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Abstract The emergence of new genes and functions is of paramount importance in the emergence of new animal species. For example, the insertion of the mobile element Tigger 2 into the sequence of the functional gene POU2F1 in primates led to the formation of a new chimeric primate-specific isoform POU2F1Z, the translation of which is activated under cellular stress. Its mRNA was found in all species of monkeys, starting with macaques. Analysis of the fragments of the Tigger2 copy corresponding to the human exon Z showed that the splicing sites of exon Z are homologous in humans and in most monkeys, with the exception of lemurs and galagos. The stop codon introduced into the mRNA by the Tigger2 sequence is present in all primates, starting with macaques. The internal ATG codon is also present in all primates, with the exception of lemurs and galagos. In the course of evolution, other MGEs, mainly of the SINE type, were inserted into the Tigger2 copy. In the course of evolution, both the location and the number of mobile SINE elements within the POU2F1 gene changed. Starting with macaques, the pattern of the arrangement of SINE elements within the Tigger2 copy in the studied region of the POU2F1 gene was fixed and then remained unchanged in other primates and humans, which may indicate its functional significance.

Neuroblastoma is one of the most common cancers in infants and is often multidrug-resistant. One of the methods of treating neuroblastomas is to create conditions for their differentiation. In this work, we performed a full-transcriptome analysis of gene expression in an undifferentiated and differentiated in vitro human neuroblastoma cell line IMR-32 and identified the signaling pathways and biological processes that undergo the greatest changes during differentiation. The results obtained show that a complex heterogeneous population of nerve cells is formed at different stages of differentiation. In the cell population of differentiating neuroblastoma, the expression of genes in which cortical neuronal progenitor cells are enriched increases; at the same time, there are cells expressing markers of early postmitotic neurons. Cells differentiate in several different directions according to the type of synaptic mediator. At the same time, the differentiation of IMR-32 cells is accompanied by an increase in the transcription of genes that suppress the differentiation of nerve cells, Sox2 and PROM1, the expression of which is normally suppressed during in vivo differentiation.

Here we attempt to reconstruct the sequence of events that led to the formation of three regulatory piRNA clusters, namely, 20A, 38C and flamenco in the Drosophila melanogaster genome. Both the 38C and flamenco clusters include inverted sequences, which potentially form double-stranded RNA hairpins. We present evidence in favor of the well-known hypothesis of piRNA clusters as “transposon traps”. According to this model, the presence of the only copy of the transposon in the genome indicates that its expression is suppressed by an RNA-interference mechanism immediately after the mobile element enters the piRNA cluster. We also discuss high the structural variability of piRNAs in Drosophila clusters and cases of horizontal transmobile elements between related species.

Abstract Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt’s lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.

— The paper discusses the techniques which are currently implemented for vaccine production based on virus-like particles (VLPs). The factors which determine the characteristics of VLP monomers assembly are provided in detail. Analysis of the literature demonstrates that the development of the techniques of VLP production and immobilization of target antigens on their surface have led to the development of universal platforms which make it possible for virtually any known antigen to be exposed on the particle surface in a highly concentrated form. As a result, the focus of attention has shifted from the approaches to VLP production to the development of a precise interface between the organism’s immune system and the peptides inducing a strong immune response to pathogens or the organism’s own pathological cells. Immunome-specified methods for vaccine design and the prospects of immunoprophylaxis are discussed. Certain examples of vaccines against viral diseases and cancers are considered.

Neuroblastoma is one of the most common cancers in infants and is often multidrug-resistant. One of the methods of treating neuroblastomas is to create conditions for their differentiation. In this work, we performed a full-transcriptome analysis of gene expression in an undifferentiated and differentiated in vitro human neuroblastoma cell line IMR-32 and identified the signaling pathways and biological processes that undergo the greatest changes during differentiation. The results obtained show that a complex heterogeneous population of nerve cells is formed at different stages of differentiation. In the cell population of differentiating neuroblastoma, the expression of genes in which cortical neuronal progenitor cells are enriched increases; at the same time, there are cells expressing markers of early postmitotic neurons. Cells differentiate in several different directions according to the type of synaptic mediator. At the same time, the differentiation of IMR-32 cells is accompanied by an increase in the transcription of genes that suppress the differentiation of nerve cells, Sox2 and PROM1, the expression of which is normally suppressed during in vivo differentiation.

