[Show abstract][Hide abstract] ABSTRACT: Here we investigated dynamic properties of the piNG-body, large perinuclear granule that was discovered previously in spermatocytes of Drosophila. The piNG-body contains ribonucleoprotein complexes involved in piRNA-silencing of genome repeats including transposons in premeiotic spermatocytes with aid of short piRNAs. Confocal microscopy of fixed and native preparations demonstrates that the piNG-body is mobile structure which does not occupy a stationary position near nuclear surface relative to chromosomal territories. FRAP-analysis reveals a high exchange rate of RNA helicase Vasa in the piNG-body and small perinuclear granules with the cytozol Vasa pool. Disruption of microtubule assembly of cytoskeleton does not affect to stability of the piNG-body and small granules. We suppose that the combination of piNG-body mobility and permanent molecular exchange of Vasa protein provides an efficient "scanning" of total volume of the cytoplasm of primary spermatocytes and timely recognition and destruction of unwanted transcripts of the repetitive elements of genome.
No preview · Article · Apr 2015 · Molekuliarnaia biologiia
[Show abstract][Hide abstract] ABSTRACT: The review summarizes a current knowledge about a role of RNA helicases in the development and maintenance of gamenogenesis in eukaryotes. We focused on three RNA helicase family members—Vasa/DDX4, Belle/DDX3, and Spindle-E/TDRD9—that contain characteristic amino acid sequence motifs (DEAD box) and perform substantial conserved functions in the germinal tissues of various species from Drosophila to human. These enzymes are involved in a broad range of activities associated with the regulation of transcription, splicing, nuclear export and, especially, with translation initiation. Expression of genes required for gametogenesis is regulated mainly at the transcriptional level. RNA helicases are involved in the formation of cytoplasmic ribonucleoprotein (RNP) granules and RNA silencing. A highly conserved central domain is characteristic of DEAD-box RNA helicases and determines their basic biological activity in ATP-dependent unwinding of short RNA duplexes.
No preview · Article · Jan 2014 · Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Drosophila testes are generally considered as a useful model for studying the fundamental developmental processes of heterogametic organisms. However, immunostaining of the whole Drosophila testes is often associated with insufficient resolution at the subcellular level, poor reproducibility and incomplete staining of fixed preparations. The main problem for adequate staining is poor permeability of the organs for antibodies and antibody-coupled fluorophores. To overcome this problem we developed a protocol for the whole mount testis immunostaining yeilding high quality preparations for confocal microscopy. Many subcellular structures can be successfully resolved, such as spectrosome, fusome, nuage granules, apoptotic bodies and protein crystals. This method preserves the inner architecture of the testes enabling the 3D image reconstruction of from a set of confocal sections. It allows to combine the simultaneous detection of fluorescently tagged and immunostained proteins as well as TUNEL analysis for apoptosis detection.
No preview · Article · Jan 2013 · Analytical Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Tandem Stellate genes organized into two clusters in heterochromatin and euchromatin of the X-chromosome are part of the Ste-Su(Ste) genetic system required for maintenance of male fertility and reproduction of Drosophila
melanogaster. Stellate genes encode a regulatory subunit of protein kinase CK2 and are the main targets of germline-specific piRNA-silencing; their derepression leads to appearance of protein crystals in spermatocytes, meiotic disturbances, and male sterility. A short promoter region of 134 bp appears to be sufficient for testis-specific transcription of Stellate, and it contains three closely located cis-regulatory elements called E-boxes. By using reporter analysis, we confirmed a strong functionality of the E-boxes in the Stellate promoter for in vivo transcription. Using selective mutagenesis, we have shown that the presence of the central E-box 2 is preferable to maintain a high-level testis-specific transcription of the reporter gene under the Stellate promoter. The Stellate promoter provides transcription even in heterochromatin, and corresponding mRNAs are translated with the generation of full-size protein products in case of disturbances in the piRNA-silencing process. We have also shown for the first time that the activity of the Stellate promoter is determined by chromatin context of the X-chromosome in male germinal cells, and it increases at about twofold when relocating in autosomes.
