Name: genetic mapping of thermotolerance differences between species of saccharomyces yeast via genome-wide reciprocal hemizygosity analysis
Uploaded: 2019-08-12T14:30:00.000+00:00
Duration: 10 min 8 s
Description: Watch this Scientific Journal Video about Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis at JoVE.com

We thank J. Roop, R. Hackley, I. Grigoriev, A. Arkin and J. Skerker for their contributions to the original study, F. AlZaben, A. Flury, G. Geiselman, J. Hong, J. Kim, M. Maurer, and L. Oltrogge for technical assistance, D. Savage for his generosity with microscopy resources, and B. Blackman, S. Coradetti, A. Flamholz, V. Guacci, D. Koshland, C. Nelson, and A. Sasikumar for discussions; we also thank J. Dueber (Department of Bioengineering, UC Berkeley) for the PiggyBac plasmid. This work was supported by R01 GM120430-A1 and by Community Sequencing Project 1460 to RBB at the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility. The work conducted by the latter was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

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The authors have nothing to disclose.

The advantages of RH-seq over previous statistical-genetic methods are several-fold. In contrast to linkage and association analysis, RH-seq affords single-gene mapping resolution; as such, it will likely be of significant utility even in studies of trait variation across individuals of a given species, as well as interspecific differences. Also, previous attempts at genome-wide reciprocal hemizygosity analysis used collections of gene deletion mutants, some of which harbor secondary mutations that can lead to false positive results9,10. The RH-seq strategy sidesteps this issue by generating and phenotyping many hemizygote mutants in each gene in turn, such that the background of any individual mutant clone contributes only marginally to the final result. In principle, RH-seq also affords the study of noncoding loci, although in the current work we focused exclusively on genes.
There are a few quirks to RH-seq, some biological and some technical, that a successful practitioner will deal with up front to maximize the utility of the approach and accelerate the path to best results. Biologically, RH-seq only makes sense as a technique if the two target species can be mated to form a stable, viable hybrid that can be genetically manipulated. Thus we cannot envision applying RH-seq to species so divergent that they fail to fuse into a karyotypically stable diploid. On the other hand, if the two parents of the diploid hybrid are too similar at the DNA level, most reads from the transposon insertion sequencing cannot be mapped allele-specifically to just one of the two parent genomes and will be unusable; thus, a given RH-seq experiment will be most successful when the parents have high-quality reference genomes available and hit a &ldquo;sweet spot&rdquo; of sequence divergence. As a separate point of consideration, given an RH-seq project formulated to dissect the genetic basis of a trait difference between the parent species, results are likely to be much more interpretable when, for the trait of interest, the biology of the hybrid serves as a reasonable representative of that of the parents. Extreme phenotypes unique to the hybrid (heterosis) could influence or obscure the effects of genes of interest underlying the phenotype as it differs between the parents. Any genes mapped through reciprocal hemizygosity analysis must be validated by independent allele-swap experiments in the genetic backgrounds of the purebred parent species.
As for technical issues in an RH-seq experiment, our experience has highlighted several potential sources of noise and provided workable solutions. Noise manifests as disagreement among the sequencing-based estimates of fitness of the hemizygotes harboring transposon insertions in a given allele of a given gene. This can derive from differing secondary mutations in the backgrounds of transposon mutants (see below); variability in the efficiency of the PCR amplifying different insertion sites; low representation of a given mutant in the bulk pool, leading to low sequencing coverage which weakens precision; and differences in position of the transposon insertion within the gene (e.g., transposons inserting at a 3&rsquo; gene end may have minimal phenotypic effect). For all these reasons, we consider it critical to generate very large transposon mutant pools and, in the final analysis, to exclude from testing any gene without a reasonable number of mutants in each of the two alleles. We note that, although we have not implemented it here, a barcoded transposon system7 could further help resolve issues of PCR bias and cut down on the cost and labor of an RH-seq experiment.
In conclusion, we have established a straightforward workflow for RH-seq, and have specified caveats of the approach. We find that the latter does not significantly compromise the utility of RH-seq; we consider that it holds great promise for high-resolution, genome-scale dissection of the phenotypic consequences of genetic variation, including differences between species that have been reproductively isolated for millions of years.

