Search for functional amyloid structures in chicken and fruit fly female reproductive cells.

We conducted a cytological search for amyloid structures in female reproductive cells of Gallus gallus domesticus and Drosophila melanogaster. We have shown that the amyloid-specific dye, Thioflavin S, but not Congo red, stains some cytoplasmic and even nuclear structures in chicken ovaries. In fruit fly eggs both Thioflavin S and Congo red specifically stain eggshell structures such as micropyle, dorsal appendages and pillars. Moreover, these structures, when stained with Congo red, demonstrate birefringence in polarized light, which is a characteristic feature of all classical amyloids. Our data show that female reproductive cells during evolution began to use amyloid fibrils to form various functional structures necessary for development under certain environmental conditions.

Amyloid properties of the yeast cell wall protein Toh1 and its interaction with prion proteins Rnq1 and Sup35.

Amyloids are non-branching fibrils that are composed of stacked monomers stabilized by intermolecular ß-sheets. Some amyloids are associated with incurable diseases, whereas others, functional amyloids, regulate different vital processes. The prevalence and significance of functional amyloids in wildlife are still poorly understood. In recent years, by applying new approach of large-scale proteome screening, a number of novel candidate amyloids were identified in the yeast Saccharomyces cerevisiae, many of which are localized in the yeast cell wall. In this work, we showed that one of these proteins, Toh1, possess amyloid properties. The Toh1-YFP hybrid protein forms detergent-resistant aggregates in the yeast cells while being expressed under its own PTOH1 or inducible PCUP1 promoter. Using bacterial system for generation of extracellular amyloid aggregates C-DAG, we demonstrated that the N-terminal Toh1 fragment, containing amyloidogenic regions predicted in silico, binds Congo Red dye, manifests 'apple-green' birefringence when examined between crossed polarizers, and forms amyloid-like fibrillar aggregates visualized by TEM. We have established that the Toh1(20-365)-YFP hybrid protein fluorescent aggregates are co-localized with a high frequency with Rnq1C-CFP and Sup35NM-CFP aggregates in the yeast cells containing [PIN+] and [PSI+] prions, and physical interaction of these aggregated proteins was confirmed by FRET. This is one of a few known cases of physical interaction of non-Q/N-rich amyloid-like protein and Q/N-rich amyloids, suggesting that interaction of different amyloid proteins may be determined not only by similarity of their primary structures but also by similarity of their secondary structures and of conformational folds.

Prions and Non-infectious Amyloids of Mammals - Similarities and Differences.

Amyloids are highly ordered aggregates of protein fibrils exhibiting cross-ß structure formed by intermolecular hydrogen bonds. Pathological amyloid deposition is associated with the development of several socially significant incurable human diseases. Of particular interest are infectious amyloids, or prions, that cause several lethal neurodegenerative diseases in humans and can be transmitted from one organism to another. Because of almost complete absence of criteria for infectious and non-infectious amyloids, there is a lack of consensus, especially, in the definition of similarities and differences between prions and non-infectious amyloids. In this review, we formulated contemporary molecular-biological criteria for identification of prions and non-infectious amyloids and focused on explaining the differences between these two types of molecules.

Proteomic Analysis of Escherichia coli Protein Fractions Resistant to Solubilization by Ionic Detergents.

Amyloids are protein fibrils adopting structure of cross-beta spine exhibiting either pathogenic or functionally significant properties. In prokaryotes, there are several groups of functional amyloids; however, all of them were identified by specialized approaches that do not reveal all cellular amyloids. Here, using our previously developed PSIA (Proteomic Screening and Identification of Amyloids) approach, we have conducted a proteomic screening for candidates for novel amyloid-forming proteins in Escherichia coli as one of the most important model organisms and biotechnological objects. As a result, we identified 61 proteins in fractions resistant to treatment with ionic detergents. We found that a fraction of proteins bearing potentially amyloidogenic regions predicted by bioinformatics algorithms was 3-5-fold more abundant among the identified proteins compared to those observed in the entire E. coli proteome. Almost all identified proteins contained potentially amyloidogenic regions, and four of them (BcsC, MukB, YfbK, and YghJ) have asparagine- and glutamine-rich regions underlying a crucial feature of many known amyloids. In this study, we demonstrate for the first time that at the proteome level there is a correlation between experimentally demonstrated detergent-resistance of proteins and potentially amyloidogenic regions predicted by bioinformatics approaches. The data obtained enable further comprehensive characterization of entirety of amyloids (or amyloidome) in bacterial cells.

