADS5 polyclonal antibody or pre-immune serum, respectively. PCR reaction was performed using primers amplifying the CArG-box motif region of the promoters of the indicated ripening specific genes. Actin was used as control. Representative image from at least three independent experiments are shown for Figure b. c DNA sequences of the CArG-boxes within the promoter of different ripening specific genes of banana and the relative binding ability of recombinant MA-MADS5 to these sequences have been indicated by + and – symbols respectively. d Gel mobility shift assay using 5′-end labeled synthetic oligonucleotide containing CArG-box motif 1 as probe. No protein extract was added in lane 1, while 2 µg recombinant MA-MADS5 protein was added in lanes 2–5. 50 and 100-molar excess of unlabeled CArG-box motif 1 was added in lanes 3 and 4 as competitor. Lane 5 contained 100 molar excess of unlabeled corresponding mutant version of CArG-box motif 1 (m1) as competitor. e–h Gel mobility shift assays using 5′-end labeled synthetic oligonucleotide containing CArG-box motif 2, 5, 7 and 8 respectively as probe. No protein extract was added in lane 1, while 2 µg recombinant protein was added in lanes 2–4. 100-molar excess of unlabeled respective CArG-box motif and unlabeled corresponding mutant version of CArG-box motifs of 2, 5, 7 and 8, respectively (m2, m5, m7, m8) were added in lanes 3 and 4 respectively. i. Gel shift assay using 5′-end labeled synthetic oligonucleotide containing CArG-box motifs 3 (lane 1), 4 (lane 2), 6 (lane 3) and 9 (lane 4) as probe. 2 µg recombinant proteins were added in lanes 1–4. Representative images from three independent trials have been shown for Figures d–i. RP-recombinant proteins; Com-competitor.