Discrimination between Paralogs using Microarray Analysis: Application to the Yap1p and Yap2p Transcriptional Networks

Ohno [Ohno, S. (1970) in Evolution by Gene Duplication, Springer, New York] proposed that gene duplication with subsequent divergence of paralogs could be a major force in the evolution of new gene functions. In practice the functional differences between closely related homologues produced by duplications can be subtle and difficult to separate experimentally. Here we show that DNA microarrays can distinguish the functions of two closely related homologues from the yeast Saccharomyces cerevisiae, Yap1p and Yap2p. Although Yap1p and Yap2p are both bZIP transcription factors involved in multiple stress responses and are 88% identical in their DNA binding domains, our work shows that these proteins activate nonoverlapping sets of genes. Yap1p controls a set of genes involved in detoxifying the effects of reactive oxygen species, whereas Yap2p controls a set of genes over represented for the function of stabilizing proteins. In addition we show that the binding sites in the promoters of the Yap1p-dependent genes differ from the sites in the promoters of Yap2p-dependent genes and we validate experimentally that these differences are important for regulation by Yap1p. We conclude that while Yap1p and Yap2p may have some overlapping functions they are clearly not redundant and, more generally, that DNA microarray analysis will be an important tool for distinguishing the functions of the large numbers of highly conserved genes found in all eukaryotic genomes

CASP, the Alternatively Spliced Product of the Gene Encoding the CCAAT-Displacement Protein Transcription Factor, Is a Golgi Membrane Protein Related to Giantin

Large coiled-coil proteins are being found in increasing numbers on the membranes of the Golgi apparatus and have been proposed to function in tethering of transport vesicles and in the organization of the Golgi stack. Members of one class of Golgi coiled-coil protein, comprising giantin and golgin-84, are anchored to the bilayer by a single C-terminal transmembrane domain (TMD). In this article, we report the characterization of another mammalian coiled-coil protein, CASP, that was originally identified as an alternatively spliced product of the CUTL1 gene that encodes CCAAT-displacement protein (CDP), the human homologue of the Drosophila homeodomain protein Cut. We find that the Caenorhabditis elegans homologues of CDP and CASP are also generated from a single gene. CASP lacks the DNA binding motifs of CDP and was previously reported to be a nuclear protein. Herein, we show that it is in fact a Golgi protein with a C-terminal TMD and shares with giantin and golgin-84 a conserved histidine in its TMD. However, unlike these proteins, CASP has a homologue in Saccharomyces cerevisiae, which we call COY1. Deletion of COY1 does not affect viability, but strikingly restores normal growth to cells lacking the Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor Gos1p. The conserved histidine is necessary for Coy1p's activity in cells lacking Gos1p, suggesting that the TMD of these transmembrane Golgi coiled-coil proteins is directly involved in their function

Short Telomeres Induce a DNA Damage Response in Saccharomyces cerevisiae

Telomerase-deficient Saccharomyces cerevisiae cells show a progressive decrease in telomere length. When grown for several days in log phase, the tlc1Δ cells initially display wild-type growth kinetics with subsequent loss of growth potential after which survivors are generated via RAD52-dependent homologous recombination. We found that chromosome loss in these telomerase-deficient cells only increased after a significant decline in growth potential of the culture. At earlier stages of growth, as the telomerase-deficient cells began to show loss of growth potential, the cells arrested in G2/M and showed RNR3 induction and Rad53p phosphorylation. These responses were dependent on RAD24 and MEC1, suggesting that short telomeres are recognized as DNA damage and signal G2/M arrest