Abstract POU2F1 (Oct-1) is a transcription factor, the overexpression of which is found in many human malignant tumors; a significant increase in its level in cells determines the malignant potential of the tumor. POU2F1 is represented in cells by several isoforms that are transcribed from alternative promoters. In Burkitt’s B-cell lymphoma Namalwa, the concentration of tissue-specific isoform Oct-1L is several times higher than in normal B cells. We tested the potential to inhibit the transcription of individual Oct-1 isoforms using the GSK3 kinase inhibitor CHIR, an aminopyrimidine derivative. We have shown that CHIR specifically affects the expression of the tissue-specific isoform Oct-1L, significantly reducing the level of mRNA and Oct-1L protein. However, CHIR does not change the amount of mRNA and protein of the ubiquitous isoform Oct-1A in Namalwa tumor cells. The results obtained show that it is possible to develop a system for selective inhibition of Oct-1 transcription factor isoforms in human cells to suppress drug resistance of tumor cells with a high POU2F1 content.

Here we attempt to reconstruct the sequence of events that led to the formation of three regulatory piRNA clusters, namely, 20A, 38C and flamenco in the Drosophila melanogaster genome. Both the 38C and flamenco clusters include inverted sequences, which potentially form double-stranded RNA hairpins. We present evidence in favor of the well-known hypothesis of piRNA clusters as "transposon traps". According to this model, the presence of the only copy of the transposon in the genome indicates that its expression is suppressed by an RNA-interference mechanism immediately after the mobile element enters the piRNA cluster. We also discuss high the structural variability of piRNAs in Drosophila clusters and cases of horizontal transmobile elements between related species.

Thapsigargin, the SERCA ATPase inhibitor, effectively suppresses the expression of metastasis marker S100A4 in breast cancer cells MDA-MB231. It has been demonstrated that transcription of the S100A4 gene is controlled by Ca2+-signaling pathways. It has been shown that synthesis of S100A4 mRNA and protein in the MDA-MB231 cell line is effectively inhibited by thapsigargin at a concentration of 0.4-4 µM, while preserving cell survival. We assume that a change in gene transcription in response to the disruption of Ca2+ homeostasis plays a direct role in the remodeling of Ca2+-signaling pathways.

Thapsigargin, the SERCA ATPase inhibitor, effectively suppresses the expression of metastasis marker S100A4 in breast cancer cells MDA-MB231. It has been demonstrated that transcription of the S100A4 gene is controlled by Ca 2+ -signaling pathways. It has been shown that synthesis of S100A4 mRNA and protein in the MDA-MB231 cell line is effectively inhibited by thapsigargin at a concentration of 0.4-4 µM, while preserving cell survival. We assume that a change in gene transcription in response to the disruption of Ca 2+ homeostasis plays a direct role in the remodeling of Ca 2+ -signaling pathways.

Thapsigargin (SERCA ATPase inhibitor) inhibited the S100A4 metastatic marker expression in MDA-MB231 breast cancer cells. We found that S100A4 gene transcription is regulated by Ca

Changes in the expression level of Oct-1A, Oct-1L, Oct-1X, and Oct-1Z isoforms and CD14 surface antigen during differentiation of HL-60 monocytic cells induced in vitro by dimethyl sulfoxide were studied, and the expression level of the four Oct-1 isoforms in vivo in human monocytes was determined.

The structural protein (Gag) of Drosophila retrovirus gypsy contains capsid and nucleocapsid domains. Gag forms virus-like particles in a bacterial cell; furthermore, its capsid alone is able to form aggregates. However, aggregates assembled from the capsid vary in size and are less organized than particles formed by a full-length Gag. The nucleocapsid determines the organization and structure of the particles, which is ensured by the amino acid residues at its N-terminal (a nucleocapsid proximal part). The assembly of the particle occurs in the presence of any RNAs or single-stranded DNA oligonucleotides.

