Detection of antagonistic cellular regulatory functions by the gene-gene interference method in yeast

Abstract

It was previously assumed that a new genetic method in yeast, termed gene-gene interference, leads to the selection of genes that antagonize, and/or are antagonized by, the particular reference gene used for their selection (Daniel 1993). In this paper two pieces of evidence are advanced in favour of this view. Firstly, the reconstitution of a system of known antagonistic genes was shown to be detectable by the gene-gene interference method. Secondly, since ART1, a new gene selected in reference to the protein kinase A gene, has been shown to contain in its deduced polypeptide a putative site for phosphorylation by protein kinase A, a mutagenesis study directed toward this putative site has been performed. Two phenotypes-in vivo filamenting activity and gene-gene interference relative to the protein kinase A gene-were tested with the various mutations thus obtained and found to be consistent with the hypothesis that, under physiological conditions, phosphorylation by protein kinase A exerts an inhibitory effect on Art1 activity. The relevance of these findings on the mechanisms and potential applications of the gene-gene interference phenomenon is discussed.

Sir-dependent downregulation of various aging processes

“…Indeed, in the first FIG experiment made with TPK1 as the reference gene (encoding a protein kinase A), a couple of selected candidates chosen for further study were found to be genes, SWI4 and MID2, that encode crucial positive regulators of the cell integrity pathway and the cell cycle Start control (Daniel 1993). These findings, which are in striking agreement with the known negative effect of protein kinase A in the Start control, were corroborated by mutational studies showing that the phosphorylation by Tpk1p of a putative site in Swi4p, has a negative impact on its activity in vivo (Daniel 1996a). More generally, the FIG approach enables a deeper understanding of in vivo functional interactions occurring within the cell's macromolecular networks (manuscript in preparation; unpublished results with various reference genes), and thus was used here in the search of genes antagonized (i.e., repressed) by the Sir complexes.…”

“…Moreover, FIG is based on the general observation that gene overexpression affects cell fitness negatively, though the extent of this effect varies from one gene to the next. As cell fitness is linked only to proliferation rate (Daniel 1993(Daniel , 1996a(Daniel , 1996b, genes affecting aging should not differ from any other genes in this respect. Indeed, overexpression of the Sir4p C-terminal fragment used for this selection results in a (moderate) ''toxic effect'' on cell fitness (see ''Materials and methods''), although it has a very significant effect on extending the life span.…”

“…Nevertheless, this represented only 26% of the total insertions, leading the authors to propose that the remaining ones might ''identify other sites at which silent chromatin assembles in genetically aged cells.'' This interesting possibility, which could have immense implications for the mechanism of action of the Sir complexes in protecting against aging, can be tested by the fitness-based interferential genetics (FIG) approach (Daniel 1993(Daniel , 1996a(Daniel , 1996b. FIG makes it possible to select for genes (or gene products) that are in antagonistic relationships with a given reference gene (or gene product).…”

A fitness-based interferential genetics approach using hypertoxic/inactive gene alleles as references

“…After puriWcation of the double transformant, a two-streak test was performed and the ratio of "white" to total colonies was counted (Daniel 1993(Daniel , 1996a. For the Trap-FIG detection test, the same procedure was applied except that plasmid YEp21-bA-PKC1 was replaced with YEp21-bA-pkc1-3.…”

“…Fitness-based interferential genetics (FIG) has been shown to be an eYcient approach for in-vivo exploration of the largely unknown domain of functional macromolecular networks in cells (Daniel 1993(Daniel , 1996a(Daniel , b, 2005(Daniel , 2007. Basically, FIG relies on the general observation that genes overexpressed in yeast cells lead to some cell toxicity, i.e., negatively aVect the cell's Wtness, even though the extent of this eVect varies from gene to gene.…”