Exploiting Spore-Autonomous Fluorescent Protein Expression to Quantify Meiotic Chromosome Behaviors in Saccharomyces cerevisiae

Abstract

The budding yeast Saccharomyces cerevisiae has proven to be a rich source of information about the mechanisms and regulation of homologous recombination during meiosis. A common technique for studying this process involves microdissecting the four products (ascospores) of a single meiosis and analyzing the configuration of genetic markers in the spores that are viable. Although this type of analysis is powerful, it can be laborious and time-consuming to characterize the large numbers of meioses needed to generate statistically robust data sets. Moreover, the reliance on viable (euploid) spores has the potential to introduce selection bias, especially when analyzing mutants with elevated frequencies of meiotic chromosome missegregation. To overcome these limitations, we developed a versatile, portable set of reporter constructs that drive fluorescent protein expression specifically in only those spores that inherit the reporter. These spore-autonomous fluorescence constructs allow direct visualization of inheritance patterns in intact tetrads, eliminating the need for microdissection and permitting meiotic segregation patterns to be ascertained even in aneuploid spores. As proof of principle, we demonstrate how different arrangements of reporters can be used to quantify crossover frequency, crossover interference, gene conversion, crossover/noncrossover ratios, and chromosome missegregation.

High Throughput Sequencing Reveals Alterations in the Recombination Signatures with Diminishing Spo11 Activity

“…Using a novel color system to study CO homeostasis in tetrads, Thacker et al [18] were unable to detect CO homeostasis in a flanking marker assay in the spo11- HA mutant which has little impact on DSB levels. The authors argued that tract-length alterations might obscure NCO/CO ratios.…”

“…The analysis of flanking marker exchange among prototrophs in the ARG4 region was used to substantiate the existence of CO homeostasis [15], [18]. Increasingly defective spo11 alleles generated prototrophs with increasingly higher levels of CO association (i.e., flanking marker exchange in selected prototrophs), suggesting that, as DSBs become scarce, a given recombination event is more likely to be repaired as a CO. A second way to define CO homeostasis was described [19] where the fluctuation of CO number remained low compared to larger fluctuations of total recombination events in wild-type meioses.…”

High-throughput tetrad analysis

“…2 ). Several reporter genes have been used to fluorescently label tetrads or individual spores 13-15 . We chose the SPS2-GFP fusion, because it has been successfully used to quantitate sporulation in a number of genetically diverse, non-laboratory strains.…”

Use of fluorescent protein to analyse recombination at three loci in Neurospora crassa

“…Shotgun transformation of seeds with ECFP (enhanced cyan fluorescent protein), EYFP (enhanced yellow fluorescent protein) or DsRed (Discosoma red) constructs yielded plants with fluorescent genes in multiple locations, allowing study of crossing over and gene conversion. In addition, CFP (m-Cerulean), RFP (tdTomato), and GFP markers have recently been used to make a set of portable constructs for insertion into S. cerevisiae chromosomes (Thacker et al, 2011). The system has been tested by analysis of recombination at arg4 and of the effect of spo11 deletion on crossover homeostasis in two intervals (Thacker et al, 2011).…”

“…In addition, CFP (m-Cerulean), RFP (tdTomato), and GFP markers have recently been used to make a set of portable constructs for insertion into S. cerevisiae chromosomes (Thacker et al, 2011). The system has been tested by analysis of recombination at arg4 and of the effect of spo11 deletion on crossover homeostasis in two intervals (Thacker et al, 2011). …”