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. 2001 Mar;183(6):1909–1920. doi: 10.1128/JB.183.6.1909-1920.2001

FIG. 3.

FIG. 3

Analysis of pms gene expression by RT-PCR. (A) Scheme representing positions of the primers used in this study, as well as lengths of the expected PCR products. Restriction sites are abbreviated as in Fig. 1. (B) RT-PCR products obtained from cDNA samples obtained after total RNA isolation of bacterial cultures grown under low- and high (Fe3+)-iron conditions. Experiments were performed as indicated in the Materials and Methods. One microliter of cDNA mixture was used in each reaction in lanes 1 to 18; 1 μl (2 ng) of DNA was used in control reactions (lanes 19 to 23). Lanes: M, size markers; 1, WCS358 (primer pair SAL01-SAL02); 2, WCS358 (Fe3+) (SAL01-SAL02); 3, WCS358(KM01/KM02); 4, WCS358 (Fe3+) (KM01-KM02); 5, WCS374 (SAL01-SAL02); 6, WCS374 (Fe3+) (SAL01-SAL02); 7, WCS374 (KM01-KM02); 8, WCS374 (Fe3+) (KM01-KM02); 9, 374-08 (SAL01-SAL02); 10, 374-08 (Fe3+) (SAL01-SAL02); 11, 374-08 (HDC01-SAL02); 12, 374-08 (Fe3+) (HDC01-SAL02); 13, 374-08 (DHB01-DHB02); 14, 374-08 (Fe3+) (DHB01-DHB02); 15, 374-08 (KM01-KM02); 16, 374-08 (Fe3+) (KM01-KM02); 17, WCS374 (DHB01-DHB02); 18, WCS374 (Fe3+) (DHB01-DHB02); 19, 374-08 DNA (SAL01-SAL02); 20, 374-08 DNA (HDC01-SAL02); 21, 374-08 DNA (DHB01-DHB02); 22, 374-08 DNA (KM01-KM02); 23, WCS374 DNA (KM01-KM02); 24, WCS374 DNA (2 μg) treated with RNase-free DNase (SAL01-SAL02); 25, 374-08 DNA (2 μg) treated with RNase-free DNase (KM01-KM02). (C) Dilution series of cDNA samples (1 μl) used in PCR experiments to estimate repression level upon Fe3+ addition. Primers used were SAL01-SAL02 for pmsB expression (214-bp PCR band) and KM01-KM02 for neo expression (405-bp PCR band). All lanes correspond to cDNA obtained from total RNA of 374-08. Odd-numbered lanes correspond to minimal-iron conditions; even-numbered lanes to 100 μM Fe3+. Lanes 1 to 4, 1 μl of the cDNA original mixture; lanes 5 to 8, 5-fold-diluted samples; lanes 9 to 12, 25-fold-diluted samples; lanes 13 to 16, 125-fold-diluted samples; lanes 17 to 20, 625-fold-diluted samples; lanes 21 to 24, 3,125-fold-diluted samples.