Vol 53, No 1 (2019)

Tea plant (Camellia sinensis [L.] O. Kuntze) is a woody crop of high economic importance worldwide; however, information on the molecular mechanisms underlying the regulation of flower development in this species is limited. In the present study, two GLOBOSA (GLO)-like MADS-box genes, CsGLO1 and CsGLO2, were isolated from C. sinensis ‘Ziyangzhong’ and were characterized to elucidate their roles in flower development. We found that CsGLO1 and CsGLO2 are nuclear-localized transcription factors without transactivation ability but with a robust interaction. They have similar patterns of expression, both mainly restricted to petals and stamens. Moreover, ectopic expression of either CsGLO1 or CsGLO2 in Arabidopsis thaliana resulted in a partial conversion of sepals to petals, suggesting full GLOBOSA functional activity. Our results indicate that CsGLO1 and CsGLO2 paralogs might redundantly contribute to petal and stamen, providing the first insight into their role in tea plant flower development.

The affinity tags in fusion proteins are extensively used in protein expression techniques. The most common affinity tags, such as glutathione S-transferase (GST), poly-histidine, maltose binding protein (MBP), and streptavidin tags, are routinely used for increasing expression, improving solubility, and facilitating protein purification. The large affinity tags (MBP, GST) are known to influence the conformational homogeneity and, therefore, the three-dimensional structure of in vivo folded proteins. The current study described in vivo effects of small affinity fusion 6×His tag on the growth of cells. Hexa-histidine tagged full length β-clamp and non-hexa-histidine tagged β-clamp were over-expressed and co-expressed in possible combinations with truncated DnaE in E. coli expression strain. After the induction with IPTG, the protein expression was assessed by SDS PAGE. The comparative analysis of the growth curves generated for the induced and un-induced cells demonstrated a decrease in growth rates of the cells over-expressing non-6×His tagged β-clamp as compared to 6×His tagged β-clamp. Based on the analysis of the soluble and insoluble protein fractions by SDS PAGE gels and published His-tagged β-clamp structure (PDB: 4K74) we propose that N-terminal 6×His Tag on β-clamp occludes its Gly66 to ultimately affect its ability to interact with the δ subunit of the clamp loader.

The previously developed technology RNA enhancement (RNAe) is reported to increase specific gene expression at post-transcriptional level via a long noncoding RNA (lncRNA). The mechanism for SINEB2-dependent enhancement of translation remains not well understood. Here we present the result of experiments with the folded states of lncRNA in doubly deionized water obtained by slowcool method. These lncRNA were used in RNA pull-down assay that yielded six lncRNA-binding proteins potentially involved in RNAe. The direct interactions of lncRNA with interleukin enhancer-binding factor 3 (ILF3) and eukaryotic initiation factor 4A-I (eIF4A1) in vivo and in vitro were confirmed in RNA-binding protein affinity experiment and electrophoretic mobility shift assay (EMSA), respectively. These observations could explain RNAe phenomenon through lncRNA-dependent guiding of ILF3 protein, which, in turn, recruits polysomes or the factors for translation initiation, and attracting eIF4A1 proteins accelerating the unwinding of the secondary structure at the 5'-end of mRNA during translation initiation. Therefore, the hypothetical mechanism through which lncRNAs may regulate the translation of a specific mRNA is proposed.

Carbon tetrachloride is a well-studied hepatotropic poison. Animal models of exposure to carbon tetrachloride resemble acute liver damage in humans. This paper presents the study of the expression of genes related to cell cycle control, apoptosis, and oxidative stress in a model of carbon tetrachloride-induced toxic hepatitis in rats. White mongrel male rats were injected with a 50% oil solution of carbon tetrachloride at a dose of 0.125–4.000 g/kg (experimental group) or olive oil (control group). The animals were decapitated 24 and 72 h after the administration of carbon tetrachloride, and the qRT-PCR expression levels of the genes encoding hemoxygenase-1 (Hmox1), cell cycle checkpoint kinase-1 (Chek1), and caspase-7 (Casp7) in the liver were analyzed. The increase in the expression levels of Hmox1 and Chek1 after exposure was detected. These genes may either play a role in promoting pathological oxidative stress in the liver or be a part of a stress response. We have concluded that the major pathway of the liver damage in carbon tetrachloride exposed animals is necrosis rather than apoptosis.