Sr33 & Sr35 gene homolog indentification in genomes of cereals related with Aegilops tauschii & Triticum monococcum.

Using bioinformatics analysis, the homologues of the genes Sr33 and Sr35 were identifed in the genomes of Triticum aestivum, Hordeum vulgare and Triticum urartu. It is known that these genes provide resistance to hightly virulent wheat stem rust races (Ug99). To identify important for resistance amino acid sites, the comparison of the founded homologues with the Sr33 and Sr35 protein sequences was performed. It was found that the sequences S5DMA6 and E9P785 are the closest homologues of RGA1e protein ­ a product of the Sr33 gene, and the sequences M7YFA9 (CNL-C) and F2E9R2 are the homologues of CNL9 ­ a product of the gene Sr35. It is assumed that the homologues of the genes Sr33 and Sr35, which derived from the wild relatives of wheat and barley, can provide resistance to various forms of a stem rust and can be used in the future breeding programs for wheat improvement.

[Prion-like determinant [NSI+] decreases expression of the SUP45 gene in Saccharomyces cerevisiae].

Previously, we described and characterized yeast non-chromosomal determinant [NSI+], possessing prion properties. This determinant causes a decrease in translation termination fidelity, which is phenotypically detectable as the nonsense suppression in the strains with decreased functional activity of eRF3 release factor. As a result of geneticscreen, we demonstrated that an increase in the expression of SUP45 encoding the eRF1 release factor (Sup45), masks, but does not eliminate nonsense suppression in the [NSI+] strains. In the present study, we first demonstrated the direct cause for the nonsense suppression in [NSI+] strains. We demonstrated that [NSI+] decreases the relative amounts of SUP45 mRNA that causes a decrease in the amounts of Sup45 protein that is detectable in the stationary growth phase. The data obtained suggest the structural protein of [NSI+] seems to be either a transcription factor or participates in the regulation of cellular mRNA stability.

[Interactions of [NSI+] determinant with SUP35 and VTS1 genes in Saccharomyces cerevisiae].

Previously we characterized [NSI+] determinant, that possesses the features of a yeast prion. This determinant causes the nonsense suppression in strains that bear different N-substituted variants of Sup35p, which is a translation release factor eRF3. As a result of the genomic screen, we identified VTS1, the overexpression of which is a phenotypic copy of [NSI+]. Here, we analyzed the influence of SUP35 and VTS1 on [NSI+]. We demonstrated nonsense suppression in the [NSI+] strains, which appears when SUP35 expression was decreased or against a background of general defects in the fidelity of translation termination. [NSI+] has also been shown to increase VTS1 mRNA amounts. These findings facilitate the insight into the mechanisms of nonsense suppression in the [NSI+] strains and narrow the range of candidates for [NSI+] determinant.

Inducing effect of PGRs on small regulatory si/miRNA in resistance to sugar beet cyst nematode.

