[Relationship between Type I and Type II Template Processes: Amyloids and Genome Stability].

Classical views of hereditary mechanisms consider linear nucleic acids, DNA and RNA, as template molecules wherein genetic information is encoded by the sequence of nitrogenous bases. The template principle embodied in the central dogma of molecular biology describes the allowed paths of genetic information transfer from nucleic acids to proteins. The discovery of prions revealed an additional hereditary mechanism whereby the spatial structure is transmitted from one protein molecule to another independently of the sequence of nitrogenous bases in their structural genes. The simultaneous existence of linear (type I) and conformational (type II) templates in one cell inevitably implies their interaction. The review analyzes the current data confirming the idea that protein amyloid transformation may influence the genome stability and considers potential mechanisms of interactions between type I and type II template processes. Special attention is paid to the joint contribution of the two process to tumor "evolution" and the mechanisms of genome destabilization due to amyloid transformation of proteins in Alzheimer's and Parkinson's diseases and Down syndrome.

Distinct Mechanisms of Phenotypic Effects of Inactivation and Prionization of Swi1 Protein in Saccharomyces cerevisiae.

Prions are proteins that under the same conditions can exist in two or more conformations, and at least one of the conformations has infectious properties. The prionization of a protein is typically accompanied by its functional inactivation due to sequestration of monomers by the prion aggregates. The most of prions has been identified in the yeast Saccharomyces cerevisiae. One of them is [SWI+], a prion isoform of the Swi1 protein, which is a component of the evolutionarily conserved chromatin remodeling complex SWI/SNF. Earlier, it was shown that the prionization of [SWI+] induces a nonsense suppression, which leads to weak growth of the [SWI+] strains containing mutant variants of the SUP35 gene and the nonsense allele ade1-14UGA on selective medium without adenine. This effect occurs because of [SWI+] induction that causes a decrease in the amount of the SUP45 mRNA. Strains carrying the SWI1 deletion exhibit significantly higher suppression of the ade1-14UGA nonsense mutation than the [SWI+] strains. In the present study, we identified genes whose expression is altered in the background of the SWI1 deletion using RNA sequencing. We found that the ade1-14UGA suppression in the swi1Δ strains is caused by an increase in the expression of this mutant allele of the ADE1 gene. At the same time, the SUP45 expression level in the swi1Δ strains does not significantly differ from the expression level of this gene in the [swi-] strains. Thus, we have shown that the phenotypic effects of Swi1 prionization and deletion are mediated by different molecular mechanisms. Based on these data, we have concluded that the prionization of proteins is not only unequal to their inactivation, but also can lead to the acquisition of novel phenotypic effects and functions.

EVALUATION OF EFFECTIVENESS OF SYNCHRONIZATION METHODS OF CELL DIVISION IN YEAST SACCHAROMYCES CEREVISIAE.

Synchronization of cell division in cultures of yeast Saccharomyces cerevisiae is widely used in research on the regulation of gene expression and biochemical processes in eukaryotes at different stages of the cell cycle. Here, we compare the efficiency of modern most commonly used methods to achieve and assess the degree of synchronization of cell division in yeast. Block-and-release methods with alpha-factor, hydroxyurea, nocodazole, cdc28-4 mutation are described in detail with practical notes.

Amyloids: from Pathogenesis to Function.

The term "amyloids" refers to fibrillar protein aggregates with cross-ß structure. They have been a subject of intense scrutiny since the middle of the previous century. First, this interest is due to association of amyloids with dozens of incurable human diseases called amyloidoses, which affect hundreds of millions of people. However, during the last decade the paradigm of amyloids as pathogens has changed due to an increase in understanding of their role as a specific variant of quaternary protein structure essential for the living cell. Thus, functional amyloids are found in all domains of the living world, and they fulfill a variety of roles ranging from biofilm formation in bacteria to long-term memory regulation in higher eukaryotes. Prions, which are proteins capable of existing under the same conditions in two or more conformations at least one of which having infective properties, also typically have amyloid features. There are weighty reasons to believe that the currently known amyloids are only a minority of their real number. This review provides a retrospective analysis of stages in the development of amyloid biology that during the last decade resulted, on one hand, in reinterpretation of the biological role of amyloids, and on the other hand, in the development of systems biology of amyloids, or amyloidomics.

[From Chromosome Theory to the Template Principle].

The template principle has originated from the chromosome theory of inheritance and claims to be the universal paradigm of modern biology. It considers the mechanisms of inheritance and different types of variability from a unified standpoint. The type I template processes (TP I) operate with linear templates: DNA and RNA. TP II deal with spatial, or conformational, templates of protein nature. TP II are based on variation and reproduction of the spatial structure of proteins and do not affect their primary structure. They are involved in many pathological and adaptive processes in living systems. The universal properties of TP I, ambiguity and repair (correction), are common to all three stages of each template process-initiation, elongation, and termination. These properties are typical for TP II as well. Ambiguity and correction at stages of initiation and termination of TP are prerequisites for the regulation of template processes. The variation in this regulation underlies the complexity and progressive evolution of living-systems.

Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae.

The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read-through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage-dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains' tolerance of a number of transition metal salts.

[The template principle: paradigm of modern genetics].

The idea of continuity in living systems, which was initially developed in mid-19th century, reached its peak in 1928 thanks to N.K. Koltsov, who proposed the template principle in chromosome reproduction. The determination of genetic functions of nucleic acids and the advent of molecular genetics led to F. Crick's statement of the central dogma of molecular biology in 1958. This dogma became a contemporary version of the template principle (templates of the first order). The discovery of "protein inheritance" underlay the notion of steric or conformational templates (second order) for reproducing conformation in a number of proteins. The template principle supplemented by this notion claims to be the main paradigm of modern genetics.

[Yeast prions as a model of neurodegenerative infectious amyloidoses in humans].

Several neurodegenerative diseases (so-called age-related diseases) in humans are associated with development of protein aggregates--amyloids. Prion diseases--kuru, Kreutzfeldt-Jakob and Gerstmann-Straussler-Sheinker diseases, fatal familial insomnia, etc.--are examples of infectious amyloidoses. A model system for investigation of mechanisms of amyloidogenesis and of its infectious nature had been developed as a result of yeast prion discovery. The existence of a prion network as an interaction of different prions identified in yeast is being confirmed recently as an interaction of different anyloids in humans. The potential danger of amyloidoses is conditioned by the very structure of almost all proteins containing fragments capable to be organized as beta-sheets, which lead to their aggregation being exposed. Meanwhile, there are several well-defined examples of the adaptive value of amyloid aggregates: cytoplasmic incompatibility factor in Podospora anserina, spider silk, cytoplasmic stress granules in mammals, prion form of CPEB protein responsible for the neuron activity in Aplisia, etc. These facts should be taken into consideration when seeking antiamyloid drugs. Discovery of protein inheritance in lower eukaryotes modifies our knowledge of the template principle significance in biology and adds a concept of conformational templates (II order templates) involved in reproduction of the three-dimensional structure of the supramolecular complexes in the cell.

Participation of translesion synthesis DNA polymerases in the maintenance of chromosome integrity in yeast Saccharomyces cerevisiae.

We employed a genetic assay based on illegitimate hybridization of heterothallic Saccharomyces cerevisiae strains (the α-test) to analyze the consequences for genome stability of inactivating translesion synthesis (TLS) DNA polymerases. The α-test is the only assay that measures the frequency of different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously. All these events are manifested as illegitimate hybridization and can be distinguished by genetic analysis of the hybrids and cytoductants. We studied the effect of Polζ, Polη, and Rev1 deficiency on the genome stability in the absence of genotoxic treatment and in UV-irradiated cells. We show that, in spite of the increased percent of accurately repaired primary lesions, chromosome fragility, rearrangements, and loss occur in the absence of Polζ and Polη. Our findings contribute to further refinement of the current models of translesion synthesis and the organization of eukaryotic replication fork.

[The role of metabolic activation of promutagens in the genome destabilization under pheromonal stress in the house mouse (Mus musculus)].

The hypothesis on a relationship between the high frequency of mitotic disturbances in bone marrow cells and the change in the activity of the S9 liver fraction containing promutagen-activating enzymes under olfactory stress in the house mouse Mus musculus has been tested. For this purpose, the effect of the pheromone 2,5-dimethylpyrazine on the frequency of mitotic disturbances in mouse bone marrow cells has been measured by the anaphase-telophase assay. The Ames test using Salmonella typhimurium has been employed to compare the capacities of the S9 liver fractions from stressed and intact mice for activating the promutagen 2-aminofluorene. It has been demonstrated that the increased frequency of mitotic disturbances in bone marrow cells induced by the pheromonal stressor in male house mice is accompanied by an increased promutagen-activating capacity of the S9 liver fraction. The model system used in the study allowed the genetic consequences of the exposure to the olfactory stressor to be estimated and the possible mechanisms of genome destabilization to be assumed.

[Phenotypic manifestation and trans-conversion of primary genetic material damages considered in the alpha-test on the yeast Saccharomyces cerevisiae].

Primary (spontaneous and externally induces) damages to genetic material frequently lead to heritable changes (gene mutations, chromosome aberrations and nondisjunction), which may cause cancer inherent and inborn diseases. It is suggested that primary damages may affect a phenotype until they are repaired or become mutations during inaccurate repair The alpha-test on the yeast Saccharomyces cerevisiae can answer the fundamental questions as the nature of primary damages that can be phenotypically manifested, their occurrence, conversion to each other and repair or conversion to heritable changes in genetic material.