Amino acid sequence of the drosophila retrovirus MDG4 (gypsy) structural protein Gag does not contain a canonical motif known for the majority of vertebrate retroviruses. Moreover, protein translation can theoretically begin with two separated initiation codons located within its unique open reading frame. We designed constructs for expression of two theoretically possible variants of Gag polypeptide and investigated the ability of each product to form virus-like particles in the bacterial cell, i.e., in the absence of eukaryotic cell factors. The results obtained showed that both variants of the gypsy protein Gag form globular particles in the bacterial cell.

42 Mobile genetic elements account for a considerable part of eukaryotic genome and significantly affect its stability. They are involved in mutagenesis, regulation of gene expression, and occurrence of genomic rearrangements. In view of this, the study of the mechanisms by which the activity of transposable elements is regulated by cellular factors is of great interest. For this purpose, it is convenient to use model objects in the genome of which mobile genetic elements are transposed and amplified. The behavior of mobile genetic elements in different Drosophila species has been described sufficiently well so far. There are many Drosophila strains characterized by genetic instability caused by amplification and transposition of mobile genetic elements. In such strains, an increased frequency of mutations and reversions as well as occurrence of chromosomal rearrangements is observed. A characteristic feature of D. melanogaster laboratory strain G32 is unusual behavior of some mobile elements. In particular, this strain characterized by substantial amplification of the gtwin retrotransposon, which is closely related to gypsy , one of the most comprehensively studied mobile genetic elements. Evidences indicating that currently gtwin undergoes transpositions have not been obtained [1]. In addition, we detected a large chromosomal aberration in this linea with an increased number of sites where gtwin and some other mobile genetic elements are located [2]. It is known from published data that mobile genetic elements are inhomogeneously distributed over the genome, which leads to occurrence of clusters playing a major role in the regulation of transposition activity [3]. Taking into account this information and the fact that the number of gtwin copies in the G32 strain was approximately 30 (which is one order of magnitude larger than the commonly detected number of copies of this element), we assumed that different gtwin copies may be located in the immediate vicinity of one another. In this study, we tested a series of D. melanogaster strains for the presence of neighboring gtwin copies by PCR using genomic DNA as a template. The annealing sites for PCR primers were located at both ends of gtwin in the vicinity of its long terminal repeats (LTRs). Synthesis proceeded in the direction of the genomic environment of the element.

The invalidism structure was analyzed for patients, suffering consequences of traumas and diseases of a knee joint (KJ). The primary invalidism level because of traumas and diseases of a KJ have constituted in 2013 yr 12.4%. The cause of invalidism in men is predominantly a one-side gonarthrosis, and in women--a bilateral one--due to concurrent aggravating causes (dishormonal changes, excessive body mass). The invalidism indices enhancement is caused by absence of a dispensary follow-up, insufficient treatment on various stages of the disease course, severity and irreversibility of pathological process in a KJ, socio-economic factors, low rehabilitational potential, prognosticated impossibility to conduct a professional-labour rehabilitation in a prepensionable and pensionable age.

Current views of retrotransposons possessing long terminal repeats (LTRs) are described. The existing classification and element types isolated by genome organization are considered. Experimental data are summarized to demonstrate that the replicative cycle of a retrotransposon is not restricted to a single cell and that LTR retrotransposons are transferred between somatic cells with a rate comparable with the element transposition rate within the genome of one cell. The major mechanisms mediating the role of LTR retrotransposons in reorganization of the genome are considered with regard to the strategies of their horizontal and vertical transfer.

A search for noncanonical variants of the gypsy retrotransposon ( MDG4 ) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF2 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. Hydei or D. Virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.

The view on Drosophila long terminal repeat (LTR) retrotransposons, which have three reading frames, as endogenous retroviruses or errantiviruses (ERVs, according to the latest ICTV nomenclature) is discussed. Data on the biology of ERVs and the mechanisms of their involvement in genetic instability of Drosophila are considered.

Since retrovirus-like particles of gypsy (mdg4) are capable of interspecific transfer, other Drosophila melanogaster gypsy-related retrotransposons were tested for this property. As a donor and a recipient, D. melanogaster and D. virilis cultured cells were used. Recipient cell DNA was analyzed with probes directed to mdg1, mdg3, 17.6, 297, 412, or B104/roo. Transfer was demonstrated for mdg3, which lacks env. The possible mechanism of transfer is discussed.

