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Methods. 2006 May;39(1):35-42.

Reporter assay systems for [URE3] detection and analysis.

Author information

1
Department Biologie I, Ludwig-Maximilians-Universit√§t, Maria-Ward-Str. 1a, 80638 M√ľnchen, Germany. a.brachmann@lrz.uni-muenchen.de

Abstract

The Saccharomyces cerevisiae prion [URE3] is the infectious amyloid form of the Ure2p protein. [URE3] provides a useful model system for studying amyloid formation and stability in vivo. When grown in the presence of a good nitrogen source, [URE3] cells are able to take up ureidosuccinate, an intermediate in uracil biosynthesis, while cells lacking the [URE3] prion can not. This ability to take up ureidosuccinate has been commonly used to assay for the presence of [URE3]. However, this assay has a number of practical limitations, affecting the range of experiments that can be performed with [URE3]. Here, we describe recently developed alternative selection methods for the presence or absence of [URE3]. They make use of the Ure2p-regulated DAL5 promoter in conjunction with ADE2, URA3, kanMX, and CAN1 reporter genes, and allow for higher stringency in selection both for and against [URE3], nonselective assay of prion variants, and direct transformation of prion filaments. We discuss advantages and limitations of each of these assays.

PMID:
16762564
DOI:
10.1016/j.ymeth.2006.04.008
[Indexed for MEDLINE]

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