Since the dawn of the field, it has been a prime goal in genetics to understand the mechanistic basis of variation across wild individuals. As we map loci underlying a trait of interest, the emergent genes can be of immediate use as targets for diagnostics and drugs, and can shed light on the principles of evolution. The industry standard toward this end is to test for a relationship between genotype and phenotype across a population via linkage or association1. Powerful as these approaches are, they have one key limitation&mdash;they rely on large panels of recombinant progeny from crosses between interfertile individuals. They are of no use in the study of species that cannot mate to form progeny in the first place. As such, the field has had little capacity for unbiased dissection of trait differences between reproductively isolated species2.
In this work we report the technical underpinnings of a new method, RH-seq3, for genome-scale surveys of the genetic basis of trait variation between species. This approach is a massively parallel version of the reciprocal hemizygote test4,5, which was first conceived as a way to evaluate the phenotypic effects of allelic differences between two genetically distinct backgrounds at a particular locus (Figure 1A). In this scheme, the two divergent individuals are first mated to form a hybrid, half of whose genome comes from each of the respective parents. In this background, multiple strains are generated, each containing an interrupted or deleted copy of each parent&rsquo;s allele of the locus. These strains are hemizygous since they remain diploid everywhere in the genome except at the locus of interest, where they are considered haploid, and are referred to as reciprocal since each lacks only one parent&rsquo;s allele, with its remaining allele derived from the other parent. By comparing the phenotypes of these reciprocal hemizygote strains, one can conclude whether DNA sequence variants at the manipulated locus contribute to the trait of interest, since variants at the locus are the only genetic difference between the reciprocal hemizygote strains. In this way, it is possible to link genetic differences between species to a phenotypic difference between them in a well-controlled experimental setup. To date the applications of this test have been in a candidate-gene framework&mdash;that is, cases in which the hypothesis is already in hand that natural variation at a candidate locus might impact a trait.
In what follows, we lay out the protocol for a genome-scale reciprocal hemizygosity screen, using yeast as a model system. Our method creates a genomic complement of hemizygote mutants, by generating viable, sterile F1 hybrids between species and subjecting them to transposon mutagenesis. We pool the hemizygotes, measure their phenotypes in sequencing-based assays, and test for differences in frequency between clones of the pool bearing the two parents&rsquo; alleles of a given gene. The result is a catalog of loci at which variants between species influence the trait of interest. We implement the RH-seq workflow to elucidate the genetic basis of thermotolerance differences between two budding yeast species, Saccharomyces cerevisiae and S. paradoxus, which diverged ~5 million years ago6.

