Bianco Shepherd posted an update 1 month, 3 weeks ago
In brief, cells from a 5 ml saturated YPD culture have been diluted into one hundred ml SPS at a density of 105 cells/ml and grown to 2.107 cells/ml at 30 with shaking at 250 rpm. The cells had been washed and diluted into 200 ml sporulation medium (1 Potassium Acetate and expected amino-acid supplements) inside a 2 l flask and shaken at 250 rpm at 30 to induce sporulation.Monitoring of meiotic landmarksMeiotic progression was monitored by fixing 500 l of cells in 1.25 ml ethanol and staining the nuclei with 0.five g/ml 4′,6-diamidino-2-phenylindole (DAPI) for 30 minutes. Fluorescence microscopy was then employed to determine the fraction of bi-nucleate (post-MI) and tetra-nucleate (post-MII) cells. Following 48 h in sporulation medium, the sporulation efficiency was determined by phase contrast microscopy as the percentage of tetrads in the culture. Spore viability was measured by dissection of four-spore tetrads. The kinetics of recombination was monitored physically and genetically utilizing the arg4-Bgl and arg4-RV heteroalleles . For the physical assay, meiotic chromosomal DNA was extracted, digested with EcoRV and BglII, and analyzed by Southern blot as described , working with the EcoRV glII (1,016 bp) ARG4 internal fragment as probe. The production of Arg+ cells was monitored by the RTG plating assay (see under).Isolation of RTG cellsTo isolate RTG cells, samples of the sporulation culture had been harvested at many time points from 0 to 24 h immediately after transfer into the sporulation media, and RTG cells isolated utilizing two complementary techniques illustrated in Fig 1. The first strategy named “isolation by prototroph selection”, corresponds for the standard RTG plating assay [11,13] based on the choice of intragenic recombinants after transfer of heteroallelic auxotrophic cells (here arg4-RV and arg4-Bgl heteroalleles) from a sporulation time course towards the selective medium (SC-arginine). The meiotic cells had been taken at different times, washed and diluted in H2O, and plated onto SC-Arg and YPD plates ( 104 and 102 cell/plate, respectively). The plates have been incubated three days at 30 . For every time point, the frequency of heteroallelic recombination at the ARG4 locus was determined by the ratio of Arg+ colonies on SC-Arg/colonies on YPD plates. Because the entry into sporulation and also the synchrony within the cell population isn’t absolute, this mode of choice does not distinguish among: (i) fast sporulating cells that passed the commitment point to sporulation and produced recombinant spores, (ii) a recombinant RTG cell that entered the meiotic prophase-I and Title Loaded From File returned to growth prior to commitment or, (iii) a mitotic recombination in a cell that didn’t enter sporulation. Recombinant RTG cells and recombinant spores may be differentiated since RTG cells outcome from an equational chromosome segregation and for that reason are diploid, when spores that completed meiosis are haploid. In thePLOS Genetics | DOI:10.1371/journal.pgen.February 1,19 /Recombination upon Reversion of Meiosisabsence of recombination in between the MAT locus as well as the centromere (chr. III), mitotic and RTG cells stay heterozygous for MAT and thus may be screened as non-maters while haploid spores are either a-mater or -mater.