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. 2005 Nov;25(22):10171–10182. doi: 10.1128/MCB.25.22.10171-10182.2005

FIG. 5.

FIG. 5.

The Pds gene is a novel target of TTF-1. (A) Sequence of the −657 bp of the rat PDS 5′ UTR. Intron sequences (430 bp) as deduced by RT-PCR and RACE are boxed. TSSs as deduced by 5′ RACE are indicated by arrows. (B) RACE products (35 cycles), from reverse-transcribed total mRNA (1 μg) from FRTL-5 cells, were subjected to PCR with nested oligonucleotides as described (see Material and Methods), revealed by ethidium bromide staining, and photographed under UV light. The figure refers to one PCR, which was run twice with the same material with comparable results. M, 100-bp DNA ladder. Lanes 1 to 3 show the results of PCRs with nested oligonucleotides. Lane 1, RACE products obtained by using the DNAs from first PCR as template; lane 2, same as lane 1, but no DNA from first PCR used as template; lane 3, no cDNA. DNA products purified from lane 1 were subcloned into TOPO-TA vector, and 15 colonies were sequenced in double orientations. (C) Activities of various PDS promoter constructs. Luciferase reporter constructs (3 μg) containing various lengths of PDS promoter were cotransfected in HeLa cells with a plasmid encoding TTF-1 (0.2 μg/60-mm-diameter dish) or CMV-Flag together with 0.3 μg of Rous sarcoma virus-Renilla as an internal control. The relative activities of each Luc-reporter plasmid with cotransfected TTF-1 or CMV-Flag expressed as arbitrary units are also indicated. Deletion of the promoter region between 3.0 and 2.2 kb significantly decreased the relative activation (n-fold) of the promoter activity by TTF-1. Mutation of site E (CAAG to CGTG) in the context of the rPDSLuc3.0 construct was obtained by site-directed mutagenesis (rPDSLuc3.0 M) and analyzed in similar experiments. A plasmid with three-E tandem motifs upstream to the thymidine-kinase minimal promoter (3E-TKLuc) or its mutated counterpart (3mE-TKLuc) was tested in similar experiments. White bars indicate the increase (n-fold) in induction when 0.2 μg of TTF-1 RNAi plasmid was cotransfected (together with TTF-1) as a control of specificity. The relative activity of each luciferase reporter plasmid with cotransfected TTF-1 or CMV-Flag, expressed in arbitrary units, is also indicated. The data shown are means ± SD of the luciferase/Renilla ratios from four experiments performed in duplicate.