A novel assay for investigating transcriptional fidelity in Saccharomyces cerevisiae

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	NCI	MOLBIO-2

Authors

• A Denney
• K Scibelli
• A Rattray
• B Shafer
• J Strathern

Abstract

Transcriptional fidelity in vivo has long proved difficult to investigate since transcription errors are transient unlike permanent DNA events. Here we demonstrate a novel colony-sectoring assay for transcriptional fidelity in S. cerevisiae using Cre recombinase-mediated homologous recombination of ADE6. Transcriptional slippage over a homopolymeric run has the potential to correct the reading frame of downstream Cre so that it is expressed and acts on target loxP sites flanking an antibiotic resistance cassette in ade6, restoring a functional ADE6. Since ade2 mutant cells accumulate a red pigment as part of the adenine biosynthetic pathway, cells which undergo these Cre-mediated events go from white (ade6::G418 and ade2∆) to red (ADE6 and ade2∆) and are visualized as red sectors in white colonies. Thus, a transient transcriptional error is permanently converted to a color phenotype in these cells and their descendants. Previously we have shown that this assay is exceedingly sensitive to an adenine-10-extra-base-one track. Currently we are constructing homopolymeric runs of varying track lengths and slippage directions for adenine and cytosine in an attempt to dampen the signal. Transcriptional fidelity is a vital cell process yet the consequences of infidelity remain to be fully understood in terms of genomic stability and mutagenesis.

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