Activity

Yeast (Saccharomyces cerevisiae) has been used as one of the main model systems for studying molecular mechanisms underlying cellular aging. A major technical challenge in studying aging in yeast is the isolation of aged cells from exponentially growing cell cultures, since aged cells in such cultures are rare. Several methods for isolating aged cells have been developed to achieve this. Here, we describe a biotin-streptavidin affinity purification protocol for isolating aged yeast cells. It consists of three main steps biotinylation of yeast cells, culturing cells to the desired age, and harvesting the aged cells using streptavidin-coated magnetic microbeads. The isolated aged cells can be used for microscopy, biochemistry, or molecular biology analysis.Macroautophagy, by its very nature, is a protein trafficking process. Cargos are transported and processed. Atg proteins come and go. In this chapter, we present three assays to monitor these dynamic events a non-radioactive pulse-chase labeling assay to monitor the transport of prApe1 and two fluorescent microscopy-based assays to assess the trafficking of Atg8 and Atg9.In eukaryotic cells, the genomic DNA is packaged into chromatin, the basic unit of which is the nucleosome. Studying the mechanism of chromatin formation under physiological conditions is inherently difficult due to the limitations of research approaches. Here we describe how to prepare a biochemical system called yeast nucleoplasmic extracts (YNPE). YNPE is derived from yeast nuclei, and the in vitro system can mimic the physiological conditions of the yeast nucleus in vivo. In YNPE, the dynamic process of chromatin assembly has been observed in real time at the single-molecule level by total internal reflection fluorescence microscopy. YNPE provides a novel tool to investigate many aspects of chromatin assembly under physiological conditions and is competent for single-molecule approaches.Genomic engineering methods represent powerful tools to examine chromosomal modifications and to subsequently study their impacts on cellular phenotypes. However, quantifying the fitness impact of translocations, independently from base substitutions or the insertion of genetic markers, remains a challenge. Here we report a rapid and straightforward protocol for engineering either targeted reciprocal translocations at the base pair level of resolution between two chromosomes or multiple simultaneous rearrangements in the yeast genome, without inserting any marker sequence in the chromosomes. Our CRISPR/Cas9-based method consists of inducing either (1) two double-strand breaks (DSBs) in two different chromosomes with two distinct guide RNAs (gRNAs) while providing specifically designed homologous donor DNA forcing the trans-repair of chromosomal extremities to generate a targeted reciprocal translocation or (2) multiple DSBs with a single gRNA targeting dispersed repeated sequences and leaving endogenous uncut copies of the repeat to be used as donor DNA, thereby generating multiple translocations, often associated with large segmental duplications (Fleiss, et al. Androgen Receptor Antagonist concentration PLoS Genet 15e1008332, 2019).Budding yeast, as a eukaryotic model organism, has well-defined genetic information and a highly efficient recombination system, making it a good host to produce exogenous chemicals. Since most metabolic pathways require multiple genes to function in coordination, it is usually laborious and time-consuming to construct a working pathway. To facilitate the construction and optimization of multicomponent exogenous pathways in yeast, we recently developed a method called YeastFab Assembly, which includes three steps (1) make standard and reusable genetic parts, (2) construct transcription units from characterized parts, and (3) assemble a complete pathway. Here we describe a detailed protocol of this method.Diversified genomes derived from chromosomal rearrangements are valuable materials for evolution. Naturally, chromosomal rearrangements occur at extremely low frequency to ensure genome stability. In the synthetic yeast genome project (Sc2.0), an inducible chromosome rearrangement system named Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is built to produce chromosomal rearrangements such as deletion, duplication, inversion, and translocation at high efficiency. Here, we detail the method to activate SCRaMbLE in a synthetic strain, to analyze the SCRaMbLEd genome, and to dissect the causative rearrangements for a desired phenotype after SCRaMbLEing.Budding yeast Saccharomyces cerevisiae has become a model eukaryotic microorganism for targeted genomic manipulation due to its efficient homologous recombination. A few genomic loci, including rDNA, Delta, and Ty1, can be utilized to introduce variable copies of genetic elements into the yeast genome. Here we describe a method that combines in vitro Golden Gate Assembly to assemble one or a complex genetic element in an orderly manner and then integrate it into predetermined multi-copy loci through homologous recombination. Different transformants may contain different copy numbers, which allows the selection of desired levels of target gene expression.The successful assembly of nucleosomes following DNA replication is critically important for both the inheritance of epigenetic information and the maintenance of genome integrity. This process, termed DNA replication-coupled (RC) nucleosome assembly, requires that DNA replication and nucleosome assembly function in a highly coordinated fashion to transmit both genetic and epigenetic information. In this chapter, we describe a genome-wide method for measuring nucleosome occupancy patterns on nascent strands, which we have termed Replication-Intermediate Nucleosome Mapping (ReIN-Map), to monitor the RC nucleosome assembly level genome-wide in vivo. This method takes advantage of next-generation sequencing and in vivo labeling of newly synthesized DNA using a thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU), and involves parallel analyses of the nucleosome formation using micrococcal nuclease (MNase) digestion of chromatin (MNase-seq) and of the newly synthesized DNA levels using sonication shearing of chromatin s (Sonication-seq).