Sugar beet cyst nematode Heterodera schachtii Schmidt is an economically important plant parasite of sugar beet in Ukraine. The pest control options are limited. Sugar beet cyst nematode resistant varieties are not available on the market. Carbamate and organophosphate pesticides have been banned due to the high toxicity. The problem is aggravated by continuously increasing of oilseed rape (which is suitable host for H. schachtii) growing area due to biofuel demands. Several studies' results indicate that PGRs have role in management of plant parasitic nematodes but for sugar beet it is not studied well. We had an objective- studying of the role of four compositional PGRs created based of avermectin in suppression of sugar beet cyst nematode population on sugar beet and oilseed rape caused by enhancing of endogenous si/miRNA complementary to H. schachtii mRNA. Laboratory study was conducted in 2011 with using method DOT-blot hybridization si/miRNA with mRNA and by testing inhibitory activity in cell free system protein biosynthesis. That was shown that application of the PGRs enhances sugar beet and oilseeds rape plant immune-protective properties and resistance against plant-parasitic nematode Heterodera schochtii through enhancement of synthesis of small regulatory si/miRNA related (complementary) to an mRNA structure of the parasitic organisms. As a result, translation of mRNA of the nematode is blocked and causes the mortality of plant parasite juveniles.

[Yeast chaperone Hspl04 regulates gene expression on the posttranscriptional level].

Yeast chaperon Hsp104 is known as a protein which is able to dissociate aggregates of the heat damaged proteins and prion aggregates into smaller pieces or monomers. In our work the effects of Hsp104 on the PrP-GFP and GFP proteins have been analyzed. The PrP-GFP protein forms the high molecular weight aggregates, whereas GFP is unable to aggregate in yeast cell. We have shown that Hsp104 regulates the amount of PrP-GFP and GFP in yeast cells and direction of chaperone action depends on promoter controlling production of these proteins. The overproduction of Hsp104 increases the amount of PrP-GFP and GFP proteins when the corresponding genes are under control of CUP1 promoter. In contrast, the overproduction of Hsp104 decreases the amount of PrP-GFP and GFP is case of their expression under control of GPD promoter. The effects of Hspl04 are not related with any changes in mRNA content of the genes under investigation and with ability of the proteins to form aggregates. Thus, the functions of this chaperon are not restricted by dissociation of the protein aggregates. Our data show that Hsp104 regulates the gene expression on the posttranscriptional level.

[Yeast prions, mammalian amyloidoses, and the problem of proteomic networks].

Prion proteins are infective amyloids and cause several neurodegenerative diseases in humans and animals. In yeasts, prions are expressed as cytoplasmic heritable determinants of a protein nature. Yeast prion [PSI], which results from a conformational rearrangement and oligomerization of translation termination factor eRF3, is used as an example to consider the structural--functional relationships in a potentially prion molecule, specifics of its evolution, and interactions with other prions, which form so-called prion networks. In addition, the review considers the results of modeling mammalian prion diseases and other amyloidoses in yeast cells. A hypothesis of proteomic networks is proposed by analogy with prion networks, involving interactions of different amyloids in mammals.

[Localization of cohesin complexes of polytene chromosomes of Drosophila melanogaster located on interbands]. / Lokalizatsiia kompleksov kogezii v politennykh khromosomakh Drosophila melanogaster sviazana s mezhdiskami.

The distribution of cohesin complex in polytene chromosomes of Drosophila melanogaster was studied. Cohesin is a complicated protein complex which is regulated by the DRAD21 subunit. Using immunostaining for DRAD21p, the cohesins were shown to be preferentially located in the interband regions. This specificity was not characteristic for puffs, where uniform staining was observed. The presence of a few brightly fluorescent regions (five to ten per chromosome arm) enriched with cohesin complexes was shown. Some of these regions had permanent location, and the others, variable location. No antibody binding was detected in the chromocenter. Immunostaining of interphase nuclei of neuroblasts revealed large cohesin formations. On the polytene chromosomes of D. melanogaster, the Drad21 gene was mapped to the chromocentric region (81) of the L arm of chromosome 3.

Evolutionary conservation of prion-forming abilities of the yeast Sup35 protein.

Saccharomyces cerevisiae prion [PSI ] is a self-propagating isoform of the eukaryotic release factor eRF3 (Sup35p). Sup35p consists of the evolutionary conserved release factor domain (Sup35C) and two evolutionary variable regions - Sup35N, which serves as a prion-forming domain in S. cerevisiae, and Sup35M. Here, we demonstrate that the prion form of Sup35p is not observed among industrial and natural strains of yeast. Moreover, the prion ([PSI + ]) state of the endogenous S. cerevisiae Sup35p cannot be transmitted to the next generations via heterologous Sup35p or Sup35NM, originating from the distantly related yeast species Pichia methanolica. This suggests the existence of a 'species barrier' in yeast prion conversion. However, the chimeric Sup35p, containing the Sup35NM region of Pichia, can be turned into a prion in S. cerevisiae by overproduction of the identical Pichia Sup35NM. Therefore, the prion-forming potential of Sup35NM is conserved in evolution. In the heterologous system, overproduction of Pichia Sup35p or Sup35NM induced formation of the prion form of S. cerevisiae Sup35p, albeit less efficiently than overproduction of the endogenous Sup35p. This implies that prion induction by protein overproduction does not require strict correspondence of the 'inducer' and 'inducee' sequences, and can overcome the 'species barrier'.

[Genetic effects of destabilizing selection for adaptively important traits in Drosophila melanogaster lines]. / Genetickie éffekty destabiliziruiushchego otbora pri selektsii po adaptivno vazhnym priznakam v liniiakh Drosophila melanogaster.

Related lines of Drosophila melanogaster selected for reproductive activity for more than 750 generations were studied. Results of the long-term selection experiment support D.K. Belyaev's concept of destabilizing selection. Selection for a behavioral trait (male mating activity) affected the intrinsic structure of the organism. Flies of the low-activity line (LA) exhibited numerous morphological, biochemical, and physiological alterations. Variation rate in the selected lines was accelerated; appearing mutations were often allelic and nonrandomly distributed over chromosomes. Selection was shown to mediate novel sources of variability, e.g., induction and repression of the hobo hybrid dysgenesis. In the process of selection, a genetically determined program of hobo transpositions in the genome was formed.

[Directed transpositions in the genome of the mobile hobo element in a long-term selected strain of Drosophila melanogaster]. / Napravlennye peremeshcheniia po genomu mobil'nogo élementa khobo v dlitel'no selektiruemoi linii na Drosphila melanogaster.

The object of our investigation was an inbred LA strain of Drosophila melanogaster. This strain was selected during more than 700 generations for low male mating activity and was characterized by complex abnormalities and the remarkably high level of spontaneous mutability. The last property is determined by hobo system of hybrid dysgenesis. LA strain's hobo activity potential in H-E system of hybrid dysgenesis comprise 30-40%. It may be drastically increased up to 70-80% through LA-strain chromosome isogenization in dysgenic crosses. In genomes of independent isogenic LA substrains, numerous directional transpositions of hobo element take place. In spite of it, the retention of hobo element insertion sites inherent in original LA persist. The obtained data open up a new possibilities to study the genome instability mechanisms.

[Directed character of genetic variation during long-term selection of Drosophila melanogaster strains for adaptively important characters]. / Napravlennyi kharakter geneticheskikh izmenenii pri dlitel'nom otbore linii Drosophila melanogaster po adaptivno vazhnym priznakam.

Related fly strains that were selected for differences in reproductive function for about 700 generations were studied. The low-active (LA) strain differs from other related and highly active strains of interest at a set of characters. Qualitative differences in the spectrum of viability mutations are retained in strains selected in opposite directions. Low-active strains carry a great number of deleterious mutations. Such mutations are rare in the related highly active strains. Moreover, about 50% of chromosomes in the highly active strains carry supervital mutations, which increase viability. Recombination analysis showed the presence of at least several viability mutations in each chromosome of the strains studied. Unlike highly active strains, the low-active ones are characterized by an extremely high spontaneous mutation rate. The conservation of genetic heterogeneity observed in long-term selected strains, as well as the high mutation rate, must be connected with regular changes in the spectrum of retrotransposons (MDG1, MDG3) and hobo elements characteristic of the strains studied. A special crossing system that induces a sharp increase in LA induction potential in the H-E system of hybrid dysgenesis was developed.

