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Genome-wide array-CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts.
Get PDFIndustrial yeasts, economically important microorganisms, are widely used in diverse biotechnological processes including brewing, winemaking and distilling. In contrast to a well-established genome of brewer's and wine yeast strains, the comprehensive evaluation of genomic features of distillery strains is lacking. In the present study, twenty two distillery yeast strains were subjected to electrophoretic karyotyping and array-based comparative genomic hybridization (array-CGH). The strains analyzed were assigned to the Saccharomyces sensu stricto complex and grouped into four species categories: S. bayanus, S. paradoxus, S. cerevisiae and S. kudriavzevii. The genomic diversity was mainly revealed within subtelomeric regions and the losses and/or gains of fragments of chromosomes I, III, VI and IX were the most frequently observed. Statistically significant differences in the gene copy number were documented in six functional gene categories: 1) telomere maintenance via recombination, DNA helicase activity or DNA binding, 2) maltose metabolism process, glucose transmembrane transporter activity; 3) asparagine catabolism, cellular response to nitrogen starvation, localized in cell wall-bounded periplasmic space, 4) siderophore transport, 5) response to copper ion, cadmium ion binding and 6) L-iditol 2- dehydrogenase activity. The losses of YRF1 genes (Y' element ATP-dependent helicase) were accompanied by decreased level of Y' sequences and an increase in DNA double and single strand breaks, and oxidative DNA damage in the S. paradoxus group compared to the S. bayanus group. We postulate that naturally occurring diversity in the YRF1 gene copy number may promote genetic stability in the S. bayanus group of distillery yeast strains
KAEA (SUDPRO), a member of the ubiquitous KEOPS/EKC protein complex, regulates the arginine catabolic pathway and the expression of several other genes in Aspergillus nidulans
Get PDFThe kaeAKAE1 (suDpro) gene, which was identified in Aspergillus nidulans as a suppressor of proline auxotrophic mutations, encodes the orthologue of Saccharomyces cerevisiae Kae1p, a member of the evolutionarily conserved KEOPS/EKC (Kinase, Endopeptidase and Other Proteins of Small size/Endopeptidase-like and Kinase associated to transcribed Chromatin) complex. In yeast, this complex has been shown to be involved in tRNA modification, transcription, and genome maintenance. In A. nidulans, mutations in kaeA result in several phenotypic effects, the derepression of arginine catabolism genes, and changes in the expression levels of several others, including genes involved in amino acid and siderophore metabolism, sulfate transport, carbon/energy metabolism, translation, and transcription regulation, such as rcoATUP1, which encodes the global transcriptional corepressor
Dolichol phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme.
Get PDFBackground: Dolichol phosphate mannose synthase (DPMS) is a key enzyme in N- and O-linked glycosylations and glycosylphosphatidylinositol (GPI)-anchor synthesis. DPMS generates DPM, the substrate for mentioned processes, by the transfer of mannosyl residue from GDP-Man to dolichol phosphate. Here we describe the role of DPMS for Candida albicans physiology with emphasis on the cell wall composition and morphogenesis.
Methods: C. albicans genes for DPMS subunits were cloned, tagged and expressed in Saccharomyces cerevisiae. The C. albicans strains with controlled expression of DPM genes were constructed and analyzed. Gene expression and enzyme activities were measured using RT-PCR and radioactive substrate. Sensitivities against chemical agents
were tested with microdilution method. The composition of the cell wall was estimated by HPLC. Glycosylation status of the marker protein was analyzed by Western blot. Morphological differentiation of the strains was checked on the media promoting hyphae and chlamydospore formation.
Results: We demonstrate that C. albicans DPMS consists of three interacting subunits, among which Dpm1 and Dpm3 are indispensable, whereas Dpm2 increases enzymatic activity. Lowered expression of DPMS genes results in decreased DPMS activity, increased susceptibility to cell wall perturbing agents and in altered cell wall composition. Mutants Tetp-DPM1 and Tetp-DPM3 show defective protein glycosylation and are impaired in hyphae and chlamydospore formation.
Major conclusion: DPMS from C. albicans, opposite to S. cerevisiae, belongs to the family of DPMS with multimeric protein structure.