We studied the frequencies of these variants of the retrovirus-like transposable element MDG4 ( gypsy ) in D. melanogaster strains obtained from wild populations. We examined 44 strains for the presence of subfamilies homologous with respect to the Hind III site and determined the number of copies in them. The Hind III site is a marker that permits distinguishing the two MDG4 variants with different retrotransposition activities. The data obtained allow us to suggest that the inactive transposable element has emerged in the D. melanogaster genome considerably earlier than the active one. In five strains, MDG4 was amplified, which might be related to genetic instability caused by its transpositions. These strains may serve as a model for further studying the genetic control of retrotransposition. Southern blotting was used for screening of the

The distribution of two variants of MDG4 (gypsy) was analyzed in severalDrosophila melanogasterstrains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutantflamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovoD test.

To study the mechanism of precise excision ofgypsy from genomic sites, the integrase domain ofgypsy pol was cloned and expressed inEscherichia coli. The endonuclease activity of recombinant integrase was assayed with synthetic substrates corresponding to 3′-U5 ofgypsy LTR and to the known genomic insertion sites ofgypsy. Integrase nicked the 5′-A ⇓ YR-3′ triplet in the (+) strand of the double-stranded substrates; cleavage of a single-stranded substrate was nonspecific. Cleavage proved to be affected by the local conformation of the substrate: the (+) strand was cleaved more efficiently when the (−) strand had an unpaired base in the triplet and was not cleaved when the (−) strand was interrupted or branched. The triplet corresponded to the consensus region ofgypsy insertion (5′-YRYR ⇓ YR-3′), the site of cleavagein vitro coinciding with the site of insertionin vivo. The unique mechanism ofgypsy excision was assumed to depend to a great extent on the enzymic properties of its integrase.

The genetically unstable Mutator Strain of D. melanogaster is characterised by a high frequency of spontaneous mutations and their reversions. Three forked mutants were obtained independently and several reversions arose spontaneously with frequency of 10(-3)-10(-4). The sites of integration and excision of the gypsy retrotransposon were analysed by Southern blot analysis and sequencing of PCR fragments. In all cases gypsy had inserted at the end of the third exon of the major transcript of the forked gene, causing the duplication of TCCA target sequence. All the reversions resulted from precise excision of the gypsy. A double mutant containing ct6 and f1, caused by gypsy insertions into untranslated regions of the corresponding genes, was constructed. Two spontaneous ct6f+ revertants as well as one ct+f1 revertant were obtained from this line. Sequence analysis of gypsy integration and excision sites revealed that in all cases gypsy excision was also precise. These experiments constitute the first demonstration of precise excision of LTR-containing elements from their host genomes.

A sequence 30 bp downstream from the start site of the Drosophila melanogaster retrotransposon mdg1 is shown to be responsible for correct and precise initiation of mdg1 RNA synthesis in combination with the RNA start-site sequence TCAGTT. A sequence-specific DNA binding protein is demonstrated to interact with the +30 sequence, and the efficient binding of this factor is necessary for in vivo transcriptional activity of the plasmid constructs containing mdg1 promoter fragments. The nucleotides -8/+34 of mdg1 represent a minimal promoter which is able to provide correct initiation of transcription by RNA polymerase II at basal levels. A comparison with properties of some other retrotransposable elements and several developmentally regulated cellular genes allows us to conclude that together they form a specific class of RNA polymerase II promoter. This promoter class characteristically lacks upstream sequences necessary for transcription initiation, such as TATA boxes, but requires a specific downstream promoter element within 40 bp downstream of the RNA start site. The level of transcription can, however, be modulated by upstream regulatory elements. The identified sequence-specific downstream initiation factor may be responsible for transcription initiation on promoters of some genes which belong to this class.

The paper describes preparation and performance of single lateral mode buried heterostructure InGaAsP/InP, λ=1.3 μm, laser diodes. It is shown that an improvement in the mesa etching and regrowth techniques permits obtaining laser diodes whose threshold current densities are about 1 kA/cm2 and are practically independent of stripe width. The diodes thus prepared had threshold currents of 8–20 mA at a cavity length L=200–500 μm and a stripe width about 2–3 μm. The maximum continuous wave power in single lateral mode operation was 160 mW.

