26 research outputs found
Modularity of the Hrd1 ERAD complex underlies its diverse client range
Additional factors combine with the core Hrd1 complex in a modular fashion, enabling it to recognize a variety of substrates
Surrogate-Based and One-Shot Optimization Methods for PDE-Constrained Problems with an Application in Climate Models
Deletion analysis of LSm, FDF, and YjeF domains of Candida albicans Edc3 in hyphal growth and oxidative-stress response
Application of the reuseable, KanMX selectable marker to industrial yeast: construction and evaluation of heterothallic wine strains of Saccharomyces cerevisiae, possessing minimal foreign DNA sequences
The characterisation of wine yeasts and the complex metabolic processes influencing wine fermentation and the quality of wine might best be achieved by exploiting the standard classical and recombinant genetic techniques which have been successfully used with laboratory strains. However, application of these techniques to industrial strains has been restricted because such strains are typically prototrophic and often polyploid. To overcome this problem, we have identified commercial wine strains with good mating and sporulation properties from which heterothallic derivatives were constructed by disruption of the HO gene. Consequently, these haploids are amenable to genetic analysis, whilst retaining desirable wine-making properties. The approach used was an adaptation of a previously published gene disruption procedure for laboratory yeast and is based on the acquisition of geneticin resistance from a removable KanMX marker. The present work is the first report of the application of a construct of this type to the disruption of the HO gene in wine yeasts that are in common commercial use. Most of the 4.9-kb disruption construct was successfully removed from the genome of the haploid derivative strains by loop-out of the KanMX marker through meiotic recombination. Sequencing of the HO region confirmed the reduction of foreign sequences to a 582-bp fragment comprised largely of a single direct repeat at the target gene. The removal of the active foreign gene (conferring antibiotic resistance) allows the application of other constructs based on the KanMX module without the need to resort to other selectable marker systems. Laboratory-scale fermentation trials typically showed minimal differences between the HO disruptants and the parental wine strains in terms of fermentation kinetics and formation of key metabolites.Michelle E. Walker, Jennie M. Gardner, Andrea Vystavelova, Colin McBryde, Miguel de Barros Lopes and Vladimir Jirane
The Cell Surface Flocculin Flo11 Is Required for Pseudohyphae Formation and Invasion by Saccharomyces cerevisiae
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N-terminal Domain of Yeast Telomerase Reverse Transcriptase: Recruitment of Est3p to the Telomerase Complex
Telomerase is a reverse transcriptase that maintains chromosome ends. The N-terminal half of the catalytic protein subunit (TERT) contains three functional domains (I, II, and III) that are conserved among TERTs but not found in other reverse transcriptases. Guided by an amino acid sequence alignment of nine TERT proteins, mutations were introduced into yeast TERT (Est2p). In support of the proposed alignment, mutation of virtually all conserved residues resulted in loss-of-function or temperature sensitivity, accompanied by telomere shortening. Overexpression of telomerase component Est3p led to allele-specific suppression of the temperature-sensitive mutations in region I, suggesting that Est3p interacts with this protein domain. As predicted by the genetic results, a lethal mutation in region I resulted in loss of Est3p from the telomerase complex. We conclude that Est2p region I is required for the recruitment of Est3p to yeast telomerase. Given the phylogenetic conservation of region I of TERT, this protein domain may provide the equivalent function in all telomerases