<table><tbody><tr><td>1-2 plasmid Gigaprep kits</td><td>Zymo Research</td><td>D4204</td><td>The number of kits required depends on how efficient your preps are in each kit. This kit comes with 5 individual plasmid prep columns. Run 1 L of saturated E. coli culture through each prep column, as using more than 1 L per column can cause clogging of the prep filter, leading to low yield and poor quality DNA.</td></tr><tr><td>10X Tris-EDTA (TE) buffer (100 mM Tris-HCl and 10 mM EDTA)</td><td>Any</td><td>N/A</td><td>Filter sterilize through a 0.22 &amp;mu;m filter before use.</td></tr><tr><td>1M LiOAc</td><td>Any</td><td>N/A</td><td>Filter sterilize through a 0.22 &amp;mu;m filter before use.</td></tr><tr><td>300 mg/mL Geneticin (G418)</td><td>Gibco</td><td>11811023</td><td></td></tr><tr><td>52% polyethylene glycol (PEG) 3350</td><td>Sigma</td><td>1546547</td><td>Dissolve in water and filter sterilize through a 0.22 &amp;mu;m filter before use. 1X trafo mix: 228 uL 52% PEG, 36 uL 1M LiOAc, 36 uL 10X TE buffer</td></tr><tr><td>Autoclaved LB liquid broth</td><td>BD Difco</td><td>244620</td><td>Make LB liquid broth using your powder from any brand, and milliQ water. Autoclave it before use.</td></tr><tr><td>Carbenicillin stock in water (100 mg/mL)</td><td>Any</td><td>N/A</td><td>Filter sterilize through a 0.22 &amp;mu;m filter before use.</td></tr><tr><td>Complete synthetic agar plates (24.1cm x 24.1cm) with 5-fluoroorotic acid (5-FOA) [0.2% drop-out amino acid mix without uracil or yeast nitrogen base (YNB), 0.005% uracil , 2% D-glucose, 0.67% YNB without amino acids, 0.075% 5-FOA]</td><td>5-FOA: Zymo Research, Drop-out mix: US Biological, Uracil: Sigma, D-glucose: Sigm), YNB: Difco</td><td>5-FOA: F9001-5, Drop-out mix: D9535, Uracil: U0750, D-glucose: G8270, YNB: DF0919</td><td></td></tr><tr><td>DMSO</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>E. coli strain carrying pJR487 (CEN-/ARS+ piggyBac-containing plasmid)</td><td>N/A</td><td>N/A</td><td>Request from Brem lab.</td></tr><tr><td>Hybrid yeast strain JR507 (S. cerevisiae DBVPG1373 x S. paradoxus Z1, URA-/URA-)</td><td>N/A</td><td>N/A</td><td>Request from Brem lab.</td></tr><tr><td>Illumina Hiseq 2500</td><td></td><td></td><td>used for SE-150 reads</td></tr><tr><td>Large shaking incubators with variable temperature settings</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>LB + carbenicillin agar plates (100 &amp;mu;g/mL)</td><td>Agar: BD Difco</td><td>Agar: 214010</td><td>Make LB agar plates as normal and add carbenicillin to 100 &amp;mu;g/mL before drying.</td></tr><tr><td>Nanodrop spectrophotometer</td><td>Thermo Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Qubit Fluorimeter</td><td>Thermo Scientific</td><td>Q33240</td><td></td></tr><tr><td>Salmon sperm DNA</td><td>Invitrogen</td><td>15632011</td><td></td></tr><tr><td>Water bath at 39&amp;deg;C</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>Yeast fungal gDNA prep kit</td><td>Zymo Research</td><td>D6005</td><td></td></tr><tr><td>Yeast peptone dextrose (YPD) liquid media</td><td>BD Difco</td><td>Peptone: 211677, Yeast Extract: 212750</td><td>Add filter-sterilized D-glucose to 2% after autoclaving.</td></tr><tr><td>YPD + G418 agar plates (300 &amp;mu;g/mL)</td><td>Agar: BD Difco</td><td>Agar: 214010</td><td>Make YPD agar plates as normal and add G418 to 300 &amp;mu;g/mL before drying.</td></tr><tr><td>YPD agar plates</td><td>Agar: BD Difco</td><td>Agar: 214010</td><td></td></tr></tbody></table>

genetic mapping of thermotolerance differences between species of saccharomyces yeast via genome-wide reciprocal hemizygosity analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

