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Adaptive response to chronic mild ethanol stress involves ROS, sirtuins and changes in chromosome dosage in wine yeasts
Get PDFIndustrial yeast strains of economic importance used in winemaking and beer production are genomically diverse and subjected to harsh environmental conditions during fermentation. In the present study, we investigated wine yeast adaptation to chronic mild alcohol stress when cells were cultured for 100 generations in the presence of non-cytotoxic ethanol concentration. Ethanol-induced reactive oxygen species (ROS) and superoxide signals promoted growth rate during passages that was accompanied by increased expression of sirtuin proteins, Sir1, Sir2 and Sir3, and DNA-binding transcription regulator Rap1. Genome-wide array-CGH analysis revealed that yeast genome was shaped during passages. The gains of chromosomes I, III and VI and significant changes in the gene copy number in nine functional gene categories involved in metabolic processes and stress responses were observed. Ethanol-mediated gains of YRF1 and CUP1 genes were the most accented. Ethanol also induced nucleolus fragmentation that confirms that nucleolus is a stress sensor in yeasts. Taken together, we postulate that wine yeasts of different origin may adapt to mild alcohol stress by shifts in intracellular redox state promoting growth capacity, upregulation of key regulators of longevity, namely sirtuins and changes in the dosage of genes involved in the telomere maintenance and ion detoxification
Convenient broad-host-range unstable vectors for studying stabilization cassettes in diverse bacteria
Get PDFBackground: Low-copy-number vectors of potential wide application in biotechnology need to encode stabilization
modules ensuring their stable inheritance. The efficiency of stabilization may vary depending on the plasmid host so a
thorough analysis of stabilization functions is required before use.
Results: To facilitate such analysis highly unstable, mobilizable, broad-host-range (BHR) vectors based on RK2 replicon
were constructed. The vectors are suitable for testing of various stabilization functions, including plasmid and
chromosomal partitioning cassettes encoding ParB homologues capable of spreading on DNA. The xylE or lacZ
reporter systems facilitate easy monitoring of plasmid segregation.
Conclusion: The range of BHR vectors with different reporter cassettes and alternative mobilization systems expands
their application in diverse bacterial species
Synthesis, Biological Activity, and NMR-Based Structural Studies of Deltorphin I Analogs Modified in Message Domain with a New a,a-Disubstituted Glycines
Get PDFThis article describes new deltorphin I analogs in which
phenylalanine residues were replaced by the corresponding
(R) or (S)-a-benzyl-b-azidoalanine, a-benzyl-b-
(1-pyrrolidinyl)alanine, a-benzyl-b-(1-piperidinyl)alanine,
and a-benzyl-b-(4-morpholinyl)-alanine residues. The
potency and selectivity of the new analogs were evaluated
by a competitive receptor binding assay in the rat
brain using [3H]DAMGO (a l ligand) and [3H]DELT (a d
ligand). The affinity of analogs containing (R) or (S)-abenzyl-
b-azidoalanine in position 3 to d-receptors
strongly depended on the chirality of the a,a-disubstituted
residue. The conformational behavior of peptides
modified with (R) or (S)-a-benzyl-b-(1-piperidinyl)Ala,
which displays the opposite selectivity, was analyzed
by 1H and 13C NMR. The l-selective Tyr-D-Ala-(R)-
a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 lacks
the helical conformation observed in the d-selective Tyr-
D-Ala-(S)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-
NH2. Our results support the proposal that differences
between d- and l-selective opioid peptides are attributable
to the presence or absence of a spatial overlap
between the N-terminal message domain and the
C-terminal address domain
Functional characterization of the RNA guided DRM2 DNA methyltransferase. Research done in the Faculty of Biology UW and IBB PAS. M.Sc. Thesis Supervisor: prof. dr hab. Ewa Bartnik.