[A comparative analysis of the proteins from a cell culture and from field plants of the ecdysteroid producer Serratula coronata L]. / Sravnitel'nyi analiz belkov kletochnoi kul'tury i polevykh rastenii produtsenta ékdisteroidov Serratula coronata L.

Differences in the composition and amount of proteins synthesized in the cell culture and leaves of field plants Serratula coronata have been shown. They proceed from differences in intensity of synthesis of secondary metabolites, ecdysteroids, whose content in the cell culture is considerably lower.

[Cloning of tobacco DNA fragment with promoter properties in a transgenic plant]. / Klonirovanie fragmenta DNK tabaka, obladaiushchego svoistvami promotora v transgennom rastenii.

A selection system for isolating DNA sequences with transcription-promoting activity by their functioning in bacterial cells has been proposed. Tobacco nuclear DNA fragments were inserted in front of the promoterless neomycin 3'-phosphotransferase II (NPT-II) gene and promoter-like sequences were identified by their ability to restore NTP-II activity in E. coli cells. One of these recombinant plasmids was introduced in tobacco protoplasts by direct gene transfer and transformed calli were isolated by kanamycin selection. The NTP-II expression in regenerated transgenic plants were highest in root, slightly lower in stem and were practically absent in leaf. Sequence analysis of cloned segment showed the presence of conserved sequences essential for promoter activity in eukaryotic cells. A transcription start site was observed by S1 mapping. The size of protected fragments corresponds to the initiation of transcription 176 and 179 base pairs upstream the initiation codon in tobacco and 75 base pairs in E. coli.

[Isolation of a biologically active poly(A)+RNA from tissues enriched with polysaccharides]. / Poluchenie biologicheski aktivnoi poli(A)+RNK iz tkanei, obogashchennykh polisakharidami.

A special technique is developed for isolation of biologically functional poly(A)+RNA from higher plant cells to start with some procedures used to isolate RNA of the eucaryote cells. The technique includes cell disruption with a 4 M guanidine thiocyanate solution, hot phenol extraction of RNA following proteinase K digestion and methoxyethanol treatment to remove polysaccharides. Finally, poly(A)+RNA was purified by oligo(dT)-cellulose chromatography. Using this technique poly(A)+RNA preparations were obtained from French bean embryo axis, maize and sugar beet seedings, potato tubers and seedlings. It is shown that poly(A)+RNA isolated from the plant cells directs protein synthesis in the cell-free system. This RNA may be also used as a matrix for synthesis of cDNA in the reverse transcription reaction.

[Extraction of native preparations of mRNP-particles from the nuclei of kidney bean cells]. / Poluchenie nativnykh preparatov m-RNP-chastits iz iader kletok semian fasoli.

A soft technique is suggested for extracting the mRNP-particles from nuclei. It is based on the incubation of nuclei with ATP. Advantages of the technique, as compared with other described in literature, are as follows: contamination-free mRNP preparations without any DNP fragments are more homogeneous in respect to the CsCl gradient buoyant density and a higher percentage of poly(A)+-mRNP in the total quantity of RNP extracted from nuclei.

[A non-damaging procedure for DNA isolation from insect cell nuclei]. / Shchadiashchii metod vydeleniia DNK iz iader kletok nasekomykh.

A gentle procedure is described for DNA isolation from Galleria mellonella cell nuclei. Nuclei lysis was performed by SDS in final concentration of 3% which was followed by DNA extraction from chromatin by a stepwise rise of NaCl concentration up to 2.5 M. After removal of nuclei insoluble components by centrifugation the native DNA was purified using the hydroxyapatite column. The method proposed provides a higher yield of the native DNA as compared with the phenol-detergent method.