Identification and Expression Analysis of Chitinase Genes in Pitchers of Nepenthes sp. during Development.

Fifteen chitinases of classes I-V were identified in the transcriptomes of pitchers and adult leaves of the carnivorous plant Nepenthes sp. Ten of these chitinases were identified for the first time, including the chitinases of classes II and V. The expression levels of all found chitinase genes in leaves and at three stages of pitcher development were determined. The maximum level of transcriptional activity in an open pitcher was observed for the genes encoding chitinase NChi4 (class II) and its isoforms. The expression levels of these genes significantly increased as the pitcher developed. In addition, for the first time, transcription of the genes encoding chitinases of all five classes was detected in the leaves of this plant.

Search for potential reading frameshifts in cds from Arabidopsis thaliana and other genomes.

A new mathematical method for potential reading frameshift detection in protein-coding sequences (cds) was developed. The algorithm is adjusted to the triplet periodicity of each analysed sequence using dynamic programming and a genetic algorithm. This does not require any preliminary training. Using the developed method, cds from the Arabidopsis thaliana genome were analysed. In total, the algorithm found 9,930 sequences containing one or more potential reading frameshift(s). This is ∼21% of all analysed sequences of the genome. The Type I and Type II error rates were estimated as 11% and 30%, respectively. Similar results were obtained for the genomes of Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Rattus norvegicus and Xenopus tropicalis. Also, the developed algorithm was tested on 17 bacterial genomes. We compared our results with the previously obtained data on the search for potential reading frameshifts in these genomes. This study discussed the possibility that the reading frameshift seems like a relatively frequently encountered mutation; and this mutation could participate in the creation of new genes and proteins.

A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX.

Hemophilia B patients suffer from an inherited blood-clotting defect and require regular administration of blood-clotting factor IX replacement therapy. Recombinant human factor IX produced in cultured CHO cells is nearly identical to natural, plasma-derived factor IX and is widely used in clinical practice. Development of a biosimilar recombinant human factor IX for medical applications requires the generation of a clonal cell line with the highest specific productivity possible and a high level of specific procoagulant activity of the secreted factor IX. We previously developed plasmid vectors, p1.1 and p1.2, based on the untranslated regions of the translation elongation factor 1 alpha gene from Chinese hamster. These vectors allow one to perform the methotrexate- driven amplification of the genome-integrated target genes and co-transfect auxiliary genes linked to various resistance markers. The natural open reading frame region of the factor IX gene was cloned in the p1.1 vector plasmid and transfected to CHO DG44 cells. Three consecutive amplification rounds and subsequent cell cloning yielded a producer cell line with a specific productivity of 10.7 ± 0.4 pg/cell/day. The procoagulant activity of the secreted factor IX was restored nearly completely by co-transfection of the producer cells by p1.2 plasmids bearing genes of the soluble truncated variant of human PACE/furin signal protease and vitamin K oxidoreductase from Chinese hamster. The resulting clonal cell line 3B12-86 was able to secrete factor IX in a protein-free medium up to a 6 IU/ml titer under plain batch culturing conditions. The copy number of the genome- integrated factor IX gene for the 3B12-86 cell line was only 20 copies/genome; the copy numbers of the genome-integrated genes of PACE/furin and vitamin K oxidoreductase were 3 and 2 copies/genome, respectively. Factor IX protein secreted by the 3B12-86 cell line was purified by three consecutive chromatography rounds to a specific activity of up to 230 IU/mg, with the overall yield > 30%. The developed clonal producer cell line and the purification process employed in this work allow for economically sound industrial-scale production of biosimilar factor IX for hemophilia B therapy.

Gene Expression in the Three-Spined Stickleback (Gasterosteus aculeatus) of Marine and Freshwater Ecotypes.

Three-spine stickleback (Gasterosteus aculeatus) is a well-known model organism that is routinely used to explore microevolution processes and speciation, and the number of studies related to this fish has been growing recently. The main reason for the increased interest is the processes of freshwater adaptation taking place in natural populations of this species. Freshwater three-spined stickleback populations form when marine water three-spined sticklebacks fish start spending their entire lifecycle in freshwater lakes and streams. To boot, these freshwater populations acquire novel biological traits during their adaptation to a freshwater environment. The processes taking place in these populations are of great interest to evolutionary biologists. Here, we present differential gene expression profiling in G. aculeatus gills, which was performed in marine and freshwater populations of sticklebacks. In total, 2,982 differentially expressed genes between marine and freshwater populations were discovered. We assumed that differentially expressed genes were distributed not randomly along stickleback chromosomes and that they are regularly observed in the "divergence islands" that are responsible for stickleback freshwater adaptation.

