SWM1, a Developmentally Regulated Gene, Is Required for Spore Wall Assembly in Saccharomyces cerevisiae

Expression of the SWM1 gene during the sporulation process. (A) Meiotic time course of SWM1expression. RNA purified from wild-type cells (strain AP1a/α) at the indicated times after transfer to sporulation medium was hybridized with a radioactively labeledSWM1 probe. The top panel represents the percentage of mature asci at each time point. (B) Expression of SWM1 inMATα/MATα cells. RNA purified from strain AP1a/α at 0 or 10 h (lanes 1 and 2, respectively) after transfer to sporulation medium or from strain AP1α/α at 0 or 10 h (lanes 3 and 4, respectively) was hybridized with the SWM1 probe. In both experiments, theACT1 probe was used to test for equal loading of RNA in all lanes.

Time course of meiosis in swm1 cells. Samples of cells from sporulating cultures of the wild-type strain YPA24 (A) or the swm1Δ mutant YPA207 (B) were fixed, stained with DAPI, and examined by fluorescence microscopy to determine the percentage of cells that had completed meiosis I (●) or meiosis II (○). Cells that appeared to be binucleate, trinucleate, or tetranucleate by DAPI staining were considered to have completed meiosis I. Cells that appeared to be trinucleate or tetranucleate were considered to have completed meiosis II. Samples were also examined by phase-contrast microscopy to monitor ascus formation (□).

Microscopic appearance of wild-type and swm1mutant asci during the sporulation process. Rows 1 and 3, differential interference contrast microscopy (DIC) photomicrographs; rows 2 and 4, corresponding photomicrographs of DAPI-stained cells. Wild-type cells (strain YPA24) at 8, 12, and 24 h after transfer to sporulation medium present a characteristic tetranuclear organization and birefringent spore walls. swm1Δ cells (strain YPA207) at 8, 12, and 24 h after transfer to sporulation medium do not display discernible spore walls by DIC microscopy. These mutant cells are clearly tetranucleate after 12 h in sporulation medium.

Resistance of wild-type and swm1Δ asci to prolonged incubations in sporulation medium, exposure to heat shock, or enzymatic (glusulase) digestion. (A) Wild-type cells from the strain YPA24 (○) or the isogenic swm1Δ strain YPA207 (●) were incubated in sporulation medium for the indicated times (at 30°C) before the plating efficiencies were assayed. (B and C) Wild-type (strain YPA24 [○]) or swm1Δ cells (strain YPA207 [●]) were incubated in sporulation medium for 24 h and then assayed for plating efficiency after exposure to 55°C (B) or to glusulase (C) for the indicated times (see Materials and Methods).

FACS analysis shows thatswm1Δ/swm1Δ cells are able to generate haploid progeny upon germination. Cells were grown in YEPD, fixed, and stained with propidium iodide prior to analysis in a Becton Dickinson FACSort. (A) Scan of haploid SWM1 cells (W303-1A). The peaks of cells marked 1N and 2N represent cells in the G₁ and the G₂ phases of the cell cycle, respectively. (B) Scan of haploid swm1Δ cells (YPA203). (C) Scan of diploid wild-type strain YPA24. The peaks of cells marked 2N and 4N represent cells in the G₁ and the G₂ phases of the cell cycle, respectively. (D) Scan of diploid mutant strain YPA207. (E and F) Scan of two of the cycloheximide-resistant clones obtained after sporulation and germination of theswm1Δ/swm1Δ diploid strain YPA207. The graphs depict relative DNA content ( x axis) versus cell number ( y axis).

Electron microscopy of wild-type and swm1Δ mutant cells incubated for 24 h in sporulation medium. (A and B) wild-type strain YPA24. (C to H) swm1Δ mutant strain YPA207. Single asci at low magnification are depicted in panels A, C, E, G, and H, while higher magnifications of spore walls are shown in panels B, D, and F. In panel A, a typical ascus of the wild-type strain YPA24 showing three of the four spores clearly presents a well-defined spore wall surrounding all of them. In panel B, one portion of the wall of the three spores present in the ascus at higher magnification clearly shows the inner, electron-transparent components, which appear as a single layer followed by the chitin-chitosan layer closely juxtaposed next to the dityrosine coat. In panels C to H, a variety of spore wall defects are apparent in swm1Δ mutant asci. Mutant spores are surrounded by an electron-dense region with a heterogeneous structure (see the text for a detailed description of the aberrant morphology in the mutant). Scale bars, 1 μm (A, C, E, G, and H) and 0.4 μm (B, D, and F).

Expression of sporulation-specific genes in wild-type and swm1Δ mutant cells. RNA was purified from wild-type (strain YPA24) and swm1Δ cells (strain YPA207) at the indicated times after transfer to sporulation medium. RNA blots were sequentially hybridized with the following radioactively labeled gene-specific probes: SWM1, HOP1,SPO12, SSG1, DIT1, andSPS100. The ACT1 gene was used to test for equal loading of RNA in all lanes.

(A) Expression of SMK1 and SPS1 in wild-type and swm1Δ mutant cells. The same RNA blots used in Fig. 8 were stripped and sequentially hybridized with radioactively labeled probes for SPS1 and SMK1. For a better comparison, the panels corresponding to SWM1 andACT1 expression are also shown. (B) Expression ofSWM1 in wild-type and mutant smk1Δ cells. RNA purified from wild-type (strain NKY278) and smk1Δ (strain LAKY30) at the indicated times after transfer to sporulation medium was hybridized with an SWM1 radioactively labeled probe, and theACT1 gene was used to test for equal loading of RNA in all lanes.

(A) Expression of SPS100 in wild type (WT) and in single- and double-deletion mutants. RNA from strains YPA24 (WT), YPA207 (swm1Δ), LS35 (smk1Δ), LS36 (sps1Δ), LS34 (smk1Δ swm1Δ), LS37 (swm1Δ sps1Δ), and LS45 (smk1Δ sps1Δ) that had been incubated in sporulation medium for the indicated time periods was hybridized with a radioactively labeled probe specific for the SPS100 gene. After the probe stripping, the ACT1 gene was applied to same filters to test for equal loading of RNA in all lanes. (B) Resistance of wild-type and single- and double-mutant cells to prolonged incubations in sporulation medium. Wild-type cells from the strain YPA24 (∗) or the isogenic strains YPA207 (swm1Δ [□]), LS35 (smk1Δ [○]), LS36 (sps1Δ [▵]), LS34 (swm1Δ smk1Δ [■]), LS37 (sps1Δ swm1Δ [●]) and LS45 (sps1Δ smk1Δ [▴]) were incubated in sporulation medium for the indicated times before direct assay of the plating efficiencies.