The Effect of Selected Polymorphisms of the ACTN3, ACE, HIF1A and PPARA Genes on the Immediate Supercompensation Training Effect of Elite Slovak Endurance Runners and Football Players.

We aimed to evaluate the effect of selected polymorphisms of the ACTN3, ACE, HIF1A and PPARA genes on the immediate supercompensation training effect of elite Slovak endurance runners and football players compared with a sedentary control group. Adaptation effect levels were evaluated by 10 s continuous vertical jump test parameters measured by Optojump. Genetic polymorphisms were determined by PCR and Sanger sequencing. We found significant differences in the effect of PPARA genotypes in the experimental group. C allele genotypes represented an advantage in immediate supercompensation (p < 0.05). We observed a significant combined effect of multiple genes on immediate supercompensation (p < 0.05): the RR genotype of the ACTN3 gene, the ID genotype of the ACE gene, the Pro/Pro genotype of HIF1A, and the GC and GG genotypes of PPARA genes. In the control group, we found a significant effect (p < 0.05) on immediate supercompensation of the II genotype of the ACE gene and the Pro/Ser genotype of the HIF1A gene. We found significant differences in genotype frequency of ACE (p < 0.01) and PPARA (p < 0.001) genes. We confirmed that individual genetic polymorphisms of ACTN3, ACE, HIF1A and PPARA genes have a different effect on the level of immediate supercompensation of the lower limbs depending on the training adaptation of the probands and the combination of genotypes.

Insertional mutagenesis in Chlamydomonas reinhardtii: An effective strategy for the identification of new genes involved in the DNA damage response.

The formation of double-strand breaks in DNA represents a serious stress for all types of organisms and requires a precisely regulated and organized DNA damage response (DDR) to maintain genetic information and genome integrity. Chlamydomonas reinhardtii possesses the characteristics of both plants and animals and is therefore suitable for the identification of novel genes connected to a wide spectrum of metabolic pathways, including DDR. One very effective tool for the detection and subsequent characterization of new mutants in C. reinhardtii is insertional mutagenesis. We isolated several insertion mutants sensitive to DNA-damaging agents that had disrupted or completely deleted genes with putative functions in the DDR. In most of the analysed mutants, we identified various changes at both ends and even inside the inserted cassette. Using recent information from databases, we were also able to supplement the characteristics of the previously described mutant with a pleiotropic phenotype. In addition, we confirmed the effectiveness of hairpin-PCR as a strategy for the identification of insertion flanking sites and as a tool for the detection of changes at the site of insertion, thus enabling a better understanding of insertion events.

Intentionally flawed manuscripts as means for teaching students to critically evaluate scientific papers.

As future scientists, university students need to learn how to avoid making errors in their own manuscripts, as well as how to identify flaws in papers published by their peers. Here we describe a novel approach on how to promote students' ability to critically evaluate scientific articles. The exercise is based on instructing teams of students to write intentionally flawed manuscripts describing the results of simple experiments. The teams are supervised by instructors advising the students during manuscript writing, choosing the 'appropriate' errors, monitoring the identification of errors made by the other team and evaluating the strength of their arguments in support of the identified errors. We have compared the effectiveness of the method with a journal club-type seminar. Based on the results of our assessment we propose that the described seminar may effectively complement the existing approaches to teach critical scientific thinking. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):22-30, 2018.

Characterization of DNA repair deficient strains of Chlamydomonas reinhardtii generated by insertional mutagenesis.

While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.

Studies on recombination processes in two Chlamydomonas reinhardtii endogenous genes, NIT1 and ARG7.

Integration of exogenous DNA in the unicellular green alga Chlamydomonas reinhardtii is principally carried out by mechanisms involving non-homologous recombination (NHR), rather than homologous recombination (HR). Homologous recombination is, however, the mechanism of choice when it comes to gene targeting. Unfortunately, attempts to establish this method in Chlamydomonas have had limited success. In this study we compared two endogenous genes, NIT1 and ARG7, and their HR/NHR ratios when different types of fragments were used as donors of homologous sequences. Transformation of the auxotrophic strain containing the inactivating point mutation arg7-8 with nonfunctional ARG7 gene fragments overlapping this mutation showed increased HR efficiencies when linearized plasmids were used. Efficiency went down rapidly with decreasing length of ARG7 homology. After identification of the inactivating 6726(G→A) point mutation in nit1-305 strains, an analogous set of experiments was performed. In the case of NIT1, overall efficiency of recombination was 10 to 100 fold lower than with ARG7. In order to better demonstrate HR we introduced three silent mutations close to the position of the point mutations in our transforming plasmids. Sequencing of transformants indicated homologous recombination over a short interval.

Complementation of the Chlamydomonas reinhardtii arg7-8 (arg2) point mutation by recombination with a truncated nonfunctional ARG7 gene.

Chlamydomonas reinhardtii arg7-8 (arg2) mutant strains carrying a hitherto undescribed mutation in their argininosuccinate lyase gene (ARG7) that leads to arginine auxotrophy have been used together with the corresponding wild-type gene as a very reliable transformation system since 1989. In this study, we finally identify the molecular nature of the arg7-8 mutation as a (6073)G to A transition in exon 9 of ARG7 leading to a (288)Gly to Ser exchange near the active site of the protein. The same mutation was found in the ARG7 genes of three commonly used C. reinhardtii laboratory strains, namely cw15-302 arg2, CC-48, and CC-1618. We did not observe exact spontaneous reversion of the arg7-8 allele in our study, but did identify two different and rare intragenic suppressor mutations, (27)Leu to Phe and (285)Tyr to Phe. In our hands, only transformation of the arg7-8 strain with a truncated nonfunctional wild-type ARG7 gene lacking 124 codons at its 5' end led to exact reversion of the mutant base (6073)A to the wild-type (6073)G, presumably by recombination. This system offers a positive selection scheme for homologous recombination (HR) and may, therefore, be useful to the methodical improvement of recombination in Chlamydomonas.

The alga Chlamydomonas reinhardtii UVS11 gene is responsible for cell division delay and temporal decrease in histone H1 kinase activity caused by UV irradiation.

The aim of the present work was to study the possible role of the UVS11 gene of the alga Chlamydomonas reinhardtii, in regulation of the cell cycle. To characterize the defect of a uvs11 mutant in respect to DNA damage-dependent cell cycle arrest, we examined first the influence of the tubulin-destabilizing drug methyl benzimidazole-2-yl-carbamate (MBC) on inhibition of mitosis in response to UV 254nm. Then the growth and reproductive processes and activity of cyclin-dependent kinases (CDK)-like kinases during the cell cycle of C. reinhardtii were investigated. In both, the wild type and the uvs11 mutant strain were compared under standard conditions and after DNA damage caused by UV 254nm. We assume the green alga C. reinhardtii possesses control mechanisms allowing to stop the cell cycle progression before mitosis in response to DNA damage. The results indicate that the uvs11 mutant is not able to stop the cell cycle after UV irradiation. We suggest that a product of the UVS11 gene affects cell response to DNA damage and influences a decrease in histone H1 kinase activity.