General significance: This work provides important data about factors required for a proper protein glycosylation and for morphogenesis of pathogenic yeast C. albicans
Combined Therapies for Lysosomal Storage Diseases
Get PDFLysosomal storage diseases (LSDs) is a group consisting of over 50 disorders caused mostly by dysfunctions of lysosomal proteins and resultant accumulation of particular compounds inside cells and extracellular volumes in affected organisms. Genetic diseases are among the most difficult targets for medical treatment. Nevertheless, understanding of molecular bases of LSDs made it possible to develop novel procedures of treatment, employing molecular medicine. Although various therapeutic approaches have been proposed, and some of them were introduced into clinical practice, none of them was found to be effective in correcting all symptoms in treated patients. Central nervous system and skeleton appear to be the most difficult targets to be improved. Therefore, a proposal appeared that perhaps no single therapeutic procedure may be fully effective in treatment of LSD patients, and only combination of two or more approaches could be a successful therapy. In this review, we present and discuss current stage of various combination therapies for LSDs, based on already available published data
UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24B, 933W, P22, P27 and P32
Get PDFShiga toxin-converting bacteriophages (Stx phages) are present as prophages in
Shiga toxin-producing Escherichia coli (STEC) strains. Theses phages can be transmitted to
previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as
they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions
to various conditions is important in both natural processes of spreading of these viruses and
potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report
we provide evidence that virions of Stx phages are significantly more sensitive to UV
irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of
50 J/m2, their infectivity dropped by 1–3 log10, depending on the kind of phage. Under these
conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of
E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of
expression of N and cro genes, crucial for lytic development. We conclude that inactivation
of Stx virions caused by relatively low dose of UV light is due to damage of capsids that
prevents effective infection of the host cells
A small, microRNA-size, ribonucleic acid regulating gene expression and development of Shiga toxin-converting bacteriophage Φ24Β
Get PDFA microRNA-size (20-nt long) molecule has been identified in Escherichia coli after induction of
Shiga toxin-converting bacteriophage Φ24B. This small RNA, named 24B_1, is encoded in the lomvb_
24B_43 region of the phage genome, and apparently it is produced by cleavage of a larger
transcript. A phage devoid of 24B_1 revealed decreased efficiency of lysogenization, quicker
prophage induction after provoking the SOS response, higher efficiency of progeny phage production
during the lytic cycle and less efficient adsorption on the host cells. Expression of most of phage
genes was drastically increased after infection of E. coli by the Φ24BΔ 24B_1 phage. Since 24B_1 may
impair expression of the d_ant gene, coding for an anti-repressor, these results may explain the
mechanism of regulations of the physiological processes by this small RNA due to impaired activity
of the cI repressor and changed expression of vast majority of phage genes. To our knowledge, this is
the first example of functional microRNA-size molecule in bacterial cells
Rab geranylgeranyl transferase β subunit is essential for male fertility and tip growth in Arabidopsis
Get PDFRab proteins, key players in vesicular transport in all eukaryotic cells, are post-translationally modified by lipid moieties.
Two geranylgeranyl groups are attached to the Rab protein by the heterodimeric enzyme Rab geranylgeranyl
transferase (RGT) αβ. Partial impairment in this enzyme activity in Arabidopsis, by disruption of the AtRGTB1 gene, is
known to influence plant stature and disturb gravitropic and light responses. Here it is shown that mutations in each
of the RGTB genes cause a tip growth defect, visible as root hair and pollen tube deformations. Moreover, FM 1–43 styryl dye endocytosis and recycling are affected in the mutant root hairs. Finally, it is demonstrated that the double mutant, with both AtRGTB genes disrupted, is non-viable due to absolute male sterility. Doubly mutated pollen is shrunken, has an abnormal exine structure, and shows strong disorganization of internal membranes, particularly of the endoplasmic reticulum system
Posttranslational Regulation of Human DNA Polymerase ι.
Get PDFHuman DNA polymerases (pols) η and ι are Y- family DNA polymerase paralogs that facilitate translesion synthesis (TLS) past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines, may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the PCNA-interacting region, the Rev1-interacting region, as well as its Ubiquitin Binding Motifs, UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA damaging agents such as UV- light (generating UV-photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand crosslinks), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via K11- and K48- linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle, as no polyubiquitination was observed after treatment with rotenone, or antimycin A, which inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase, KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at K11- and K48- rather than oxidative damage per se
Short-chain polyisoprenoids in the yeast Saccharomyces cerevisiae - new companions of the old guys
Get PDFDolichols are, among others, obligatory cofactors of protein glycosylation in eukaryotic cells. It is well known that yeast cells accumulate a family of dolichols with Dol-15/16 dominating while upon certain physiological conditions a second family with Dol-21 dominating is noted. In this report we identified the presence of additional short-chain length polyprenols - all-trans Pren-7 in three yeast strains (SS328, BY4741 and L5366), Pren-7 was accompanied by traces of putative Pren-6 and -8. Moreover, in two of these strains a single polyprenol mainly-cis-Pren-11 was synthesized at the stationary phase of growth. Identity of polyprenols was confirmed by HR-HPLC/MS, NMR and metabolic labeling. Additionally, simvastatin inhibited their biosynthesis
Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity.
Get PDFDuffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone