No abstract

We investigated the genetic causes of ethanol tolerance by characterizing mutations selected in Saccharomyces cerevisiae W303‐1A under the selective pressure of ethanol. W303‐1A was subjected to three rounds of turbidostat, in a medium supplemented with increasing amounts of ethanol. By the end of selection, the growth rate of the culture has increased from 0.029 to 0.32 h−1. Unlike the progenitor strain, all yeast cells isolated from this population were able to form colonies on medium supplemented with 7% ethanol within 6 days, our definition of ethanol tolerance. Several clones selected from all three stages of selection were able to form dense colonies within 2 days on solid medium supplemented with 9% ethanol. We sequenced the whole genomes of six clones and identified mutations responsible for ethanol tolerance. Thirteen additional clones were tested for the presence of similar mutations. In 15 of 19 tolerant clones, the stop codon in ssd1‐d was replaced with an amino acid‐encoding codon. Three other clones contained one of two mutations in UTH1, and one clone did not contain mutations in either SSD1 or UTH1. We showed that the mutations in SSD1 and UTH1 increased tolerance of the cell wall to zymolyase and conclude that stability of the cell wall is a major factor in increased tolerance to ethanol.

MAPKs (mitogen-activated protein kinases) are key components in cell signalling pathways. Under optimal growth conditions, their activity is kept off, but in response to stimulation it is dramatically evoked. Because of the high degree of evolutionary conservation at the levels of sequence and mode of activation, MAPKs are believed to share similar regulatory mechanisms in all eukaryotes and to be functionally substitutable between them. To assess the reliability of this notion, we systematically analysed the activity, regulation and phenotypic effects of mammalian MAPKs in yeast. Unexpectedly, all mammalian MAPKs tested were spontaneously phosphorylated in yeast. JNKs (c-Jun N-terminal kinases) lost their phosphorylation in pbs2Delta cells, but p38s and ERKs (extracellular-signal-regulated kinases) maintained their spontaneous phosphorylation even in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Kinase-dead variants of ERKs and p38s were phosphorylated in strains lacking a single MEK (MAPK/ERK kinase), but not in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Thus, in yeast, p38 and ERKs are phosphorylated via a combined mechanism of autophosphorylation and MEK-mediated phosphorylation (any MEK). We further addressed the mechanism allowing mammalian MAPKs to exploit yeast MEKs in the absence of any activating signal. We suggest that mammalian MAPKs lost during evolution a C-terminal region that exists in some yeast MAPKs. Indeed, removal of this region from Hog1 and Mpk1 rendered them spontaneously and highly phosphorylated. It implies that MAPKs possess an efficient inherent autoposphorylation capability that is suppressed in yeast MAPKs via a C-terminal domain and in mammalian MAPKs via as yet unknown means.

Understanding the genetic basis of the yeast ability to proliferate and ferment in the presence of restrictive concentrations of ethanol is of importance to both science and technology. In this study, we searched for genes that improve ethanol tolerance in ethanol-sensitive strains. To screen for suppressors of ethanol sensitivity, we introduced a 2µ-based genomic library, prepared from the ethanol-tolerant yeast S288C, into the ethanol-sensitive strain W303-1A. Two genomic fragments from this library rescued the ethanol sensitivity of W303-1A. One contained the PDE2 gene, which when over-expressed, conferred ethanol tolerance. Surprisingly, the effect of PDE2 was not mediated via MSN2/MSN4 transcription factors, as it was able to improve ethanol tolerance in msn2Δmsn4Δ strain. In the second genomic fragment, it was the N-terminal region of the SSD1 gene that carried the ethanol-tolerant phenotype. The SSD1-V allele of the polymorphic SSD1 gene expressed from a low-copy number plasmid also resulted in the tolerant phenotype. Both SSD1 and PDE2 seemed to improve ethanol tolerance by maintaining robustness of the yeast cell wall.

In sweet potato, an anti-virus defense mechanism termed reversion has been postulated to lead to virus freedom from once infected plants. The objectives of this study were to identify anti-virus defense genes and evaluate their segregation in progenies. Reference genes from different plant species were used to assemble transcript sequences of each sweet potato defense gene in silico. Sequences were used for evaluate phylogenetic relationships with similar genes from different plant species, mining respective defense genes and thereafter developing simple sequence repeats (SSRs) for segregation analysis. Eight potential defense genes were identified: RNA dependent RNA polymerases 1, 2, 5, and 6; Argonaute 1, and Dicer-like 1, 2, and 4. Identified genes were differentially related to those of other plants and were observed on different chromosomes. The defense genes contained mono-, di-, tri-, tetra, penta-, and hexa-nucleotide repeat motifs. The SSR markers within progenies were segregated in disomic, co-segregation, nullisomic, monosomic, and trisomic modes. These findings indicate the possibility of deriving and utilizing SSRs using published genomic information. Furthermore, and given that the SSR markers were derived from known genes on defined chromosomes, this work will contribute to future molecular breeding and development of resistance gene analogs in this economically important crop.