<i>SIR</i>-regulated heat shock transgene system.

Abstract

<p>(a) Summary of <i>hsp82</i> transgenes used in this study with location, orientation and dosage of integrated <i>HMRE</i> silencers indicated by arrows (see Inset for location of silencer binding proteins ORC, Rap1 and Abf1). Transgenes occupy the native chromosomal <i>HSP82</i> locus (located ∼95 kb from TEL16L) and contain the indicated silencer insertions with no extraneous DNA sequence (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#s4" target="_blank">Materials and Methods</a>). Note that the transcription start site lies 60 bp upstream of the start codon and the 3′ integration site lies ∼50 bp 3′ of the mapped transcription termination site <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen.1004202-Farrelly1" target="_blank">[74]</a>. The ORF is indicated as a black rectangle; coordinates are numbered relative to the ATG codon. (b) Transcriptional output of <i>hsp82</i> transgenes under non-inducing (30°C) and inducing conditions (20 min heat shock at 39°C). Depicted are bar graph summaries of Northern analyses of <i>SIR⁺</i> and <i>sir4Δ</i> cells bearing the indicated <i>hsp82</i> transgenes (arrows symbolize integrated silencers as in A); <i>HSP82</i>⁺ was analyzed in the parental strain. Transcript abundance was normalized to <i>ACT1</i> and is presented relative to the non-induced <i>HSP82</i>⁺ level set at 100 (depicted are means ± S.D.; N = 2).</p