Full-text · Article · Nov 2012 · Biochemistry (Moscow)
[Show abstract][Hide abstract] ABSTRACT: Testis-specific tandemly repeated Stellate genes are part of the Ste-Su(Ste) genetic system required for male fertility in Drosophila melanogaster. Stellate genes encode a functional homolog of the β-subunit of protein kinase CK2. Derepression of Stellate results in their over-expression, meiotic disturbances and male sterility. Stellate genes are represented by clustered copies in the X chromosome and carry promoters shared with another X-chromosome cluster, βNACtes genes, encoding putative β-subunits of the nascent polypeptide-associated complex. Using Electrophoretic Mobility Shift Assay, we revealed in the Stellate promoter three cis-acting elements, E-boxes, the loss of which greatly diminished the reporter gene expression in Drosophila testes. We identified that these E-boxes were recognized by helix-loop-helix protein, dUSF (Drosophila ortholog of mammalian USF) in testis nuclear extract. All three E-boxes were preserved in the promoters of both euchromatic and heterochromatic Stellate clusters. Two analogous E-boxes were detected in the promoters of 5'-copies of the duplicated βNACtes gene pairs, whereas the 3'-copies lacked these sites but possessed a new binding site for a testis protein distinct from dUSF. Here we characterized a new type of testis-specific core promoter and identified dUSF as its interacting transcription factor.
[Show abstract][Hide abstract] ABSTRACT: Ribonucleoprotein-containing granules in the cytoplasm of germinal cells are known to be a common attribute of eukaryotic organisms. Germ granules appear to ensure the posttranscriptional regulation of germline mRNAs. Recent studies specify the participation of the germ granules in genome integrity maintenance by mechanisms involving short piRNAs. PIWI clade proteins and associated piRNAs are considered as key participants of the germline-specific piRNA pathway. Proteins of the PIWI clade, Aub and AGO3, concentrated in the germline-specific perinuclear granules called nuage, are involved in silencing of retrotransposons and other selfish repetitive elements in the Drosophila genome. In Drosophila testes, two types of perinuclear nuage granules are found: a large amount of small particles around the nuclei and significantly larger structures, the piNG-bodies. In this mini-review, we analyze the recent published data about structure and functions of Drosophila male germ granules, and especially their involvement in the piRNA silencing pathway.
[Show abstract][Hide abstract] ABSTRACT: Regulation of the testis-specific crystal-Stellate genetic system in Drosophila melanogaster is a subject of intensive investigation. The X-linked tandem testis-specific clusters of Stellate genes encode proteins homologous to the regulatory ß-subunit of CK2 protein kinase, CK2ß, and are not expressed in wild-type flies. Repression of Stellate genes is carried out with the assistance of the Y-linked crystal locus (also known as Su(Ste), Supressor of Stellate). The Su(Ste) repeats are highly homologous to Stellate genes, and normally, the expression of Stellate is repressed by short RNAs provided by anti-sense transcription of Su(Ste), via piRNA silencing.Derepression of Stellate genes in the absence of the crystal locus (crystal line) leads to the accumulation of Stellate protein crystals in spermatocytes, defects in condensation and segregation of meiotic chromosomes and also partial or complete male sterility. Functions of these genes are unclear, whereas CK2 plays an important role in the regulation of transcription and cell division in D.melanogaster and other eukaryotes. Why the crystal-Stellate system had appeared and for what function, as well as the exact mechanism of the Stellate pathogenesis, have not yet been revealed. Stellate genes are found only in D.melanogaster and are believed to originate due to the amplification of the CK2ßtes autosomal gene encoding another regulatory ß-subunit of CK2. The soluble Stellate protein exists in the nuclei of the spermatocytes of the crystal line flies, where it interacts with the catalytic α-subunit of CK2 (CK2α), causing a modulation of phosphorylation of some nuclear proteins. Stellate also undergoes lysine methylation and mimics the trimethyl-H3K9 epigenetic modification of the histone H3 tail. In this chapter, we summarize the current conceptions of the origin and organization of the crystal-Stellate system, and transcriptional and post-transcriptional regulation of Stellate expression. We also discuss presumable mechanisms of pathogenesis caused by the hyper-expression of Stellate protein.
[Show abstract][Hide abstract] ABSTRACT: Proteins of the PIWI subfamily Aub and AGO3 associated with the germline-specific perinuclear granules (nuage) are involved in the silencing of retrotransposons and other selfish repetitive elements in the Drosophila genome. PIWI proteins and their 25- to 30-nt PIWI-interacting RNA (piRNAs) are considered as key participants of the piRNA pathway. Using immunostaining, we found a large, nuage-associated organelle in the testes, the piNG-body (piRNA nuage giant body), which was significantly more massive than an ordinary nuage granule. This body contains known ovarian nuage proteins, including Vasa, Aub, AGO3, Tud, Spn-E, Bel, Squ, and Cuff, as well as AGO1, the key component of the microRNA pathway. piNG-bodies emerge at the primary spermatocyte stage of spermatogenesis during the period of active transcription. Aub, Vasa, and Tud are located at the periphery of the piNG-body, whereas AGO3 is found in its core. Mutational analysis revealed that Vasa, Aub, and AGO3 were crucial for both the maintenance of the piNG-body structure and the silencing of selfish Stellate repeats. The piNG-body destruction caused by csul mutations that abolish specific posttranslational symmetrical arginine methylation of PIWI proteins is accompanied by strong derepression of Stellate genes known to be silenced via the piRNA pathway.
Full-text · Article · Jul 2011 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: This review is devoted to the dramatically expanding investigations of lysine methylation on nonhistone proteins and its functional importance. Posttranslational covalent modifications of proteins provide living organisms with ability to rapidly change protein activity and function in response to various stimuli. Enzymatic protein methylation at different lysine residues was evaluated in histones as a part of the "histone code". Histone methyltransferases methylate not only histones, but also many nuclear and cytoplasmic proteins. Recent data show that the regulatory role of lysine methylation on proteins is not restricted to the "histone code". This modification modulates activation, stabilization, and degradation of nonhistone proteins, thus influencing numerous cell processes. In this review we particularly focused on methylation of transcription factors and other nuclear nonhistone proteins. The methylated lysine residues serve as markers attracting nuclear "reader" proteins that possess different chromatin-modifying activities.
Full-text · Article · May 2010 · Biochemistry (Moscow)
[Show abstract][Hide abstract] ABSTRACT: IL-4 is a pleiotropic immunoregulatory cytokine secreted by Th2 subset of CD4(+) Th cells. Several transcription factors (TFs) have been determined with various degrees of certainty to bind the IL-4 promoter and to regulate its expression in human. To investigate the mechanisms responsible for phenotypic effects of the C-33T IL-4 promoter polymorphism, we performed a search of TFs binding to this promoter locus and discriminating the -33C and -33T alleles. In silico searches suggest few factors bind this region. Using an electromobility shift assay we found that Jurkat T cells contained proteins which specifically interacted with oligonucleotide probes, corresponding to the -33 region. Considerable binding differences between C and T alleles were demonstrated using competitive conditions, the proteins bound predominantly with -33C allele. We found that the transcription factor Oct-1 produced the major shifted complex. The binding of Oct-1 was not improved using activated nuclear extracts; however, we observed increases in other shifted complexes upon cell activation. We suppose that Oct-1 occupancy may compete for binding of activator proteins to closely or overlapped binding sites. Our findings suggest that the interplay between Oct-1 and unknown TFs may be responsible for the C-33T polymorphism effects.