We mated S. cerevisiae and S. paradoxus to form a sterile hybrid, which we subjected to transposon mutagenesis. Each mutagenized clone was a hemizygote, a diploid hybrid in which one allele of one gene is disrupted (Figure 1A, Figure 2). We competed the hemizygotes against one another by growth at 39 &#176;C and, in a separate experiment as a control, at 28 &#176;C (Figure 1B), and we isolated DNA from each culture. To report the fitness of each hemizygote we quantified abundance via bulk sequencing, using a protocol in which DNA was fragmented and ligated to adapters, followed by amplification of transposon insertion positions (Figure 1C). If the primers for this amplification are distinct from, and less efficient than, those provided in the protocol, background reads will predominate in the sequencing data, leading to fewer usable reads and eroding the accuracy of fitness estimates. Similar quality issues may result from low DNA input into the sequencing library preparation.
With results in hand from our sequencing, for a given gene we compared hemizygote abundances at the two temperatures between two classes of hemizygotes: clones where only the S. cerevisiae allele was wild-type and functional, and clones relying only on the S. paradoxus allele (Figure 1D). In analysis at this stage, if the computational post-processing strategy of the protocol is not followed and genes with relatively few transposon mutants in the pool are included in the analysis, statistical power will drop and no significant gene calls will result. In our implementation, we detected strong signal at eight housekeeping genes (Figure 3). In each case, transposon insertions in the S. cerevisiae allele in the hybrid compromised growth at high temperature (Figure 3). These loci represented candidate determinants of the thermotolerance trait that distinguishes S. cerevisiae from S. paradoxus. In separate experiments reported elsewhere, we validated the impact of allelic variation at each site using standard transgenesis methods beyond the scope of the current protocol3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59972/59972fig01.jpg" /> 
Figure 1. Schematic of the RH-seq workflow. 
A. S. cerevisiae and S. paradoxus (blue and yellow respectively), are mated to form a hybrid (green) that contains a single copy of each of the parents&#8217; genomes. At a given locus in the hybrid, a transposon insertion (black box) in each species&#8217; allele in turn creates a hemizygote, which is diploid at the rest of the genome except for the locus of interest. Comparing phenotypes across hemizygotes reveals the phenotypic effects of allelic variation at the manipulated locus. B. Across many clones hemizygous at a given gene (YFG), some reach higher abundance than others in competitive culture, as quantified by sequencing. C. DNA from a hemizygote pool is sheared and ligated to adapters (red). For a given clone, the junction between the transposon (tn, black) and the genome (blue) is amplified with a transposon-specific primer (black arrowhead) and an adapter-specific primer (red arrowhead). Sequencing read counts from the amplicon report the fitness of the clone in the population. D. For an RH-seq gene hit, tabulating the proportion of hemizygote clones (y-axis) exhibiting a given fitness after competition at high temperature (x-axis) reveals a striking difference between two genotypic classes: those with a transposon insertion in the S. cerevisiae allele (with the S. paradoxus allele remaining; yellow) and those with the S. paradoxus allele disrupted (and S. cerevisiae allele remaining; blue). <a href="https://www.jove.com/files/ftp_upload/59972/59972fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59972/59972fig02.jpg" /> 
Figure 2. Selection scheme for generating a pool of genome-wide reciprocal hemizygotes with the PiggyBac plasmid-borne transposon. 
The PiggyBac plasmid (pJR487) is transformed into a URA3-/- clone of the diploid hybrid S. cerevisiae DBVPG1373 x S. paradoxus Z1 (JR507). The presence of the plasmid or transposon is selected for via growth in G418, which selects for the presence of the KanMX cassette; survivors are cells which have taken up the PiggyBac plasmid and/or harbor an integrated transposon. Cells without the latter are selected against via growth in 5-FOA, which is toxic in the presence of the URA3 cassette. Since the untransformed hybrid is URA3-/-, the only cells that will die in this step are those still containing the PiggyBac plasmid, which contains a URA3 cassette. What remains is a pool of hybrid mutant cells containing the transposon integrated into the genome. <a href="https://www.jove.com/files/ftp_upload/59972/59972fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59972/59972fig03.jpg" /> 
Figure 3. Top hits mapped by RH-seq. 
Each panel reports RH-seq data for the indicated gene from RH-seq. The x-axis reports the log2 of abundance of a transposon mutant clone after selection at 39 &#176;C, relative to the analogous quantity at 28 &#176;C. The y-axis reports the proportion of all clones bearing insertions in the indicated allele that exhibited the abundance ratio on the x, as a kernel density estimate. Underlying read and count data for insertions are reported elsewhere3. <a href="https://www.jove.com/files/ftp_upload/59972/59972fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis at JoVE.com

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

genetic-mapping-thermotolerance-differences-between-species

Nanyang Technological University

Research

JoVE Journal

Genetics

16.9K Views.  University of California Berkeley. Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

Video: Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Biology

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

BEST: Barcode Enabled Sequencing of Tetrads

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.

Barcode Enabled Sequencing of Tetrads (BEST) replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by fluorescence-activated cell sorting onto agar plates, separates the spores by agitation with glass beads, and determines which randomly arrayed colonies were derived from the same original tetrad using molecular barcodes.

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

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Chapters in this video

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Microarray Analysis for Saccharomyces cerevisiae

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

BEST: Barcode Enabled Sequencing of Tetrads

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe