Splitting the yeast centromere by recombination.

Although fusions between the centromeres of different human chromosomes have been observed cytologically in cancer cells, since the centromeres are long arrays of satellite sequences, the details of these fusions have been difficult to investigate. We developed methods of detecting recombination within the centromeres of the yeast Saccharomyces cerevisiae (intercentromere recombination). These events occur at similar rates (about 10-8/cell division) between two active or two inactive centromeres. We mapped the breakpoints of most of the recombination events to a region of 43 base pairs of uninterrupted homology between the two centromeres. By whole-genome DNA sequencing, we showed that most (>90%) of the events occur by non-reciprocal recombination (gene conversion/break-induced replication). We also found that intercentromere recombination can involve non-homologous chromosome, generating whole-arm translocations. In addition, intercentromere recombination is associated with very frequent chromosome missegregation. These observations support the conclusion that intercentromere recombination generally has negative genetic consequences.

Global genomic instability caused by reduced expression of DNA polymerase ε in yeast.

SignificanceAlthough most studies of the genetic regulation of genome stability involve an analysis of mutations within the coding sequences of genes required for DNA replication or DNA repair, recent studies in yeast show that reduced levels of wild-type enzymes can also produce a mutator phenotype. By whole-genome sequencing and other methods, we find that reduced levels of the wild-type DNA polymerase ε in yeast greatly increase the rates of mitotic recombination, aneuploidy, and single-base mutations. The observed pattern of genome instability is different from those observed in yeast strains with reduced levels of the other replicative DNA polymerases, Pol α and Pol δ. These observations are relevant to our understanding of cancer and other diseases associated with genetic instability.

Uncovering Bleomycin-Induced Genomic Alterations and Underlying Mechanisms in the Yeast Saccharomyces cerevisiae.

Bleomycin (BLM) is a widely used chemotherapeutic drug. BLM-treated cells showed an elevated rate of mutations, but the underlying mechanisms remained unclear. In this study, the global genomic alterations in BLM-treated cells were explored in the yeast Saccharomyces cerevisiae. Using genetic assay and whole-genome sequencing, we found that the mutation rate could be greatly elevated in S. cerevisiae cells that underwent Zeocin (a BLM member) treatment. One-base deletion and T-to-G substitution at the 5'-GT-3' motif represented the most striking signature of Zeocin-induced mutations. This was mainly the result of translesion DNA synthesis involving Rev1 and polymerase ζ. Zeocin treatment led to the frequent loss of heterozygosity and chromosomal rearrangements in the diploid strains. The breakpoints of recombination events were significantly associated with certain chromosomal elements. Lastly, we identified multiple genomic alterations that contributed to BLM resistance in the Zeocin-treated mutants. Overall, this study provides new insights into the genotoxicity and evolutional effects of BLM. IMPORTANCE Bleomycin is an antitumor antibiotic that can mutate genomic DNA. Using yeast models in combination with genome sequencing, the mutational signatures of Zeocin (a member of the bleomycin family) are disclosed. Translesion-synthesis polymerases are crucial for the viability of Zeocin-treated yeast cells at the sacrifice of a higher mutation rate. We also confirmed that multiple genomic alterations were associated with the improved resistance to Zeocin, providing novel insights into how bleomycin resistance is developed in cells.

Origin, Regulation, and Fitness Effect of Chromosomal Rearrangements in the Yeast Saccharomyces cerevisiae.

Chromosomal rearrangements comprise unbalanced structural variations resulting in gain or loss of DNA copy numbers, as well as balanced events including translocation and inversion that are copy number neutral, both of which contribute to phenotypic evolution in organisms. The exquisite genetic assay and gene editing tools available for the model organism Saccharomyces cerevisiae facilitate deep exploration of the mechanisms underlying chromosomal rearrangements. We discuss here the pathways and influential factors of chromosomal rearrangements in S. cerevisiae. Several methods have been developed to generate on-demand chromosomal rearrangements and map the breakpoints of rearrangement events. Finally, we highlight the contributions of chromosomal rearrangements to drive phenotypic evolution in various S. cerevisiae strains. Given the evolutionary conservation of DNA replication and recombination in organisms, the knowledge gathered in the small genome of yeast can be extended to the genomes of higher eukaryotes.

Genome-wide mapping of spontaneous genetic alterations in diploid yeast cells.

Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain of Saccharomyces cerevisiae by whole-genome sequencing of many independent isolates (n = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n = 1,337) and mitotic recombination events (n = 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.

Flavobacterium salilacus sp. nov., isolated from surface water of a hypersaline lake, and descriptions of Flavobacterium salilacus subsp. altitudinum subsp. nov. and Flavobacterium salilacus subsp. salilacus subsp. nov.

Two yellow-pigmented, Gram-stain-negative, aerobic, rod-shaped bacteria were isolated from the water of the hypersaline Chaka Salt Lake (strain SaA2.12T) and sediment of Qinghai Lake (strain LaA7.5T), PR China. According to the 16S rRNA phylogeny, the isolates belong to the genus Flavobacterium, showing the highest 16S rRNA sequence similarities to Flavobacterium arcticum SM1502T(97.6-97.7 %) and Flavobacterium suzhouense XIN-1T(96.5-96.6 %). Moreover, strains SaA2.12T and LaA7.5T showed 99.73 % 16S rRNA sequence similarity to each other. Major fatty acids, respiratory quinones and polar lipids detected in these isolates were iso-C15 : 0, menaquinone-6 and phosphatidylethanolamine, respectively. Strains SaA2.12T and LaA7.5T showed significant unique characteristics between them as well as between the closest phylogenetic members. The highest digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between SaA2.12T and its closest neighbours were 25.3 and 82.8 %, respectively; whereas these values (highest) between LaA7.5T and its closest members were 25.2 and 82.8 %, respectively. The dDDH and ANI values between strains SaA2.12T and LaA7.5T were calculated as 75.9 and 97.2 %, respectively. Therefore, based on polyphasic data, we propose that strain SaA2.12T represents a novel species with the name Flavobacterium salilacus sp. nov., with the type strain SaA2.12T (=KCTC 72220T=MCCC 1K03618T) and strain LaA7.5T as a subspecies within novel Flavobacterium salilacus with the name Flavobacterium salilacus subsp. altitudinum subsp. nov., with the type strain LaA7.5T (=KCTC 72806T=MCCC 1K04372T). These propositions automatically create Flavobacterium salilacus subsp. salilacus subsp. nov. with SaA2.12T (=KCTC 72220T=MCCC 1K03618T) as the type strain.

Flavobacterium zhairuonensis sp. nov., a gliding bacterium isolated from marine sediment of the East China Sea.

A yellow pigmented, Gram-stain-negative, aerobic bacterium designated A5.7T was studied to evaluate the taxonomic position following the modern polyphasic approach. The strain was isolated from sediments near Zhairuo Island, which is situated in the East China Sea. Cells were non-spore forming rods without flagella but showed motility by gliding. Growth was observed at 15-35°C (optimum 28°C), pH 6.0-9.0 (optimum pH 6.5) and 0-2% (w/v) NaCl (optimum 0-0.5%) in LB broth. The major respiratory quinone of A5.7T was menaquinone 6. The major polar lipid of A5.7T was phosphatidylethanolamine and the predominant fatty acids (> 5%) were iso-C15:0, iso-C17:0 3-OH, C15:1ω6c, iso-C15:0 3-OH, iso-C15:1 G, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 9 (iso-C17:1ω9c and/or C16:010-methyl). Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belongs to the genus Flavobacterium and shares the highest sequence similarities with Flavobacterium sharifuzzamanii A7.6T (98.5%), Flavobacterium tistrianum GB 56.1T (98.3%), Flavobacterium nitrogenifigens NXU-44T (97.8%), Flavobacterium anhuiense D3T (97.6%), Flavobacterium ginsenosidimutans THG 01T (97.6%), and Flavobacterium foetidum CJ42T (97.6%). Digital DNA-DNA hybridization and average nucleotide identity values between the strain and its closest phylogenetic neighbors showed the ranges from 19.6 to 34.1% and 73.7 to 87.9%, respectively. Therefore, based on polyphasic characteristics, strain A5.7T represents a novel species of the genus Flavobacterium for which the name Flavobacterium zhairuonensis sp. nov. is proposed. The type strain is A5.7T (= KCTC 62406T = MCCC 1K03494T).

Global Analysis of Furfural-Induced Genomic Instability Using a Yeast Model.

Furfural is an important renewable precursor for multiple commercial chemicals and fuels; a main inhibitor existing in cellulosic hydrolysate, which is used for bioethanol fermentation; and a potential carcinogen, as well. Using a genetic system in Saccharomyces cerevisiae that allows detection of crossover events, we observed that the frequency of mitotic recombination was elevated by 1.5- to 40-fold when cells were treated with 0.1 g/liter to 20 g/liter furfural. Analysis of the gene conversion tracts associated with crossover events suggested that most furfural-induced recombination resulted from repair of DNA double-strand breaks (DSBs) that occurred in the G1 phase. Furfural was incapable of breaking DNA directly in vitro but could trigger DSBs in vivo related to reactive oxygen species accumulation. By whole-genome single nucleotide polymorphism (SNP) microarray and sequencing, furfural-induced genomic alterations that range from single base substitutions, loss of heterozygosity, and chromosomal rearrangements to aneuploidy were explored. At the whole-genome level, furfural-induced events were evenly distributed across 16 chromosomes but were enriched in high-GC-content regions. Point mutations, particularly the C-to-T/G-to-A transitions, were significantly elevated in furfural-treated cells compared to wild-type cells. This study provided multiple novel insights into the global effects of furfural on genomic stability.IMPORTANCE Whether and how furfural affects genome integrity have not been clarified. Using a Saccharomyces cerevisiae model, we found that furfural exposure leads to in vivo DSBs and elevation in mitotic recombination by orders of magnitude. Gross chromosomal rearrangements and aneuploidy events also occurred at a higher frequency in furfural-treated cells. In a genome-wide analysis, we show that the patterns of mitotic recombination and point mutations differed dramatically in furfural-treated cells and wild-type cells.

Flavobacterium sharifuzzamanii sp. nov., Isolated from the Sediments of the East China Sea.

A novel bacterial strain A7.6T was isolated from the sediments collected near the Zhairuo Island located in the East China Sea and characterized using a polyphasic approach. Cells were Gram-stain-negative, rod-shaped, non-spore forming, non-flagellated but motile by gliding. The strain was aerobic, positive for oxidase and catalase activities. The strain can grow at 4-35 °C, pH 5.5-9.0, and 0-3% (w/v) NaCl concentration. The major polar lipid was phosphatidylethanolamine, the predominant fatty acids (> 10%) were iso-C15:0 and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The genomic G+C content was 33.6 mol% and the major respiratory quinone was menaquinone 6. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain A7.6T belonged to the genus Flavobacterium and was closely related to Flavobacterium tistrianum GB 56.1T (98.4% similarity), F. nitrogenifigens NXU-44T (98.4%), F. ginsenosidimutans THG 01T (98.0%) and F. anhuiense D3T (97.7%). Average nucleotide identities and digital DNA-DNA hybridizations values for genomes ranged from 75.9 to 91.4% and 21.4 to 43.9% between strain A7.6T and its closest phylogenetic neighbors. The polyphasic characterization indicated that strain A7.6T represented a novel species of the genus Flavobacterium, for which the name Flavobacterium sharifuzzamanii is proposed. The type strain is A7.6T (= KCTC 62405T = MCCC 1K03485T). The NCBI GenBank accession number for the 16S rRNA gene of A7.6T is MH396692, and for the genome sequence is QJGZ00000000. The digital protologue database (DPD) Taxon Number is TA00643.

Genome Instability Induced by Low Levels of Replicative DNA Polymerases in Yeast.

Most cells of solid tumors have very high levels of genome instability of several different types, including deletions, duplications, translocations, and aneuploidy. Much of this instability appears induced by DNA replication stress. As a model for understanding this type of instability, we have examined genome instability in yeast strains that have low levels of two of the replicative DNA polymerases: DNA polymerase α and DNA polymerase δ (Polα and Polδ). We show that low levels of either of these DNA polymerases results in greatly elevated levels of mitotic recombination, chromosome rearrangements, and deletions/duplications. The spectrum of events in the two types of strains, however, differs in a variety of ways. For example, a reduced level of Polδ elevates single-base alterations and small deletions considerably more than a reduced level of Polα. In this review, we will summarize the methods used to monitor genome instability in yeast, and how this analysis contributes to understanding the linkage between genome instability and DNA replication stress.

Effects of genome duplication on phenotypes and industrial applications of Saccharomyces cerevisiae strains.

Polyploidy is common in Saccharomyces cerevisiae strains, but the physiological and phenotypic effects of ploidy changes have not been fully clarified. Here, isogenic diploid, triploid, and tetraploid S. cerevisiae strains were constructed from a haploid strain, CEN.PK2-1C. Stress tolerance and ethanol fermentation performance of the four euploid strains were compared. Each euploid strain had strengths and weaknesses in tolerance to certain stressors, and no single strain was tolerant of all stressors. The diploid had higher ethanol production than the other strains in normal fermentation medium, while the triploid strain showed the fastest fermentation rate in the presence of inhibitors found in lignocellulosic hydrolysate. Physiological determination revealed diverse physiological attributes, such as trehalose, ergosterol, glutathione, and anti-oxidative enzymes among the strains. Our analyses suggest that both ploidy parity and number of chromosome sets contribute to changes in physiological status. Using qRT-PCR, different expression patterns of genes involved in the regulation of cell morphology and the biosynthesis of key physiological attributes among strains were determined. Our data provide novel insights into the multiple effects of genome duplication on yeast cells and are a useful reference for breeding excellent strains used in specific industrial applications.

Global analysis of genomic instability caused by DNA replication stress in Saccharomyces cerevisiae.

DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G2 phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.