Full-text · Article · Oct 2009 · International Journal of Immunogenetics
[Show abstract][Hide abstract] ABSTRACT: SummaryThe X-chromosome-linked clusters of the tandemly repeated testis-specific Stellate genes of Drosophila melanogaster, encoding proteins homologous to the regulatory β-subunit of the protein kinase casein kinase 2 (CK2), are repressed in wild-type males. Derepression of Stellate genes in the absence of the Y chromosome or Y-linked crystal locus (crystal line) causes accumulation of abundant protein crystals in testes and different meiotic abnormalities, which lead to partial or complete male sterility. To understand the cause of abnormalities in chromosome behavior owing to Stellate overexpression, we studied subcellular localization of Stellate proteins by biochemical fractionation and immunostaining of whole testes. We showed that, apart from the known accumulation of Stellate in crystalline form, soluble Stellate was located exclusively in the nucleoplasm, whereas Stellate crystals were located mainly in the cytoplasm. Coimmunoprecipitation experiments revealed that the α-subunit of the protein kinase CK2 (CK2α) was associated with soluble Stellate. Interaction between soluble Stellate and CK2α in the nucleus could lead to modulations in the phosphorylation of nuclear targets of CK2 and abnormalities in the meiotic segregation of chromosomes. We also observed that Stellate underwent lysine methylation and mimicked trimethyl-H3K9 epigenetic modification of histone H3 tail.
Full-text · Article · Jun 2009 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Immunogenicity for laboratory animals (rabbits and mice) of the whole hepatitis C virus envelope proteins and their conserved
as well as hypervariable HVR1 sites has been investigated. Rabbit immune responses to HCV envelope proteins (both single E2
and E1E2 heterodimer) were shown to be much more efficient than murine immune responses. Rabbit immunization with E2 protein
caused formation of antibodies to several highly conserved linear B-epitopes of this protein as well as to the N-terminal
fragment of the hypervariable region HVR1. Epitopes in the CR2 region were determined for the first time. There was cross-reactivity
between the N-terminal fragment of the protein E2 hypervariable region HVR1 and the octapeptide fragment of the protein E1
conserved region CR1, which shared four identical amino acid residues.
Full-text · Article · Jun 2009 · Biochemistry (Moscow) Supplement Series B Biomedical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed
co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA–Aub complex is potentially
able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of β-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases β-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which
are accessible for translation.
Full-text · Article · Apr 2009 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Penetration of a virus into a host cell comprises the first step of the viral life cycle. Blockage of this process can stop or prevent the rise of the infection. In order to develop substances that show directed blocking activity, one should know which host cell and viral molecules are involved in the reciprocal recognition and interaction leading to the virus entry into the cell. This review is devoted to the problems of the identification of cell outer membrane molecules that participate in the hepatitis C virus binding and its transfer inside the cells. The putative role of these molecules as hepatitis C virus receptors and coreceptors in the beginning and development of the infection is discussed.
Full-text · Article · Sep 2008 · Biochemistry (Moscow) Supplement Series B Biomedical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Heparan sulphate is one of the candidate receptors for hepatitis C virus (HCV). Envelope glycoproteins of HCV have been proposed to be responsible for recognition and binding with cell receptors. They are characterized by great genetic polymorphism. In this study the mapping of regions with glycosaminoglycan-binding properties within HCV envelope proteins has been undertaken. We prepared a set of overlapping peptides corresponding to conserved regions of these envelope proteins and analysed them by solid phase heparin-binding assay. The search for established glycosaminoglycan-binding motifs in the HCV envelope proteins showed the absence of the sites corresponding to the glycosaminoglycan-binding patterns in consensus sequence. We identified one highly conserved and two less conserved heparin-binding sequences within the envelope protein E2 based on solid phase assay results. We did not find any differences in binding efficiency of these peptides with heparin, heparan sulphate or dextran sulphate. Our data supported the specific association between HCV envelope protein E2 and cell surface glycosaminoglycans. We hypothesize that identified regions from E2 can contribute to HCV binding to cell surface glycosaminoglycans.
