Haploid Mutants Research Articles

De Novo DNM1L Pathogenic Variant Associated with Lethal Encephalocardiomyopathy-Case Report and Literature Review.

Pathogenic variants in DNM1L, encoding dynamin-like protein-1 (DRP1), cause a lethal encephalopathy. DRP1 defective function results in altered mitochondrial networks, characterized by elongated/spaghetti-like, highly interconnected mitochondria. We validated in yeast the pathogenicity of a de novo DNM1L variant identified by whole exome sequencing performed more than 10 years after the patient's death. Meanwhile, we reviewed the broadness and specificities of DNM1L-related phenotype. The patient, who exhibited developmental delay in her third year, developed a therapy-refractory myoclonic status epilepticus, followed by neurological deterioration with brain atrophy and refractory epilepsy. She died of heart failure due to hypertrophic cardiomyopathy. She was found to be heterozygous for the DNM1L variant (NM_ 012062.5):c.1201G>A, p.(Gly401Ser). We demonstrated its deleterious impact and dominant negative effect by assessing haploid and diploid mutant yeast strains, oxidative growth, oxygen consumption, frequency of petite, and architecture of the mitochondrial network. Structural modeling of p.(Gly401Ser) predicted the interference of the mutant protein in the self-oligomerization of the DRP1 active complex. DNM1L-related phenotypes include static or (early) lethal encephalopathy and neurodevelopmental disorders. In addition, there may be ophthalmological impairment, peripheral neuropathy, ataxia, dystonia, spasticity, myoclonus, and myopathy. The clinical presentations vary depending on mutations in different DRP1 domains. Few pathogenic variants, the p.(Gly401Ser) included, cause an encephalocardiomyopathy with refractory status epilepticus.

The Cytological Mechanism of the Peach Haploid Producing Triploid Offspring

Abstract Peach is one of the most economically valuable fruit trees. Haploid peach trees occur spontaneously at very low frequencies and they are usually highly sterile. Therefore, the haploid with partial fertility is extremely rare germplasm, which is highly valuable to genetic research and breeding programmes. In this study, we investigated the cytological mechanism underlying the fertility of a peach haploid mutant ‘9-D’ derived from a spontaneous mutation. Cytologic evaluation and flow cytometry analysis demonstrated that ‘9-D’ is a pure haploid. Scanning electron microscope analysis revealed a considerable proportion of abnormal pollen grains in ‘9-D’. Pollen viability assay by Alexander staining showed that 50.4% of pollen grains from ‘9-D’ were viable. However, the pollen germination assay showed that only 7.6% of the pollen grains could germinate normally. Investigation of the chromosomal behavior of pollen mother cells at different stages of meiosis showed that pollen mother cells of ‘9-D’ lacked the process between anaphase I and prophase II of meiosis. Various types of sporophyte morphology were observed in haploid pollen mother cells at the tetrad stage. Measurement of the diameter of pollen grains indicated the presence of pollen with 2x ploidy in ‘9-D’. The offspring of ‘9-D’ were predominantly triploid or triploid aneuploid. The triploid offspring were more likely derived from the 2x male gametophyte combined with the haploid female gametophyte, which may explain the reason why ‘9-D’ has fertility. This study not only expands our understanding of haploid fertility mechanisms, but is also useful for ploid breeding programs in peach.

Evidence that DNA polymerase δ proofreads errors made by DNA polymerase α across the Saccharomyces cerevisiae nuclear genome

We show that the rates of single base substitutions, additions, and deletions across the nuclear genome are strongly increased in a strain harboring a mutator variant of DNA polymerase α combined with a mutation that inactivates the 3´-5´ exonuclease activity of DNA polymerase δ. Moreover, tetrad dissections attempting to produce a haploid triple mutant lacking Msh6, which is essential for DNA mismatch repair (MMR) of base•base mismatches made during replication, result in tiny colonies that grow very slowly and appear to be aneuploid and/or defective in oxidative metabolism. These observations are consistent with the hypothesis that during initiation of nuclear DNA replication, single-base mismatches made by naturally exonuclease-deficient DNA polymerase α are extrinsically proofread by DNA polymerase δ, such that in the absence of this proofreading, the mutation rate is strongly elevated. Several implications of these data are discussed, including that the mutational signature of defective extrinsic proofreading in yeast could appear in human tumors.

Screening and In Silico Analyses of the Yeast Saccharomyces cerevisiae Σ1278b Bank Mutants Using Citral as a Natural Antimicrobial.

The antimicrobial function of citral, one of the main compounds of the essential oils (EO) of the Citrus genus, and widely used by the food industry toward spoilage yeast, was previously proven. In this study, the possible mode of action of citral against yeast cells was evaluated by using a global deletome approach. Firstly, the suitability of Saccharomyces cerevisiae Σ1278b to serve as model yeast was assessed by determining its sensitivity to citral (MIC = 0.5 μL/mL). Subsequently, the complete library of Σ1278b haploid mutants deleted in 4019 non-essential genes was screened to identify potential molecular targets of citral. Finally, the deleted genes in the 590 mutants showing increased citral resistance was analyzed with an in-silico approach (Gene Ontology). The significantly enriched GO Terms were "cytoplasm", "vacuole", and "mitochondrion" (cellular components); "catalytic activity" (molecular function); "pseudohyphal growth" (biological process). For molecular function, resistant mutants were grouped into thiosulfate sulfur transferase activity, transferase activity, and oxidoreductase activity; for cellular components, resistant mutants were grouped as: cytoplasm, intracellular organelle, membrane-bounded organelle, mitochondrion, organelle membrane, and vacuole; and finally, with regard to biological process, deleted genes were grouped as: pseudohyphal growth, mitochondrion organization, lipid metabolic process, DNA recombination and repair, and proteolysis. Interestingly, many identified genes were associated with the cellular response to oxidative stress and ROS scavenging. These findings have important implications for the development of citral-based antimicrobials and the elucidation of its mechanism of action.

Genetic manipulation of Indian mustard genotypes with WRR-gene(s) confers resistance against Albugo candida.

Brassica species is the second most important edible oilseed crop in India. Albugo candida (Pers.) Kuntze, a major oomycete disease of oilseed brassica causing white rust, leads to 60% yield loss globally. The prevalence of A. candida race 2 (Ac2V) that specifically infects B. juncea, coupled with limitations of conventional methods has resulted in a dearth of white rust resistance resources in cultivated varieties. In an effort to develop resistant plants, Agrobacterium mediated genetic transformation of three B. juncea genotypes viz., susceptible host var. Varuna, along with its doubled haploid mutant lines C66 and C69 (showing moderate tolerance to field isolates of A. candida) was initiated to transfer resistance genes (WRR8Sf-2 and WRR9Hi-0) identified in Arabidopsis thaliana against race Ac2V, that encode for Toll-like/interleukin-1 receptor-nucleotide binding-leucine-rich repeat proteins that recognize effectors of the pathogen races. Our results demonstrate that introduction of resistance genes from a tertiary gene pool by genetic transformation enhances disease resistance in B. juncea genotypes to a highly virulent Ac2V isolate.

Mft1, identified from a genome-wide screen of the yeast haploid mutants, mediates cell cycle arrest to counteract quinoxaline-induced toxicity.

Quinoxaline is a heterocyclic compound with a two-membered ring structure that undergoes redox cycling to produce toxic free radicals. It has antiviral, antibacterial, antifungal, and antitumor activities. However, the biological functions that are involved in mounting a response against the toxic effects of quinoxaline have not been investigated. Herein, we performed a genome-wide screen using the yeast haploid mutant collection and reported the identification of 12 mutants that displayed varying sensitivity towards quinoxaline. No mutant was recovered that showed resistance to quinoxaline. The quinoxaline-sensitive mutants were deleted for genes that encode cell cycle function, as well as genes that belong to other physiological pathways such as the vacuolar detoxification process. Three of the highly sensitive gene-deletion mutants lack the DDC1, DUN1, and MFT1 genes. While Ddc1 and Dun1 are known to perform roles in the cell cycle arrest pathway, the role of Mft1 remains unclear. We show that the mft1Δ mutant is as sensitive to quinoxaline as the ddc1Δ mutant. However, the double mutant ddc1Δ mft1Δ lacking the DDC1 and MFT1 genes, is extremely sensitive to quinoxaline, as compared to the ddc1Δ and mft1Δ single mutants. We further show that the mft1Δ mutant is unable to arrest in the G2/M phase in response to the drug. We conclude that Mft1 performs a unique function independent of Ddc1 in the cell cycle arrest pathway in response to quinoxaline exposure. This is the first demonstration that quinoxaline exerts its toxic effect likely by inducing oxidative DNA damage causing cell cycle arrest. We suggest that clinical applications of quinoxaline and its derivatives should entail targeting cancer cells with defective cell cycle arrest.

The impact of the GLOSSY2 and GLOSSY2-LIKE BAHD-proteins in affecting the product profile of the maize fatty acid elongase.

The maize glossy2 and glossy2-like genes are homologs, which encode proteins that belong to the BAHD family of acyltransferases. In planta genetic studies have demonstrated that these genes may be involved in the elongation of very long chain fatty acids (VLCFAs) that are precursors of the cuticular wax fraction of the plant cuticle. VLCFAs are synthesized by a fatty acyl-CoA elongase complex (FAE) that consists of four component enzymes. Previously, we functionally identified the maize FAE component enzymes by their ability to complement haploid Saccharomyces cerevisiae strains that carry lethal deletion alleles for each FAE component enzyme. In this study we used these complemented haploid strains and wild-type diploid strains to evaluate whether the co-expression of either GLOSSY2 or GLOSSY2-LIKE with individual maize FAE component enzymes affects the VLCFA product-profile of the FAE system. Wild-type diploid strains produced VLCFAs of up to 28-carbon chain length. Co-expression of GLOSSY2 or GLOSSY2-LIKE with a combination of maize 3-ketoacyl-CoA synthases stimulated the synthesis of longer VLCFAs, up to 30-carbon chain lengths. However, such results could not be recapitulated when these co-expression experiments were conducted in the yeast haploid mutant strains that lacked individual components of the endogenous FAE system. Specifically, lethal yeast mutant strains that are genetically complemented by the expression of maize FAE-component enzymes produce VLCFAs that range between 20- and 26-carbon chain lengths. However, expressing either GLOSSY2 or GLOSSY2-LIKE in these complemented strains does not enable the synthesis of longer chain VLCFAs. These results indicate that the apparent stimulatory role of GLOSSY2 or GLOSSY2-LIKE to enable the synthesis of longer chain VLCFAs in diploid yeast cells may be associated with mixing plant enzyme components with the endogenous FAE complex.

Identification of Aly1 and Aly2 as Modulators of Cytoplasmic pH in Saccharomyces cerevisiae.

The regulation of intracellular pH in yeast (Saccharomyces cerevisiae) cells is critical for cell function and viability. In yeast, protons (H+) can be excreted from the cell by plasma membrane ATPase PMA1 and pumped into vacuoles by vacuolar H+-ATPase. Because PMA1 is critical to the survival of yeast cells, it is unknown whether other compensatory components are involved in pH homeostasis in the absence of PMA1. To elucidate how intracellular pH is regulated independently of PMA1, we employed a screening approach by exposing the yeast haploid deletion mutant library (ver 4.0) to the selective plant plasma membrane H+-ATPase inhibitor PS-1, which we previously reported. After repeated screenings and verification, we identified two proteins, Aly1 and Aly2, that play a role in the regulation of intracellular pH when PMA1 is deficient. Our research uncovers a new perspective on the regulation of intracellular pH related to PMA1 and also preliminarily reveals a role for Aly1 and Aly2 in the regulation of intracellular pH.

DNA Repair Does Not Trigger Genetic Instability of Yeast Cells

The dependence of the delayed formation of colonies on the dose of ionizing radiation (gamma rays of 60Co, dose rate 10 Gy/min) was obtained for six strains of wild-type haploid and diploid yeast, capable of recovering from radiation damage and characterized by sigmoidal survival curves as well as for six haploid and diploid radiosensitive mutants defective in reparation and characterized by exponential survival curves. The delay in the formation of colonies by survival cells after irradiation is considered as a genetic instability display. It was shown that for all diploid strains, genetic instability reached 100% with an increase in the dose of ionizing radiation, regardless of the shape of survival curves and the cell ability to recover from radiation damage. Conversely, for all haploid strains, genetic instability was only close to 20%. In contrast to traditional concepts, these data indicate that the late formation of colonies by surviving yeast cells after irradiation is determined mainly by cell ploidy and does not depend on the shape of the dose-response curves and cell radiosensitivity. This means that DNA repair does not trigger genetic instability in yeast cells.

Investigation of mutation load and rate in androgenic mutant lines of rapeseed in early generations evaluated by high-density SNP genotyping

Oilseed rape (Brassica napus) is an important oil crop distributed worldwide with a broad adaptation to different climate zones. The cultivation of rapeseed is one of the most commercially viable areas in crop production. Altogether 269,093 ha of rapeseed are cultivated in Kazakhstan. However, all rapeseed cultivars and lines cultivated in Kazakhstan on an industrial scale predominantly belong to the foreign breeding system. Therefore, the formation of a diverse genetic pool for breeding new, highly productive cultivars adopted to the environmental conditions of Kazakhstan is the most important goal in country selection programs. In this work, we have developed ethyl methanesulfonate (EMS) doubled haploid mutant lines from plant material of cultivars ‘Galant’ and ‘Kris’ to broad diversity of rapeseed in Kazakhstan. The development of mutant lines was performed via embryo callusogenesis or embryo secondary callusogenesis. Mutants were investigated by Brassica90k SNP array, and we were able to locate 24,657 SNPs from 26,256 SNPs filtered by quality control on the genome assembly (Bra_napus_v2.0). Only 18,831 SNPs were assigned to the available annotated genomic features. The most frequent combination of mutations according to reference controls was adenine with guanine (70%), followed by adenine with cytosine (28.8%), and only minor fractions were cytosine with guanine (0.54%) and adenine with thymine (0.59%). We revealed 5606.27 markers for ‘Kris’ and 4893.01 markers for ‘Galant’ by mutation occurrence. Most mutation occurrences were occupied by double mutations where progenitors and offspring were homozygous by different alleles, enabling the selection of appropriate genotypes in a short period of time. Regarding the biological impact of mutations, 861 variants were reported as having a low predicted impact, with 1042 as moderate and 121 as high; all others were reported as belonging to non-coding sequences, intergenic regions, and other features with the effect of modifiers. Protein encoding genes, such as wall-associated receptor kinase-like protein 5, TAO1-like disease resistance protein, receptor-like protein 12, and At5g42460-like F-box protein, contained more than two variable positions, with an impact on their biological activities. Nevertheless, the obtained mutant lines were able to survive and reproduce. Mutant lines, which include moderate and high impact mutations in encoding genes, are a perfect pool not only for MAS but also for the investigation of the fundamental basis of protein functions. For the first time, a collection of mutant lines was developed in our country to improve the selection of local rapeseed cultivars.

Biochemical profiling of Brassica juncea doubled haploid mutant genotypes under white rust disease stress

White rust, caused by oomycete pathogen Albugo candida, is a serious disease for the crops of Brassicaceae family. It is an obligate pathogen responsible for up to 60% yield loss with combined infection of leaves and inflorescence across the globe. Biochemical variations in host plants can alter the content of proteins, sugars, phenols, iron content etc. enables to understand the biochemistry of host-pathogen interaction. Therefore, the present study was conducted during 2017-2019 at Amity University, Noida, Uttar Pradesh to evaluate the biochemical response of Brassica juncea var. Varuna and its doubled haploid (DH) mutant genotypes infected with A. candida causing white rust at cotyledonary leaf stage. The seven mutant genotypes were tested for pathogenic response against five white rust isolates (WRI). In the challenge assay, mutant genotypes C66 and C69 showed resistance response as compared to susceptible host B. juncea var. Varuna against different isolates on the basis of disease severity. The biochemical parameters were found to be high in the uninoculated genotypes in comparison to infected cotyledons of B. juncea genotypes. The biochemical profile emphasizes significant increase in content of protein, sugar, and phenol from highest in genotype C66 positively correlating with resistance response against pathogen in comparison to other mutant genotype C69 and susceptible check Varuna. The results suggest that factors conditioning the response of mutant genotypes to WRI may differ or control in different ways and triggers novel defense signaling.

