Reversal of cell determination in yeast meiosis: postcommitment arrest allows return to mitotic growth.

Abstract

When yeast from the early stages of melosis are transferred from porton to growth medium, they can reenter the mitotic cell cldirectly. In contrast, cells from later stages of meosis (after the initiation of the first nuclear division) will The meiotic fate is triggered by deprivation of glucose and nitrogen. As is true in many types ofcell differentiation, cells in meiosis remain uncommitted through the early stages of the process-i.e., they will abort meiosis and return to mitotic growth if the cells are transferred from sporulation to growth medium (4). Significantly, genetic and cytological analyses have revealed that meiotic DNA replication, synaptonemal complex formation, chromosome pairing, and DNA recombination all occur during this uncommitted period (5-7). In contrast, cells become fillly committed to meiotic development at the initiation of chromosome segregation in meiosis I-i.e., after this point they will complete the two meiotic divisions and form spores despite the addition of glucose and nitrogen. This study presents the unexpected result that when meiotic cells are arrested after either the first or second division, cells are able to return to mitotic growth. Further experiments suggest that commitment results from a transient delay in mitotic growth rather than an irreversible inhibition of the cell cycle during sporulation. MATERIALS AND METHODSStrains. The following diploid strains were used in this study. H65 contains MATa/MATa ADE2/ade2-1 adeSi ADE5 can]/cani CYH2/cyh2 HIS] /hisi his7/HIS7 HOM3/ hom3-10 leu2-3/leu2-1 lysi/LYS) LYS2/lys2 metl4/MET14 pet8/PET8 TRPJ/trpl ura3/ura3. H91 is isogenic to H65 except for the presence of a homozygous disruption/ duplication allele of SP014 designated spol4:URA3:spol4 (8). REE1525 contains MATa/MATa arg4-1/arg4-1 cyhi-li cyhl-l HO/HO met4/met4. SH495 is congenic to REE1525 and is spo3-1/spo3-1.Media. Growth (YPDA) and acetate (YPA) media have been described (8). The sporulation media (SPII-30 and SPII-31) contain 20 g of potassium acetate per liter supplemented with 75 pg (each) ofeither adenine sulfate, L-leucine, and uracil per ml (for strains H65 and H91) or L-arginine and L-methionine (for strains REE1525 and SH493).Assaying nmark Events in Meiosls. Fifty-milliliter cultures were grown for "'20 hr at 300C in YPA to 5.0 x 107. Cells were washed three times, resuspended in 25 ml ofsporulation medium (pH 6.5), and aerated by shaking at constant temperature in a New Brunswick Scientific gyratory water bath shaker at 250 rpm. At intervals, 200-1d samples were fixed by addition of400 y4 of95% ethanol. An aliquot (6 y1) ofthis cell suspension was spotted on a microscope slide and allowed to dry. Nuclei were stained by placing 6 p4 of a solution of 1 pAg of 4',6-diamidino-2-phenylindole dihydrochloride per ml (DAPI, Boehringer Mannheim), 0.1 mg of 1,4-phenylene diamine per ml (Sigma), 50 mM sodium phosphate buffer, and 50%1 glycerol on the dried cells. Cells were visualized by Nomarski/fluorescence microscopy on a Jenalumar microscope. Two hund...

Mutation of the SPS1-encoded protein kinase of Saccharomyces cerevisiae leads to defects in transcription and morphology during spore formation.

“…The ori gin of the haploid progeny that were derived from spslarrested cells is unclear. It is possible that a haploid nu cleus that failed to be encapsulated in an immature spore migrated into a daughter bud on resumption of growth, as has been shown recently for spol4-arrested cells (Honigberg et al 1992;Honigberg and Esposito 1994). Another possibility is that the spsi prospores were re leased from the ascal sac and began to grow.…”

“…As might be expected from the observation that maximal expres sion of SPSI occurs as meiosis is nearing completion, v^e found that spsi/spsi cells could complete meiosis I and meiosis II as assayed by DAPI staining of nuclear mate rial. The mutant cells, v^hich did not form spores as as sessed by phase-contrast microscopy, were able to re enter the mitotic cycle on transfer to rich medium as has been shown for several other meiotically arrested mu tants (Davidow and Byers 1984;Shuster andByers 1989,-Bishop et al 1992;Honigberg et al 1992;Rose and Holm 1993;Sym et al 1993;Honigberg and Esposito 1994). The recovery of haploid progeny from spsi-arrested cells suggested that postmeiotic nuclear compartmentalization had occurred in a portion of the mutant cells.…”

The Ras/cAMP Pathway and the CDK-Like Kinase Ime2 Regulate the MAPK Smk1 and Spore Morphogenesis in Saccharomyces cerevisiae

“…These signals control the IME1 promoter and also regulate Ime1 post-translationally. Addition of rich media can inhibit sporulation even after meiotic S phase, genetic recombination, and synapotenemal complex formation have taken place (Sherman and Roman 1963;Simchen et al 1972;Esposito and Esposito 1974;Honigberg et al 1992;Honigberg and Esposito 1994;Zenvirth et al 1997;Friedlander et al 2006). Moreover, such refed cells can exit from the sporulation program and return to mitotic growth (RTG) as long as the commitment point (MI) has not been passed.…”

PP2ACdc55is required for multiple events during meiosis I

“…In addition, cdc55 cells remained diploid throughout meiosis and were unable to commit to proceed through meiosis I using "return to growth" assays. 73,74 Recombination bypass and checkpoint mutations do not suppress the meiosis I defect of cdc55 cells. We next asked whether any recombination bypass mutations were able to suppress the block in meiosis I. Deletions of MEI4, SPO11 or SPO13 allow mutants defective or blocked in recombination (or pachytene) to bypass meiosis I chromosome segregation and undergo a single equational division.…”

“…We determined trp1 recombination in the W303 strain described by Honigberg and colleagues. 70,71 arg4 and leu2 frequencies were determined in SK1 strains kindly provided by the laboratories of Alain Nicolas 72 and Rochelle Esposito, 73,74 respectively. Heteroalleles of trp1, leu2 or arg4 can be repaired through intragenic recombination, generating wild-type alleles.…”