A Survey of Essential Gene Function in the Yeast Cell Division Cycle

Abstract

Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO 7 promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication. INTRODUCTIONCell division is a fundamental biological process that in all organisms consists of a series of closely coordinated events. In the budding yeast Saccharomyces cerevisiae, the cell division cycle begins with an initial growth phase, G1, during which time the cell increases in mass and volume. The transition from G1 into S phase is marked by progression through Start (the point of commitment to cell division), initiation of nuclear DNA synthesis, and the emergence of a bud that will form the new daughter cell. S phase is followed by a second growth phase, G2, which is in turn followed by nuclear division, and then cell separation. A very similar series of events occurs in all eukaryotes, with the obvious exception of bud emergence. Because of the fundamental biological importance of cell cycle progression and its importance in both human development and diseases such as cancer, identification of the molecular determinants of specific stages of the eukaryotic cell cycle has been a subject of intense study for several decades.The morphological landmarks of the cell cycle stages in budding yeast, most notably the size of the bud relative to the size of the mother cell, allows the identification of mutants blocked at specific stages of the cell cycle and thereby forms the basis for the classic cell cycle screens (Hartwell et al., 1970;Culotti and Hartwell, 1971;Hartwell, 1971aHartwell, , 1971bHartwell, , 1973Moir et al., 1982). These genetic screens, using conditional temperature-sensitive mutants, identified more than 50 genes that are required for specific stages in the cell division cycle and so were termed CDC genes. On average 4.6 alleles were identified for each CDC gene in the original screens, suggesting the number of cdc mutan...

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development

“…It has to be noted that a similar phenotype was observed with weak mutations in the PAS3/GURKE gene, which codes the cytosolic acetylCoA carboxylase required for providing the malonyl-CoA to the elongase complex (26). The link between VLCFA and cell division was also reported in yeast (7,27). In plants, the overexpression of PAS2 delayed cell cycle progression, in particular during mitosis (19).…”

“…Identification of the dehydratase remained elusive until the recent identification of YJL097w/PHS1 as encoding this activity (although a role in sphingolipid biosynthesis had previously been inferred from the biochemical phenotype of phs1 mutant that accumulated the long chain base phytosphingosine [PHS]) (6). The phs1 mutant was also characterized as a cell cycle mutant defective in G2/M phase (7). Ultimate confirmation of the biochemical function of Phs1p as the elongase dehydratase was provided by in vitro activity of recombinant protein and reconstitution of the elongase complex in proteoliposomes (8).…”

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

“…Loss of Msh6 or Pms1 (msh6D or pms1D) is sufficient to extinguish Pol d proofreading-deficient (pol3-01) cells (Figure 1), the apparent consequence of mutation accumulation during DNA replication (Morrison et al 1993;Herr et al 2011a). Consistent with a mechanism of errorinduced lethality, yeast cells with Pol d proofreading and MMR defects form microcolonies of $100 cells arrested at various stages of the cell cycle (Morrison et al 1993) and with diverse cell morphologies resembling inactivation of different essential genes (Yu et al 2006). The maximum tolerated mutation rate in haploid yeast ($10 23 inactivating mutations/gene/cell division % 3 · 10 26 mutations/base pair/cell division) is consistent with random inactivation of essential genes as the cause of extinction (Herr et al 2011a).…”

cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases

“…NUS1 was described as an essential gene needed for cell division in S. cerevisiae (69) contemporaneously with the identification of NgBR in human cells (64). Yeast strains were constructed by replacement of native promoters with a TetO 7 cassette (Tet-off system), which was examined for defects after doxycycline addition.…”

“…The essential nature of cis-PT activity for dolichol biosynthesis is clear based on lethality in yeast and mice when Nus1, Rer2/Srt1, or NgBR is deleted (34,46,47,69,77). Ablation of NgBR in the mouse results in early embryonic lethality around embryonic day 6.5.…”