reporter 2

O Sir2p y Sir3p 0 Sir4p □ Sir1p rDNA array

Figure 17.4. Triple silencer yeast strains. To monitor silencing at the 3 types of silenced yeast loci in a single population of yeast cells, 3 different reporter genes are inserted into the silenced loci in the same strain. In order to monitor both increases and decreases in silencing, a mutant version of HMR can be used where the RAP1 protein binding site in the E box (Figure 17.2 top) is deleted. Because the amount of SIR silencing protein in the cell appears to be limited, such strains can monitor the redistribution of silencing function. Shortening telomeres would bind less SIR2, SIR3 and SIR4 protein, and so would free up some of these proteins that could then bind to the rDNA array or mutant silent mating type cassette.

HMLa as only about 1 in 106 cells expresses information from these cassettes and an increase in silencing is very difficult to detect. Mutated cassettes show weaker silencing such that reporter genes placed within the cassettes are expressed in 10% of the cells. Strains bearing these three types of reporters allow one to examine silencing at these three types of loci and determine the relative level of silencing using a simple technique called a spot test.

Spot tests are performed by diluting a freshly grown culture of cells, typically a single yeast colony, into sterile water and by making serial dilutions of this suspension, usually 10-fold dilutions. Aliquots of 3 to 5 microliters of each suspension are spotted onto different media to monitor the total number of cells that can grow and reporter gene expression (Gottschling et al., 1990). Markers with both positive and negative selections are used as reporters. In one triple silencer strain (Ray et al., 2003), the hmr locus was marked by the TRP1 gene. The expression of TRP1 is monitored by growth on medium lacking tryptophan, which requires the TRP1 gene product. This assay is a positive selection for TRP1 expression, so more expression leads to more growth which equals less silencing. In contrast, the telomere and rDNA array were marked, respectively, with URA3 and CAN1. Expression of these two genes can be monitored on medium containing drugs (5-fluoro orotic acid and canavanine, respectively) that kill cells which express these genes. These assays are negative selections for gene expression, so less expression leads to more growth which equals more silencing. Understanding these two different types of yeast reporters is essential for evaluating the silencing data, but once they are understood the assay becomes simple and extremely rapid. Six colonies can be assayed on one set of plates, allowing the screening of many different strains.

An important aspect of silencing is that the factors that accomplish it are limited. An excess of Sir proteins is toxic to the cells, and their levels are controlled (reviewed by Kahana and Gottschling (1999)). Consequently, concentrating more Sir proteins at one silenced locus can result in a decrease in silencing at another. The significance of this observation is that old yeast cells and some long-lived mutant yeast have a ''silencing configuration" in which the relative level of silencing function is shifted to increase silencing at the rDNA array and decrease it at telomeres and the silent mating type cassettes.

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