Lipopolysaccharides (LPS), components of the cell wall of Gram-negative bacteria, activate neutrophils that trigger pathological processes, including Gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H₂S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellular glutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by lps and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

Cruciform structures are preferential targets for many architectural and regulatory proteins, as well as a number of DNA binding proteins with weak sequence specificity. Some of these proteins are also capable of inducing the formation of cruciform structures upon DNA binding. In this paper we analyzed the amino acid composition of eighteen cruciform binding proteins of Homo sapiens. Comparison with general amino acid frequencies in all human proteins revealed unique differences, with notable enrichment for lysine and serine and/or depletion for alanine, glycine, glutamine, arginine, tyrosine and tryptophan residues. Based on bootstrap resampling and fuzzy cluster analysis, multiple molecular mechanisms of interaction with cruciform DNA structures could be suggested, including those involved in DNA repair, transcription and chromatin regulation. The proteins DEK, HMGB1 and TOP1 in particular formed a very distinctive group. Nonetheless, a strong interaction network connecting nearly all the cruciform binding proteins studied was demonstrated. Data reported here will be very useful for future prediction of new cruciform binding proteins or even construction of predictive tool/web-based application.

A good scoring function is necessary for ab inito prediction of RNA tertiary structures. In this study, we explored the power of a machine learning based approach as a scoring function. Compared with the traditional scoring functions, the present approach is more flexible in incorporating different kinds of features; it is also free of the difficult problem of choosing the reference state. Two multi-layer neural networks were constructed and trained. They took RNA a structural candidate as input and then output its likeness score that evaluates the likeness of the candidate to the native structure. The first network was working at the coarse-grained level of RNA structures, while the second at the all-atom level. We also built an RNA database and split it into the training, validation, and testing sets, containing 322, 70, and 70 RNAs, respectively. Each RNA was accompanied with 300 decoys generated by high-temperature molecular dynamics simulations. The networks were trained on the training set and then optimized with an early-stop strategy, based on the loss of the validation set. We then tested the performance of the networks on the testing set. The results were found to be consistently better than a recent knowledge-based all-atom potential.

Retroelements are considered as one of the important sources of genomic variability in modern humans. It is known that transposition activity of retroelements in germline cells generates new insertions in various genomic loci and sometimes results in genetic diseases. Retroelements activity in somatic cells is restricted by different cellular mechanisms; however, there is an evidence for it in some tissue types. Somatic insertions can trigger tumorigenesis or participate in normal functioning such as generation of neurons` plasticity. In spite of the rapid development of high-throughput sequencing methods a confident detection of somatic insertions is still quite a challenging task. That, in part, is due to the absence of adequate bioinformatic tools for the analysis of sequencing data. Here, we propose an advanced computational pipeline for the identification of somatic insertions in datasets generated by selective amplification and high-throughput sequencing of genomic regions flanking insertions of AluYa5. Particular attention is paid for the identification of various artifacts arising in course of library preparation and the parameters for their filtration. Pipeline sensitivity is confirmed by in silico experiments with artificial datasets. Using the proposed pipeline we remove at least 80% of artifacts and preserve 75% of potentially somatic insertions. The approaches used in this work can be applied for the study of other mobile elements insertion variability.

Proteome profiling of human testicular biopsies was performed using tandem mass spectrometry with electrospray ionization. Protein identification results were compared for the Mascot commercial search engine, the SearchGUI noncommercial package, and their analog IdentiProt based on the open-source IdentiPy algorithm (http://hg.theorchromo.ru/identipy). A feature of IdentiPy is an automatic optimization of MS/MS search parameters. A set of protein identifications obtained with IdentiPy was consequently greater by one third than the sets with the other search engines. For the first time, an IdentiPy/IdentiProt search was conducted within the Progenesis LC–MS framework, which allows spectrum alignment, and the proteome profile obtained with alignment was compared with that obtained using the ProteoWizard converter. A total of 16 human chromosome 18 proteins were identified, including the myelin basic protein, which is not characteristic of testicular tissue.