Full-text · Article · Dec 2005 · Journal of Viral Hepatitis
[Show abstract][Hide abstract] ABSTRACT: Development of computer methods in molecular biology and fast growth of microbial genomics data enabled new approach based on selecting in silico antigenic components to design vaccine constructs. It is expected that application of this technology will eliminate side effects of new vaccines and reduce the time consumption and financial expenses. The bioinformatics methods of sequence analysis are used to reveal the most prospective proteins or protein fragments of infectious agents as candidates for vaccine design. In these studies the specialized molecular immunology databases are widely used. The new approach ("Reverse vaccinology") could help in designing vaccines against diseases where traditional methods are not successful, e.g. when the viral genome reveals the extreme variability and permanent changes of antigenic properties that make difficulties for selection of molecular targets for medicines and candidate vaccines. A number of informational resources are already designed to collect and provide genomic data on certain microbes or viruses. The peculiarity of such resources is presentation of data, characterizing the different genomic variants of the same infectious agents. These structural data coupled with information on functional/immune features and software tools have to compose basis for constructing a new generation of vaccines against "common" and new infections such as AIDS, Hepatitis C, and SARS. The approaches published in literature, as well as the authors' original results are discussed.
Full-text · Article · Apr 2005 · Current Computer - Aided Drug Design
[Show abstract][Hide abstract] ABSTRACT: With the modern molecular biology techniques, it has been possible to detect, isolate and clone biological macromolecules, which could be used as immunogenes in artificial vaccine constructs. In the post-genomic era, the prospective immunogenic components are searched using bionformatic tools and proteomic technologies. Today it is quite realistic to combine the artificial vaccine constructs from the preselected molecular components. Existing computational methods are able to detect the potential immunogenes in genomic sequences, predict their characteristics and subcellular location. The set of methods is designed to predict the T- and B-epitopes that can be used as components of minimal vaccine constructs. The variety of systems for production and delivery of vaccines are developed and tested. These include transgenic plants, bacterial and viral vectors, DNA molecules etc. Several informational resources provide free access to molecular immunology data and deliver services on prediction of antigenic features. Several artificial vaccines have alredy been launched, but much more preparations are under preclinical and clinical trials. Computer-aided design of vaccines may significantly decrease time and costs required for their development. Modern bioinformatic technologies are now employed for discovery of more effective and potent vaccine.
[Show abstract][Hide abstract] ABSTRACT: With the modern molecular biology techniques, it has been possible to detect, isolate and clone biological macromolecules, which could be used as immunogenes in artificial vaccine constructs. In the post-genomic era, the prospective immunogenic components are searched using bionformatic tools and proteomic technologies. Today it is quite realistic to combine the artificial vaccine constructs from the preselected molecular components. Existing computational methods are able to detect the potential immunogenes in genomic sequences, predict their characteristics and subcellular location. The set of methods is designed to predict the T- and B-epitopes that can be used as components of minimal vaccine constructs. The variety of systems for production and delivery of vaccines are developed and tested. These include transgenic plants, bacterial and viral vectors, DNA molecules etc. Several informational resources provide free access to molecular immunology data and deliver services on prediction of antigenic features. Several artificial vaccines have already been launched, but much more preparations are under preclinical and clinical trials. Computer-aided design of vaccines may significantly decrease time and costs required for their development. Modern bioinformatic technologies are now employed for discovery of more effective and potent vaccine.
[Show abstract][Hide abstract] ABSTRACT: Forty-eight overlapping octapeptides covering highly conservative regions of E1 and E2 hepatitis C virus (HCV) envelope proteins were synthesized and tested by ELISA against different groups of sera obtained from HCV-infected patients. All sera from patients with acute infection, except a single case of serum reactivity with the region HINRTALN, were nonreactive with any peptide. Sera obtained from chronic patients reacted with 12 peptides from five selected regions. Two immunodominant B epitopes were found, one being the precisely mapped antigenic site RMAWDM positioned inside the earlier shown immunodominant epitope from E1, and the second site, PALSTGLIH from E2, detected for the first time. New minor antigenic site was determined as PTDCFRKH from E2. We found only minor seroreactivity for one of the putative sites involved in CD81 binding, PYCWHYAP.
Full-text · Article · Jun 2002 · Journal of Viral Hepatitis