Silencing of the reporter gene <i>URA5-miRs</i>.

Abstract

<p>(A) The upper panels show a slower growth rate of the transformants of <i>URA5-miR1</i> (two transformants was picked in each case, namely, miR1-1 and miR1-2), and <i>URA5-miR2</i> (miR2-1 and miR2-2), than the wild type B4500, and the transformants of miR1-m (<i>i.e.</i> miR1-m1 and m2) and miR2-m, on MIN agar supplemented with 100 µg/ml hygromycin B, no hygromycin for B4500 and B4500FOA. The negative control B4500FOA (<i>ura5</i>) did not grow on MIN. On MINFOA (the bottom panels), the wild type strains and transformants of miR1-m and miR2-m were killed by FOA. Transformants containing miR1 and miR2, as well as <i>ura5</i>⁻ strain B4500FOA grew properly. MIN: minimal medium, and MINFOA: minimal medium with 5-FOA and 50 mg/l uracil. For selection of transformants, 100 µg/ml hygromycin B was added to MIN or MINFOA when needed. (B) Reverse transcription-PCR confirmed the decreased mRNA level of <i>URA5</i> in <i>URA5-miR</i> transformants. <i>URA5</i> mRNA levels in the wild type and in the transformants of miR-ms were close to each other. In each assay, two transformants were picked for double examination. (C) A semi-quantification of <i>URA5</i> mRNA to <i>ACT1</i> mRNA in the strains in (B).</p