Diversification of the Homeotic AP3 Clade MADS-Box Genes in Asteraceae Species Chrysanthemum morifolium L. and Helianthus annuus L.

The structure and phylogeny of MADS-box genes HAM91 of sunflower (Helianthus annuus) and CDM115 of chrysanthemum (Chrysanthemum morifolium) were characterized. It is shown that these genes encode MADS-domain transcription factors, which are orthologs of TM6 (Solanum lycopersicum) and APETALA3 (Arabidopsis thaliana), respectively. We obtained two types of transgenic tobacco plants (Nicotiana tabacum) with constitutive expression of HAM91 and CDM115 genes. Both types of plants flowered later than the control plants and formed more flowers and seed pods. The weight of seeds of 35S::CDM115 plants was significantly lower than in the control and 35S::HAM91 plants, which may indicate to a change in the identity of ovules in 35S::CDM115.

Ectopic Expression of the Homeotic MADS-Box Gene HAM31 (Helianthus annuus L.) in Transgenic Plants Nicotiana tabacum L. Affects the Gynoecium Identity.

The structure of the MADS-box gene HAM31 of the sunflower (Helianthus annuus) was characterized. It is shown that the product of this gene is an ortholog of the B-class MADS transcription factor PISTILLATA (Arabidopsis thaliana). Two types of transgenic tobacco plants (Nicotiana tabacum) with the constitutive expression of the HAM31 gene in the sense and antisense orientation were obtained. The 35S::HAM31s plants formed flowers with an altered gynoecium identity, whereas 35S::HAM31as plants did not differ from the control.

Differential miRNA expression in the three-spined stickleback, response to environmental changes.

miRNAs play important role in the various physiological and evolutionary processes, however, there is no data allowing comparison of evolutionary differences between various ecotypes adapted to different environmental conditions and specimen demonstrating immediate physiological response to the environmental changes. We compared miRNA expression profiles between marine and freshwater stickleback populations of the three-spined stickleback to identify the evolutionary differences. To study the immediate physiological response to foreign environment, we explored the changes induced by transfer of marine sticklebacks into freshwater environment and vice versa. Comparative analysis of changes in miRNA expression suggested that they are driven by three independent factors: (1) non-specific changes in miRNA expression under different environmental conditions; (2) specific response to freshwater conditions in the marine stickleback ecotype; (3) specific response to extreme osmotic conditions for both marine and freshwater ecotypes during the contact with non-native environment. Gene Ontology enrichment analysis of differential expressed miRNA targets supports our current hypothesis.

[Identification and expression analysis of receptor-like kinase gene ERECTA in mycoheterotrophic plant Monotropa hypopitys].

The precise spatial-temporal coordination of cell division and differentiation is necessary for the correct formation of tissues, organs, and the organism as a whole. This coordination has been implemented by the intercellular communication mediated by signaling molecules and receptors that selectively recognize them. Membrane receptor kinases of ERECTA family regulate inflorescence and flower structure, the formation of root epidermis and adaptation responses. The characterization of the ERECTA genes of flowering plant pinesap Monotropa hypopitys with unique development features can enrich the knowledge about the kinase ERECTA functions and conserved development processes with their participation. Transcriptomic and genomic search with the subsequent structural-phylogenetic analysis identified the mRNA of a gene of serine-threonine kinase receptor with leucine-rich repeats of MhyERL1, which is the only ortholog of the ERECTA family kinases of pinesap. A quantitative analysis of the MhyERL1 gene transcripts has revealed its expression in all analyzed pinesap tissues with maximum levels in the flowers. MhyERL1 is probably involved in defining the inflorescence and flower architecture, and the formation of the pinesap root epidermis. The cascades involving ERL1 are apparently conserved. The exception are pathways associated with the development of above-ground vegetative structures, and the immune response to fungal pathogens probably lost in the process of the pinesap adaptation to unfavorable environmental conditions.

[Hsp70 Genes of the Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) Parasitic Wasp].

Miniaturization is an evolutionary process that is widely represented in both invertebrates and vertebrates. Miniaturization frequently affects not only the size of the organism and its constituent cells, but also changes the genome structure and functioning. The structure of the main heat shock genes (hsp70 and hsp83) was studied in one of the smallest insects, the Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) parasitic wasp, which is comparable in size with unicellular organisms. An analysis of the sequenced genome has detected six genes that relate to the hsp70 family, some of which are apparently induced upon heat shock. Both induced and constitutively expressed hsp70 genes contain a large number of introns, which is not typical for the genes of this family. Moreover, none of the found genes form clusters, and they are all very heterogeneous (individual copies are only 75-85% identical), which indicates the absence of gene conversion, which provides the identity of genes of this family in Drosophila and other organisms. Two hsp83 genes, one of which contains an intron, have also been found in the M. amalphitanum genome.

Homeotic MADS-box genes encoding LeMADS-MC orthologues in wild tomato species (genus Solanum).

New full-length genes encoding the LeMADS-MC transcription factor orthologues were identified and functionally characterized in wild tomato species S. cheesmaniae and S. habrochaites. A comparative analysis of the encoded proteins and LeMADS-MC of the cultivated tomato species S. lycopersicum revealed two major amino acid residues substitutions: V155E in the K-domain of ShaMADS-MC (S. habrochaites) and S80L in the I-region of SchMADS-MC (S. cheesmaniae). Structural differences of the C-terminal regions of MC and the canonical euAP1 proteins indicate possible chromosomal rearrangements in the Solanoideae subfamily, which, however, did not change the main known conserved euAP1 functions.

Identification and characterization of the flower meristem identity gene MhyLFY in mycoheterotrophic plant Monotropa hypopitys.

The gene encoding the transcription factor LEAFY was identified in the genome of the mycoheterotrophic plant, pinesap Monotropa hypopitys. In the transcriptomes of roots, bracts, and flowers of flowering pinesaps, the MhyLFY gene expression was absent. These data suggest the conservativeness of the LFY-dependent mechanism of flower meristem identity and flower formation in heterotrophic species with some differences associated to the specificity of development and the structure of such plants. The pinesap flowering under the control of the transcription factor MhyLFY may be initiated either in an embryonic inflorescence during spring dormancy release of adventitious root buds or in an inflorescence of a growing reproductive stem after photoperiodic induction.

Approaches to Controlled Co-Amplification of Genes for Production of Biopharmaceuticals: Study of the Insertion and Amplification Dynamics of Genetic Cassettes in the Genome of Chinese Hamster Ovary Cells during Co-Expression of Compatible Pair of Plasmids.

Plasmid vector family p1.1 based on non-coding regions of Chinese hamster housekeeping gene EEF1A and concatemer of Epstein-Barr virus terminal repeat increases the frequency of genome integration and provides rapid amplification of the target genes in the genome. For a pair of fluorescent proteins eGFP and mCherry it was shown that p1.1 vectors bearing dihydrofolate reductase and glutamine synthetase selection markers upon co-transfection into CHO DG44 cell line allow obtaining a polyclonal cell population in which ~70% of cells express both genes. The subsequent one-step gene amplification of the genome-integrated genetic cassettes under the selective pressure of increased concentrations of methotrexate can increase the expression of both integrated genes up to 8.2% eGFP and 9.9% mCherry of total protein. This approach can be used for the development of cell lines for the production of functional heterodimeric proteins, e.g. polypeptide hormones and therapeutic antibodies.

[Gene mutations in patients with hereditary cavernous malformations]. / Analiz mutatsii genov u bol'nykh s nasledstvennymi kavernoznymi mal'formatsiiami TsNS.

AIM: To identify mutations in cerebral cavernous malformation (CCM) genes in patients with hereditary and sporadic CCMs in the Russian population. MATERIAL AND METHODS: Blood samples from 73 randomly selected patients, including 29 MRI-confirmed familial cases, 8 clinically confirmed familial cases and 38 so-called sporadic cases, were examined. A search for large deletions/duplications was performed using multiplex ligation-dependent probe amplification (MPLA). For MLPA-negative samples, the whole genome sequencing was performed to search for single nucleotide polymorphisms (SNP). RESULTS: Deletions in three genes (ССМ1, ССМ2, ССМ3) were identified in 14 patients, including 5 without definitely established familial type, in whom the familial character of disease was not confirmed by clinical and neuroimaging results. SNP mutations were found in 13 patients, CCM gene mutations in 27. Mutations were detected in 91.7% of familial cases. In two patients, new CCM3 deletions were identified. Gene distribution was as follows: 60.7 for CCM1, 32.2 for CCM2 and 7.1% for CCM3. In two members of a family with hereditary CCMs, no high effect mutations in the known CCM genes were found. Patients with mutations had greater severity of disease. Two patients with CCM3 mutations demonstrated the most aggressive clinical course. De novo formation and growth of CCM were observed only in patients with mutations. CONCLUSION: The distribution of pathogenic mutations in known CCM genes is consistent with other large-scale studies. Familial CCMs are associated with more severe disease course and may be caused by mutations beyond the known CCM genes.

Identification and expression analysis of chitinase genes in parasitic plant Monotropa hypopitys.

Genes encoding six chitinases, five of which belong to classes I (MhCHI3 and MhCHI4), IV (MhCHI1), V (MhCHI5), and VII (MhCHI2), were identified in the transcriptome of the parasitic mixoheterotrophic plant Monotropa hypopitys. The transcription level of MhCHI5 and MhCHI1 was low; however, in the leaves (bracts) and roots it was higher than in flowers. MhCHI4 transcripts were detected primarily in the flowers and were almost absent in the roots, whereas the expression level of MhCHI3 was relatively high in all organs but maximum in the leaves (bracts).

Metagenomic analysis of microbial community of a parasitoid wasp Megaphragma amalphitanum.

The vast majority of multicellular organisms coexist with bacterial symbionts that may play various roles during their life cycle. Parasitoid wasp Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) belongs to the smallest known insects whose size is comparable with some bacteria. Using 16S rRNA gene sequencing and Whole Genome Sequencing (WGS), we described microbiota diversity for this arthropod and its potential impact on their lifecycle. Metagenomic sequences were deposited to SRA database which is available at NCBI with accession number SRX2363723 and SRX2363724. We found that small body size and limited lifespan do not lead to a significant reduction of bacterial symbionts diversity. At the same time, we show here a specific feature of microbiota composition in M. amalphitanum - the absence of the Rickettsiaceae family representatives that are known to cause sex-ratio distortion in arthropods and well represented in other populations of parasitoid wasps.

Search of tandem repeats with insertion and deletions in the A. thaliana genome.

A new mathematical method was used for the first time to search for tandem repeats with insertions and deletions in the full-length sequence of the A. thaliana genome. The method is based on a new algorithm for multiple alignment of sequences of certain periods without using paired comparisons of sequences. We identified 13997 periodic sites 2 to 50 characters long, only approximately 30% of which were known earlier. The possible origin and use of the identified sites with tandem repeats are discussed.

Construction of the plasmid-free strain for human growth hormone production.

The E. coli strain, overproducing human growth hormone (hGH) was made by integration of the hGH gene under the control of T7 promoter into the chromosomal LacZ gene of BL21(DE3) via lambda Red recombineering. The strain gave higher productivity (50 mg·L(-1)·OD550(-1)) and better growth characteristics than the corresponding strain in which the same hGH expression cassette was placed in a plasmid. The protein produced by the plasmid-free strain was purified and characterized to be hGH. The results demonstrates that a plasmid-free recombinant strain having a single-copy gene expression cassette in the chromosome could provide better gene activity regulation, higher productivity, superior growth characteristics, as well as more stringent control of the gene sequence invariance than a plasmid-based strain.

Pleiotropic effect of thyroid hormones on gene expression in fish as exemplified from the blue bream Ballerus ballerus (Cyprinidae): Results of transcriptomic analysis.

A pronounced pleiotropic effect of thyroid hormones on the regulation of gene expression in fish in postembryogenesis was demonstrated for the first time using larvae and juveniles of the blue bream Ballerus ballerus as an example. Genome-wide transcriptome sequencing (RNA-seq) identified 1212 differentially expressed genes in the brain and liver of fish kept in triiodothyronine solution (0.25 ng/mL). Our data show that the regulation of gene expression by thyroid hormones is widespread in nature: it involves not only the structural genes but also the regulatory genes. A significant number of genes under the control of thyroid hormones are involved in the determination of morphological traits.

Identification of novel microRNA genes in freshwater and marine ecotypes of the three-spined stickleback (Gasterosteus aculeatus).

The three-spined stickleback (Gasterosteus aculeatus L.) is an important model organism for studying the molecular mechanisms of speciation and adaptation to salinity. Despite increased interest to microRNA discovery and recent publication on microRNA prediction in the three-spined stickleback using bioinformatics approaches, there is still a lack of experimental support for these data. In this paper, high-throughput sequencing technology was applied to identify microRNA genes in gills of the three-spined stickleback. In total, 595 miRNA genes were discovered; half of them were predicted in previous computational studies and were confirmed here as microRNAs expressed in gill tissue. Moreover, 298 novel microRNA genes were identified. The presence of miRNA genes in selected 'divergence islands' was analysed and 10 miRNA genes were identified as not randomly located in 'divergence islands'. Regulatory regions of miRNA genes were found enriched with selective SNPs that may play a role in freshwater adaptation.

GMOs in Russia: Research, Society and Legislation.

Russian legislation lags behind the rapid developments witnessed in genetic engineering. Only a scientifically based and well-substantiated policy on the place of organisms that are created with the use of genetic engineering technologies and an assessment of the risks associated with them could guarantee that the breakthroughs achieved in modern genetic engineering technologies are effectively put to use in the real economy. A lack of demand for such breakthroughs in the practical field will lead to stagnation in scientific research and to a loss of expertise.