Yeast
Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two proteins with internal repeats (PIR) genes, i.e. PpPIR1 and PpPIR2, from the methylotrophic yeast Pichia pastoris has been reported. The PIR1 and PIR2 genes' open reading frames were found to contain 1068 and 972 bases, respectively. The sequence homology search showed a homologous conserved...
Laccase activity in plants results in the formation of a number of brown pigments, often referred to as tannins. Laccase‐dependent pigment production is also catalogued in numerous fungal and bacterial species. The laccase of the haploid yeast Cryptococcus neoformans forms melanin‐like pigmentation outside the cell wall in the presence of exogenous substrates. While this process is a contributing...
It has been difficult to develop molecular tools for studying the fungal pathogen Candida albicans because this species uses a non‐standard genetic code and is diploid without a complete sexual cycle. Vector systems with regulatable promoters to produce conditional mutants, epitope tags for protein detection and recyclable selection markers are useful for functional study of genes. However, most currently...
A novel reverse genetic approach termed ‘marker reconstitution mutagenesis’ was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination. Second, random...
We used soft X‐ray tomography (SXT)—a high‐resolution, quantitative imaging technique—to measure cell size and organelle volumes in yeasts. Cell size is a key factor in initiating cell division in yeasts, whereas the number and volume of the organelles have a profound impact on the function and viability of a cell. Consequently, determining these cell parameters is fundamentally important in understanding...
To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N‐terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show...
Caffeine exerts pleiotropic effects on eukaryotic cells via its ability to act as a low‐affinity adenosine analogue. Here we report that the genes HSE1, RTS3, SDS23 and SDS24 confer caffeine resistance when overexpressed in S. cerevisiae. The Hse1 protein functions in ubiquitin‐dependent vacuolar protein sorting, whereas the other proteins are poorly characterized. Bioinformatic analysis of genetic...
Sophorolipids produced by the non‐pathogenic yeast Candida bombicola ATCC 22214 are glycolipid biosurfactants applied commercially as biodegradable and eco‐friendly detergents. Their low cell toxicity, excellent wetting capability and antimicrobial activity attract the attention of high‐value markets, such as the cosmetic and pharmaceutical industries. Although sophorolipid production yields have...
Candida albicans undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments. In this organism, cAMP‐dependent protein kinase (PKA), coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1), mediates basic cellular processes, such as the yeast‐to‐hypha transition and cell cycle regulation. It is known that both Tpk isoforms...
Yeast‐based reporter assays are useful for detecting various genotoxic chemicals. We established a genotoxicity assay using recombinant strains of Saccharomyces cerevisiae, each containing a reporter plasmid with the secretory luciferase gene from Cypridina noctiluca, driven by a DNA damage‐responsive promoter of the yeast RNR3 gene. This system detected the genotoxicity of methyl methanesulphonate...
PCR‐based gene targeting technologies have previously been developed for Candida albicans molecular genetic manipulation. Modular marker plasmids for the functional analysis of C. albicans genes have been generated to delete genes, exchange promoters and tag genes with GFP. Here, we have embedded two fluorescent proteins encoded by Venus and yEmCherry into the pFA‐plasmid series and demonstrate their...
Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis...
We describe a new cloning‐free strategy to delete genes in the opportunistic pathogenic yeast Candida lusitaniae. We first constructed two ura3Δ strains in C. lusitaniae for their use in transformation experiments. One was deleted for the entire URA3 coding sequence; the other possessed a partial deletion within the coding region, which was used to determine the minimum amount of homology required...
Alcohol dehydrogenases catalyse the reversible oxidation of alcohols to aldehydes or ketones, with concomitant reduction of NAD+ or NADP+. Adh1p is responsible for the reduction of acetaldehyde to ethanol, while Adh2p catalyses the reverse reaction, the oxidation of ethanol to acetaldehyde. Lack of Adh1p shifts the cellular redox balance towards excess NADH/NADPH and acetaldehyde, while absence of...
The Rec10 protein, a component of the linear elements forming along sister chromatids in meiotic prophase of Schizosaccharomyces pombe, plays an important role in the activation of Rec12 for double‐strand break formation, and thus the initiation of recombination between homologous chromosomes. Recombination between homologous chromosomes was moderately reduced in homozygous crosses of the C‐terminal...
Scheffersomyces stipitis and the closely related yeast Candida shehatae assimilated the L‐amino acids glutamate, aspartate and proline as both carbon and nitrogen sole sources. We also found this rarely investigated ability in ascomycetous species such as Candida glabrata, C. reukaufii, C. utilis, Debaryomyces hansenii, Kluyveromyces lactis, K. marxianus, Candida albicans, L. elongisporus, Meyerozyma guilliermondii, C....
Stress granules (SGs) and processing bodies (P bodies) are cytoplasmic domains and play a role in the control of translation and mRNA turnover in mammalian cells subjected to environmental stress. Recent studies have revealed that SGs also form in the budding yeast Saccharomyces cerevisiae in response to glucose depletion and robust heat shock. However, information about the types of stress that cause...
We quantified the growth behaviour of all available single‐gene deletion and overexpression strains of budding yeast. Genome‐wide analyses enabled the extraction of the genes and identification of the functional categories for which genetic perturbation caused the change of growth behaviour. Statistical analyses revealed defective growth for 646 deletion and 1302 overexpression strains. We classified...
Local anesthetics and antipsychotic phenothiazines cause a rapid shutdown of both actin polarization and translation initiation in yeast cells, like some environmental stresses. These compounds all have an amphiphilic structure, surfactant activity and the ability to lyse yeast cells. To elucidate the structures responsible for the shutdown activity and cell lysis, we investigated a variety of amphiphiles...
Proteins exit the endoplasmic reticulum (ER) in vesicles pinching off from the membrane at sites covered by the COPII coat, which consists of Sec23/24p and Sec13/31p. We have shown that the glycoprotein Hsp150 exits the ER in the absence of Sec13p or any member of the Sec24p family. The determinant responsible for this resides in the C‐terminal domain of Hsp150 (CTD). Here, A‐ and B‐type Walker motifs...