[The origin of novel proteins by gene duplication: what is common in evolution of the color-sensitive pigment proteins and translation termination factors].

The review is discussing a role of duplications in evolution, including events from genes to genomes duplications. The important role of duplications is their participation in the block-modular reorganizations leading to a combination of fragments from various genes. Examples of gene duplications leading to occurrence of proteins with divergent functions are shown. For instance, human and Old World monkey trichromatic vision has arisen due to consecutive duplications of the genes encoding color-sensitive pigment proteins, and their subsequent divergence. Many proteins participating in regulation and the control of protein synthesis have resulted from series of gene duplications that has led to origin of modern translation elongation and termination factors. It is supposed, that proteins participating in the control of newly synthesized mRNA quality have arisen also due to duplication of the genes encoding ancient translation elongation factors. Their subsequent divergence has led to the origin of proteins with the new properties, but already unable to participate in the control of translation.

[The influence of mutations at ATG triplets of the open reading frame SUP35 on viability of the yeast Saccharomyces cerevisiae].

The open reading frame SUP35 encoding the translation termination eRF3 factor vital to life contains three ATG codons (ATG1, ATG124, and ATG254). Previously, other authors detected two SUP35 transcripts: a major one that corresponds to the full-length open reading frame and a minor transcript that corresponds to the 3' terminal site of SUP35 starting at the third ATG codon (ATG254). In this work, mutations at triplets ATG1, ATG124, and ATG254 were obtained as well as double mutations, which combine the point mutation in one of three ATG triplets and a deletion at the site for binding with the transcription factor Abf1 within the SUP35 (sup35-deltaAbf1) promoter. The influence of these mutations on the yeast viability was analyzed. Mutations at triplets ATG124 and ATG254 did not affect yeast viability in their own right or in the background of deletion sup35-deltaAbf1. Mutation sup35-AGG1 (ATG1-->AGG) causes the lethal effect in cells grown on media containing glucose as the sole source of carbon. The replacement of glucose by galactose, or histidine starvation, partially restore the viability of sup35-AGG1 mutants, but not that of double mutants sup35-deltaAbf1,AGG1. The restoration of sup35-AGG1 mutant viability under these conditions can be explained by either the appearance (or enhancement) of the production of short peptides synthesized on the mRNA triplets SUP35 AUG124 and AUG254, or by the enhanced production of the full-length SUP35 transcript coupled with translation initiation from the noncanonical AGG1 codon. These data confirm that the expression of gene SUP35 at the transcription and(or) translation level is regulated by environmental conditions.

[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.

["Do not lose the winning games". To the 100th anniversary of M.E. Lobashev].

Mikhail E. Lobashev (1907-1971), Head of the Department of Genetics and Breeding with the Leningrad (now, St. Petersburg) State University from 1957 to 1971, had traveled a long way from a homeless to an Honored Scientist of the Russian Federation. Lobashev was among the discoverers of chemical mutagenesis in Drosophila; he pioneered in connecting the mutation process and the repair of genetic material and developed the concept of signal inheritance. Through the entire Great Patriotic War, he served with the field forces, and defended his doctoral dissertation on the physiological hypothesis of mutation process in 1946 on the return to the University. In 1948, Mikhail Efimovich was discharged from the University, where he was the Dean of the Biological Faculty, as a Morganist. On his return to the University in 1957, Lobashev devoted all his energies to the restoration of genetic education in this country, wrote the first domestic genetic textbook in the post-Lysenko period, organized the research at the Department of Genetics and Breeding, and created the scientific school, whose representatives are still successfully working in the field of genetics.

[Viable nonsense mutants for the SUP45 gene in the yeast Saccharomyces cerevisiae are lethal at increased temperature].

Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination eRFI factor in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a truncated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37 degrees C. When grown under nonpermissive conditions (37 degrees C), cells of nonsense mutants sup45-104, sup45-105. and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.

[Conservation of the MC domains in eukaryotic termination factor eRF3].

Eukaryotic translation termination employs two protein factors, eRF1 and eRF3. Proteins of the eRF3 family each consist of three domains. The N and M domains vary in different species, while the C domains are highly homologous. The MC domains of Homo sapiens eRF3a (hGSPT I), Xenopus laevis eRF3 (XSup35), and Mus musculus eRF3a (mGSPTI) and eRF3b (mGSPT2) were found to compensate for the sup35-21(ts) temperature-sensitive mutation and lethal disruption of the SUP35 gene in yeast Saccharomyces cerevisiae. At the same time, strains containing the MC domains of the eRF3 proteins from different species differed in growth rate and the efficiency of translation termination.

[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.