Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that - at a subsequent stage of transfection into yeast cells - it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein's secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 µg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

Erratum to: "Gas chromatography-mass spectrometry in the taxonomy of Miscanthus".

Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2019;23(8):1076-1081 (in Russian) Page 1081, in Acknowledgements instead of This work was supported by State Budgeted Project АААА-А17-117092070032-4. should read This work was supported by State Budgeted Project 0259-2019-0011. The original article can be found under DOI 10.18699/VJ19.583.

An integrated method for taxonomic identification of microorganisms.

For accurate species-level identification of microorganisms, researchers today increasingly use a combination of standard microbiological cultivation and visual observation methods with molecular biological and genetic techniques that help distinguish between species and strains of microorganisms at the level of DNA or RNA molecules. The aim of this work was to identify microorganisms from the ICG SB RAS Collection using an integrated approach that involves a combination of various phenotypic and genotypic characteristics. Key molecular-genetic and phenotypic characteristics were determined for 93 microbial strains from the ICG SB RAS Collection. The strains were characterized by means of morphological, physiological, moleculargenetic, and mass-spectrometric parameters. Specific features of the growth of the strains on different media were determined, and cell morphology was evaluated. The strains were tested for the ability to utilize various substrates. The strains studied were found to significantly differ in their biochemical characteristics. Physiological characteristics of the strains from the collection were identified too, e.g., the relationship with oxygen, type of nutrition, suitable temperature and pH ranges, and NaCl tolerance. In this work, the microorganisms analyzed were combined into separate groups based on the similarities of their phenotypic characteristics. This categorization, after further refinement and expansion of the spectrum of taxa and their metabolic maps, may serve as the basis for the creation of an "artificial" classification that can be used as a key for simplified and quicker identification and recognition of microorganisms within both the ICG SB RAS Collection and other collections.

No DNA damage response and negligible genome-wide transcriptional changes in human embryonic stem cells exposed to terahertz radiation.

Terahertz (THz) radiation was proposed recently for use in various applications, including medical imaging and security scanners. However, there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli, and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes, which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure.

Transcription regulatory regions database (TRRD): its status in 2000.

Transcription Regulatory Regions Database (TRRD) has been developed for accumulation of experimental information on the structure-function features of regulatory regions of eukaryotic genes. Each entry in TRRD corresponds to a particular gene and contains a description of structure-function features of its regulatory regions (transcription factor binding sites, promoters, enhancers, silencers, etc.) and gene expression regulation patterns. The current release, TRRD 4.2.5, comprises the description of 760 genes, 3403 expression patterns, and >4600 regulatory elements including 3604 transcription factor binding sites, 600 promoters and 152 enhancers. This information was obtained through annotation of 2537 scientific publications. TRRD 4.2.5 is available through the WWW at http://wwwmgs.bionet.nsc.ru/mgs/dbases/trrd4/

Integrated databases and computer systems for studying eukaryotic gene expression.

MOTIVATION: The goal of the work was to develop a WWW-oriented computer system providing a maximal integration of informational and software resources on the regulation of gene expression and navigation through them. Rapid growth of the variety and volume of information accumulated in the databases on regulation of gene expression necessarily requires the development of computer systems for automated discovery of the knowledge that can be further used for analysis of regulatory genomic sequences. RESULTS: The GeneExpress system developed includes the following major informational and software modules: (1) Transcription Regulation (TRRD) module, which contains the databases on transcription regulatory regions of eukaryotic genes and TRRD Viewer for data visualization; (2) Site Activity Prediction (ACTIVITY), the module for analysis of functional site activity and its prediction; (3) Site Recognition module, which comprises (a) B-DNA-VIDEO system for detecting the conformational and physicochemical properties of DNA sites significant for their recognition, (b) Consensus and Weight Matrices (ConsFrec) and (c) Transcription Factor Binding Sites Recognition (TFBSR) systems for detecting conservative contextual regions of functional sites and their recognition; (4) Gene Networks (GeneNet), which contains an object-oriented database accumulating the data on gene networks and signal transduction pathways, and the Java-based Viewer for exploration and visualization of the GeneNet information; (5) mRNA Translation (Leader mRNA), designed to analyze structural and contextual properties of mRNA 5'-untranslated regions (5'-UTRs) and predict their translation efficiency; (6) other program modules designed to study the structure-function organization of regulatory genomic sequences and regulatory proteins. AVAILABILITY: GeneExpress is available at http://wwwmgs.bionet.nsc. ru/systems/GeneExpress/ and the links to the mirror site(s) can be found at http://wwwmgs.bionet.nsc.ru/mgs/links/mirrors.html+ ++.

Transcription Regulatory Regions Database (TRRD):its status in 1999.

The Transcription Regulatory Regions Database (TRRD) is a curated database designed for accumulation of experimental data on extended regulatory regions of eukaryotic genes, the regulatory elements they contain, i.e., transcription factor binding sites, promoters, enhancers, silencers, etc., and expression patterns of the genes. Release 4.1 of TRRD offers a number of significant improvements, in particular, a more detailed description of transcription factor binding sites, transcription factors per se, and gene expression patterns in a computer-readable format. In addition, the new TRRD release provides considerably more references to other molecular biological databases. TRRD 4.1 is installed under SRS and is available through the WWW at http://www.bionet.nsc.ru/trrd/