Actin-Related Protein 4 and Linker Histone Sustain Yeast Replicative Ageing.

Ageing is accompanied by dramatic changes in chromatin structure organization and genome function. Two essential components of chromatin, the linker histone Hho1p and actin-related protein 4 (Arp4p), have been shown to physically interact in Saccharomyces cerevisiae cells, thus maintaining chromatin dynamics and function, as well as genome stability and cellular morphology. Disrupting this interaction has been proven to influence the stability of the yeast genome and the way cells respond to stress during chronological ageing. It has also been proven that the abrogated interaction between these two chromatin proteins elicited premature ageing phenotypes. Alterations in chromatin compaction have also been associated with replicative ageing, though the main players are not well recognized. Based on this knowledge, here, we examine how the interaction between Hho1p and Arp4p impacts the ageing of mitotically active yeast cells. For this purpose, two sets of strains were used—haploids (WT(n), arp4, hho1Δ and arp4 hho1Δ) and their heterozygous diploid counterparts (WT(2n), ARP4/arp4, HHO1/hho1Δ and ARP4 HHO1/arp4 hho1Δ)—for the performance of extensive morphological and physiological analyses during replicative ageing. These analyses included a comparative examination of the yeast cells’ chromatin structure, proliferative and reproductive potential, and resilience to stress, as well as polysome profiles and chemical composition. The results demonstrated that the haploid chromatin mutants arp4 and arp4 hho1Δ demonstrated a significant reduction in replicative and total lifespan. These findings lead to the conclusion that the importance of a healthy interaction between Arp4p and Hho1p in replicative ageing is significant. This is proof of the concomitant importance of Hho1p and Arp4p in chronological and replicative ageing.

Genetic Dissection of T-DNA Insertional Mutants Reveals Uncoupling of Dikaryotic Filamentation and Virulence in Sugarcane Smut Fungus.

The biotrophic basidiomycetous fungus Sporisorium scitamineum causing smut disease in sugarcane is characterized by a life cycle composed of a yeast-like nonpathogenic haploid basidiosporial stage outside the plant and filamentous pathogenic dikaryotic hyphae within the plant. Under field conditions, dikaryotic hyphae are formed after mating of two opposite mating-type strains. However, the mechanisms underlying genetic regulation of filamentation and its association with pathogenicity and development of teliospores are unclear. This study has focused on the characterization and genetic dissection of haploid filamentous mutants derived from T-DNA insertional mutagenesis. Our results support the existence of at least three genotypes among the six haploid filamentous mutants that differentially contribute to virulence and development of the whip and teliospore, providing a novel foundation for further investigation of the regulatory networks associated with pathogenicity and teliospore development in S. scitamineum.

Investigating the Antifungal Mechanism of Action of Polygodial by Phenotypic Screening in Saccharomyces cerevisiae.

Polygodial is a “hot” peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial’s antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast.

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

Trigenic Synthetic Genetic Array (τ-SGA) Technique for Complex Interaction Analysis.

Complex genetic interactions occur when mutant alleles of multiple genes combine to elicit an unexpected phenotype, which could not be predicted given the expectation based on the combination of phenotypes associated with individual mutant alleles. Trigenic Synthetic Genetic Array (τ-SGA) methodology was developed for the systematic analysis of complex interactions involving combinations of three gene perturbations. With a series of replica pinning steps of the τ-SGA procedure, haploid triple mutants are constructed through automated mating and meiotic recombination. For example, a double-mutant query strain carrying two mutant alleles of interest, such as a deletion allele of a nonessential gene and a conditional temperature-sensitive allele of an essential gene, is crossed to an input array of yeast mutants, such as the diagnostic array set of ~1200 mutants, to generate an output array of triple mutants. The colony-size measurements of the resulting triple mutants are used to estimate cellular fitness and quantify trigenic interactions by incorporating corresponding single- and double-mutant fitness estimates. Trigenic interaction networks can be further analyzed for functional modules using various clustering and enrichment analysis tools. Complex genetic interactions are rich in functional information and provide insight into the genotype-to-phenotype relationship, genome size, and speciation.

Applications of Doubled Haploids in Plant Breeding and Applied Research.

Manifold and diverse applications of doubled haploid (DH) plants have emerged in academy and in the plant breeding industry since the first discovery of a haploid mutant in the Jimson Weed (Datura stramonium), followed by the first reports about anther culture in the same species, maternal haploids by wide crosses in tobacco (Nicotiana tabacum L.) and barley (Hordeum vulgare L.), interspecific hybridization, ovary culture (gynogenesis), isolated microspore culture, and more recently the CENH3 approach in thale cress (Arabidopsis thaliana L.) and other species. Research and development efforts were and are still significant in both user groups. Luckily, often academic and industrial partners cooperate in challenging and sometimes voluminous projects worldwide. Not only to develop innovative DH protocols and technologies per se, but also to exploit the advantages of DH plants in a huge variety of research and development experiments. This review concentrates not on the DH technologies per se, but on the application of DHs in plant-related research and development projects.

Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates.

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

Yeast Lacking the PP2A Phosphatase Regulatory Subunit Rts1 Sensitizes rad51 Mutants to Specific DNA Damaging Agents.

Rts1 is a regulatory subunit of the trimeric protein phosphatase 2A phosphatase and it participates in many biological processes by modulating the phosphorylation status of proteins. Consistent with its role, mutants lacking Rts1 display multiple phenotypes. We have previously performed a high throughput screen to search for yeast haploid mutants with altered sensitivity to the anticancer drug bleomycin, which acts by damaging the DNA to produce single and double strand breaks. RTS1 was among the genes that when singly deleted cause sensitivity to bleomycin. We investigate whether Rts1 plays a role in the repair of bleomycin-induced DNA lesions. We show that deletion of the RTS1 gene in the rad51 null background, lacking Rad51 known to be involved in the repair of bleomycin-induced DNA lesions, resulted in double mutants that were sensitized to bleomycin and not to other DNA damaging agents that creates DNA adducts. We further show that Rts1 has the ability to bind to DNA and in its absence cells displayed an increase in the frequency of both spontaneous and bleomycin-induced mutations compared to the parent. This is the first report implicating Rts1 with a role in DNA damage and repair, perhaps regulating the phosphorylation status of one or more proteins involved in the repair of DNA strand breaks.

Transcriptomic and chemogenomic analyses unveil the essential role of Com2-regulon in response and tolerance of Saccharomyces cerevisiae to stress induced by sulfur dioxide.

During vinification Saccharomyces cerevisiae cells are frequently exposed to high concentrations of sulfur dioxide (SO2) that is used to avoid overgrowth of unwanted bacteria or fungi present in the must. Up to now the characterization of the molecular mechanisms by which S. cerevisiae responds and tolerates SO2 was focused on the role of the sulfite efflux pump Ssu1 and investigation on the involvement of other players has been scarce, especially at a genome-wide level. In this work, we uncovered the essential role of the poorly characterized transcription factor Com2 in tolerance and response of S. cerevisiae to stress induced by SO2 at the enologically relevant pH of 3.5. Transcriptomic analysis revealed that Com2 controls, directly or indirectly, the expression of more than 80% of the genes activated by SO2, a percentage much higher than the one that could be attributed to any other stress-responsive transcription factor. Large-scale phenotyping of the yeast haploid mutant collection led to the identification of 50 Com2-targets contributing to the protection against SO2 including all the genes that compose the sulfate reduction pathway (MET3, MET14, MET16, MET5, MET10) and the majority of the genes required for biosynthesis of lysine (LYS2, LYS21, LYS20, LYS14, LYS4, LYS5, LYS1 and LYS9) or arginine (ARG5,6, ARG4, ARG2, ARG3, ARG7, ARG8, ORT1 and CPA1). Other uncovered determinants of resistance to SO2 (not under the control of Com2) included genes required for function and assembly of the vacuolar proton pump and enzymes of the antioxidant defense, consistent with the observed cytosolic and mitochondrial accumulation of reactive oxygen species in SO2-stressed yeast cells.