Get PDFDNA methylation in plants is in part controlled by small RNAs. A key player in RNA directed DNA methylation (RdDM) is Domain Rearranged Methyltransferase 2 (DRM2). In addition to its catalytic domain, which resembles the catalytic domain of animal DNA methyltransferases, the protein has several UBA domains of unknown biochemical significance. Genetic experiments on Arabidopsis thaliana indicate that a DRM2 variant lacking all UBA domains is similarly incapacitated as a catalytic mutant. The aim of this work was to elucidate the role of the UBA domains in DRM2. Towards this end, expression plasmids were generated, which should direct expression of DRM2 variants lacking individual UBA domains only after Agrobacterium mediated transformation. Unfortunately, protein expression of the deletion constructs was not observed, even though a reporter control protein was expressed in parallel experiments
An avian influenza H5N1 virus vaccine candidate based on the extracellular domain produced in yeast system as subviral particles protects chickens from lethal challenge
Get PDFPerlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs
Get PDFThe exosome-independent exoribonuclease DIS3L2
is mutated in Perlman syndrome. Here, we used extensive
global transcriptomic and targeted biochemical
analyses to identify novel DIS3L2 substrates in
human cells. We show that DIS3L2 regulates pol
II transcripts, comprising selected canonical and
histone-coding mRNAs, and a novel FTL short RNA
from the ferritin mRNA 5� UTR. Importantly, DIS3L2
contributes to surveillance of maturing snRNAs during
their cytoplasmic processing. Among pol III transcripts,
DIS3L2 particularly targets vault and Y RNAs
and an Alu-like element BC200 RNA, but not Alu repeats,
which are removed by exosome-associated
DIS3. Using 3� RACE-Seq, we demonstrate that
all novel DIS3L2 substrates are uridylated in vivo
by TUT4/TUT7 poly(U) polymerases. Uridylationdependent
DIS3L2-mediated decay can be recapitulated
in vitro, thus reinforcing the tight cooperation
between DIS3L2 and TUTases. Together these results
indicate that catalytically inactive DIS3L2, characteristic
of Perlman syndrome, can lead to deregulation
of its target RNAs to disturb transcriptome homeostasis
N-Homocysteinylation impairs collagen cross-linking in cystathionine β-synthase-deficient mice: a novel mechanism of connective tissue abnormalities
Get PDFCystathionine β-synthase (CBS) deficiency, a genetic disorder in homocysteine (Hcy)
metabolism in humans, elevates plasma Hcy-thiolactone and leads to connective tissue abnormalities
affecting cardiovascular and skeletal systems. However, the underlying mechanism of these
abnormalities is not understood. Hcy-thiolactone has the ability to form isopeptide bonds with
protein lysine residues, which generates N-homocysteinylated protein. Because lysine residues are
involved in collagen crosslinking, N-homocysteinylation of these lysines should impair crosslinking.
Using a Tg-I278T Cbs -/- mouse model of hyperhomocysteinamia (HHcy) that recapitulates connective
tissue abnormalities observed in CBS-deficient patients, we show that N-Hcy-collagen was elevated in
bone, tail, and heart of Cbs -/- mice, while pyridinoline crosslinks were significantly reduced. Plasma
deoxypyridinoline crosslink and crosslinked carboxyterminal telopeptide of type I collagen were also
significantly reduced in Cbs -/- mice. Lysine oxidase activity and mRNA level were not reduced by the
Cbs -/- genotype. We also show that collagen carries S-linked Hcy bound to the thiol of N-linked Hcy. In
vitro experiments show that Hcy-thiolactone modifies lysine residues in collagen type I alpha-1 chain.
Residue K160, located in the non-helical N-telopeptide region and involved in pyridinoline crosslink
formation, was also N-homocysteinylated in vivo. Taken together, our findings show that N-
homocysteinylation of collagen in Cbs -/- mice impairs its crosslinking. These findings explain at least in
part connective tissue abnormalities observed in HHcy
Electrochemical genosensor based on disc and screen printed goldelectrodes for detection of specific DNA and RNA sequences derivedfrom Avian Influenza Virus H5N1
Get PDFtThe genosensors based on thiolated ssDNA probe deposited on the two types of gold electrodes: screen-printed (miniaturized) and disc electrodes destined for determination of specific sequences of DNA andRNA derived from Avian Influenza Virus H5N1 have been proposed. The working principle of genosensor isbased on the ion-channel mechanism. The analytical signals generated upon hybridization processes wererecorded using electrochemical technique – Osteryoung square wave voltammetry in the presence of aredox active marker [Fe(CN)6]3−/4−in the sample solution. The miniaturized genosensor based on screenprinted gold electrodes was able to detect the 20-mer complementary DNA oligonucleotide sequence aswell as ∼280-mer RNA sequences containing the complementary 20-mer sequence in various positions:at 3�-terminus, at 5�-terminus and in the middle of the RNA transcript at the 1 pM concentration. Themeasuring systems were selective. Non-complementary 20-mer oligonucleotide sequence as well asRNA transcript without complementary region generated weak response. The RNA transcripts were alsotested with gold disc electrodes modified in the same manner. This device was able to detect ∼280-mer RNA sequences, but at higher concentration of 10 pM. The good discrimination of the position ofcomplementary part in the ∼280-mer RNA sequences was observed with using both types of modifiedelectrodes
Omega (ParB) binding sites together with the RNA polymerase-recognized sequence are essential for centromeric functions of the Pω region in the partition system of pSM19035
Get PDFA mild and efficient approach to the 6H-oxazolo[3,2-f]pyrimidine-5,7-dione scaffold via unexpected rearrangement of 2,3-dihydropyrimido[6,1-b][1,5,3]dioxazepine-7,9(5H,8H)-diones:synthesis, crystallographic studies, and cytotoxic activity screening
Get PDFWe report a mild and efficient approach to the optically pure 6H-oxazolo[3,2-f]pyrimidine-5,7-dione
scaffold via the unexpected rearrangement and ring contraction of 2,3-dihydropyrimido[6,1-b][1,5,3]-
dioxazepine-7,9(5H,8H)-diones derived from nucleoside precursors. The developed procedure enables
the synthesis of a wide range of compounds with great structural diversity. The structure of the obtained
compounds was confirmed by NMR spectroscopy and single crystal X-ray structural analysis. The final
products were tested for cytotoxic effect on one non-cancerous (fibroblasts) and six cancer cell lines of
different origins (colon, glioma, breast, cervix, vulvar, and lung). The synthesized products are low molecular
weight compounds with lead-like properties suitable for a medicinal chemistry optimization
program