The location of different cloned transposable elements was determined in the polytene chromosomes of gt waand Oregon RC inbred Drosophila melanogaster stocks. Each stock was found to have about 300 hybridization sites for 12 different clones. The arrangement of the clones in the chromosomes was identical in individuals of the same stock but differed significantly between the two stocks studied. Only 10% of the localisation sites of the clones did not change their location in the chromosomes. Calculations showed that each band of a polytene chromosome may contain transposable elements. Thus these elements provide a high level of structural heterozygosis in the chromosomes of hybrid individuals. There was a slight but statistically significant tendency of transposable elements to form clusters in certain chromosomal regions. The number of chromosomal regions that they occupied was 80% of the number expected for a random distribution. A concentration of transposable elements occurred in the chromosomal regions adjacent to the chromocenter, in histone and 5S RNA loci. The banding pattern of homologous chromosomal regions as revealed by electron microscopy was similar in both D. melanogaster stocks although they differed greatly in the arrangement of transposable elements. It is likely that the structural heterozygosis provided by transposable elements in heterozygous individuals influences the frequency of genetic recombination in affected chromosomal regions, disturbs the time course of DNA synthesis, and changes the efficiency of transcription.

In Drosophila melanogaster structural genes are located close to moderately reiterated sequences. One of the clones obtained contains the DNA related to intercalary heterochromatin of D. melanogaster. These are individual differences in the distribution of genetic material in polytenic chromosomes of different stocks of D. melanogaster. The techniques that allow isolation of DNA fragments containing structural genes at the beginning, in the middle, or the end of the coding strand have been elaborated.

The gypsy (mdg4) mobile element of Drosophila contains two closely spaced regions which bind proteins from nuclear extracts. One of these is an imperfect palindrome having homology with the lac-operator of Escherichia coli; the other contains a reiterated sequence (5'PyPuT/C TGCATAC/TPyPy) homologous to the octamer that is the core of many enhancers and upstream promoter elements. Transient expression of deletion mutants has shown that these DNA regions are negative and positive regulators of transcription. As was demonstrated earlier by other authors, mutations induced by the presence of gypsy in different loci are suppressed owing to either repression or activation of gypsy transcription in Drosophila strains carrying unlinked mutations in su(Hw) or su(f) genes. We have shown that binding to a negative regulator (silencer) is weakened in nuclear extracts isolated from fly stocks carrying su(f) mutations which activate gypsy transcription; therefore the su(f) gene seems to code for a protein capable of gypsy repression. Furthermore, binding to a positive regulator is weakened in nuclear extracts isolated from fly stocks carrying su(Hw) gene mutations which decrease the level of gypsy transcription; therefore, the su(Hw) gene most likely encodes a protein which activates gypsy transcription.

1. The previously described method of the isolation of long fragments of native DNA containing structural genes at the beginning, in the middle or at the end of the coding strand /1,2/ was applied to the purification of DNA fragments enriched in globin gene sequences. One cycle of purification procedure gives up to several thousand-fold enrichment in the globin gene. 2. The clones containing Drosophila melanogaster genes (Dm218, Dm225 and Dm234) which produce very abundant mRNA in cell culture were characterized. In situ hybridization experiments show that Dm225 and Dm234 are present in several tens of sites in polytene chromosomes, the great majority of which correspond to the sites of intercalary heterochromatin. There are individual differences in the distribution of genetic material in polytenic chromosomes of different stocks of Drosophila melanogaster.

The location of DNA fragments representing mobile dispersed genes (MDG) in salivary gland and midgut polytene chromosomes was compared by means of in situ hybridization. In the Drosophila stock under study the average number of hybridization sites in the polytene chromosomes of one nucleus was 20 for MDG-1 and 10 for MDG-3. The total numbers of hybridization sites and their relative positions proved to be same in the polytene chromosomes of the two tissues. These results support the idea of a stable location of the mobile dispersed genes in the course of ontogenesis.

Histone oligomers produced by formaldehyde treatment of chromatin were studied. It was shown that these histone oligomers could be converted into monomers by boiling in 0.1 NH2SO4. Dimers H2B-H4 and H2B-H2A were identified by two-dimensional polyacrylamide gel electrophoresis.