[Selection of DNA aptamers, specifically interacting with fibrillar form of the yeast Sup35 protein].

Single-stranded DNA aptamers interacting with fibers formed by the Sup35 protein of Saccharomyces cerevisiae were obtained by the SELEX procedure. Specificity of interaction with Sup35p is investigated for 10 from total of 40 selected aptamers. It is shown that 9 aptamers bind to fibrillar Sup35p and not to monomeric form of the protein. The rate of dissociation constant of aptamer-fiber complex varies from 0.1 to 1 microM for different aptamers. Selected aptamers can be used to study prionization of Sup35.

[Biological functions of amyloids: facts and hypotheses].

Amyloids are fibrous protein aggregates which arise due to polymerization of proteins accompanied by their conformational rearrangement and formation of a specific "cross-beta" structure. The interest to amyloids is caused by their relation to a vast group of human and animal diseases called amyloidoses. Some of these diseases caused by prions, a specific type of amyloids, are transmissible. Besides mammals, prion amyloids are described in lower eukaryotes, where they underlie non-chromosomal genetic determinants. Though in humans and animals amyloids are usually associated with pathologies, the increasing number of findings suggests that in some cases acquisition of amyloid or prion form by a protein may be of biological significance. Here, we summarize data on biological significance of prion and nonprion amyloids obtained in a wide range of species, from bacteria to mammals.

[Neurodegenerative amyloidoses: the yeast model].

More than 20 human diseases are related to protein misfolding which causes formation of amyloids, fibrillar aggregates of normally soluble proteins. Such diseases are called amyloid diseases or amyloidoses. Of them only prion diseases are transmissible. Amyloids of the prion type are described in lower eukaryotes. However, in contrast to mammalian prions, which cause incurable neurodegenerative diseases, prions of lower eukaryotes are related to some non-chromosomally inherited phenotypic traits. Here we summarize the results of studies of prions of the yeast Saccharomyces cerevisiae and of the use of yeast model for investigation of some human amyloidoses, such as prion diseases, Alzheimer's, Parkinson's, and Huntington's diseases.

[New approach to reveal genes that control cell wall biogenesis of the yeast Saccharomyces cerevisiae].

The Mcd4 protein of Saccharomyces cerevisiae is probably involved in addition of the phosphoethanolamine moiety to the first mannose residue of the glycosylphosphatidylinositol precursor(s). However, significance of this modification is unclear. Besides, functions of the MCD4 gene also is not completely clear, since mutations in this gene may have pleiotropic manifestations, which are not obviously related to the glycosylphosphatidylinositol biosynthesis. To clarify the functions of Mcd4p we have performed a search for genes whose mutations are lethal or semilethal in combination with the ssu21 mutation in MCD4. In total, we have isolated six mutations some of which cause sensitivity to SDS and/or calcofluor white. Genes which are able to complement two of these mutations were cloned. They were MNN9 which encodes protein involved in formation of outer chains of the N-linked glycans of secretory proteins and GWT1, encoding the protein of the endoplasmic reticulum involved in the glycosylphosphatidylinositol biosynthesis. The results obtained indicate that in both cases growth inhibition was caused by defect of cell wall biogenesis and alteration of folding of secretory proteins. Search for mutations that lethal in combination with the ssu21 is an effective approach to reveal genes involved in the control of cell wall biogenesis.

[Caffeine sensitivity of the yeast Saccharomyces cerevisiae MCD4 mutant is related to alteration of calcium homeostasis and degradation of misfolded proteins].

The MCD4 gene of Saccharomyces cerevisiae encodes a protein involved in glycosylphosphatidylinositol (GPI) biosynthesis. However, some mutations in the MCD4 gene have pleiotropic effects that may not be related to the defects in GPI anchor synthesis. The ssu21 mutation in the MCD4 gene studied here causes sensitivity to caffeine. The screen for multicopy suppressors of caffeine sensitivity caused by the ssu21 mutation revealed genes involved in aminoglycerophospholipid metabolism, unfolded protein response, and protein degradation. The suppressor effect of all isolated genes was enhanced by an increase in concentration of external calcium, which is consistent with the ability of caffeine to block calcium entry into the yeast cell. Obtained results indicate that caffeine sensitivity caused by the ssu21 mutation could be due to cytoplasmic accumulation of misfolded proteins destined for degradation.

[Yeast as a model for studying the prion and amyloid occurrence]. / Drozhzhi kak model' dlia izucheniia molekuliarnykh osnov vozniknoveniia prionov i amiloidov.

Recent data on the use of yeast as a model for studying the molecular basis of prion infection are summarized. In contrast to mammalian prions, which are related to incurable neurodegenerative diseases, yeast prions determine the appearance of non-chromosomally inherited phenotypic traits. Prions of yeast are structurally similar to amyloids of mammals and their replication involves not only growth, but also fragmentation of prion amyloid-like fibrils. In mammals the fragmentation should lead to an increase in infectious titer. The use of yeast for study of the mechanisms of human amyloidoses, development of new anti-prion drugs and search for new proteins with prion properties is described.

[A novel approach to isolation and functional characterization of genomic DNA from the methylotrophic yeast Hansenula polymorpha]. / Novyi podkhod k klonirovaniiu i funktsional'nomu analizu genomnoi DNK metilotrofnykh drozhzhei Hansenula polymorpha.

A novel approach to isolation and functional characterization of the Hansenula polymorpha genes basing on the use of two strains of different origin is described. One of these strains is better suited for the isolation of genomic DNA fragments, while the other is preferable for their functional analysis. Thirty three genomic sequences governing expression of a reporter protein have been isolated. Analysis of the sequence encoding a homolog of the Saccharomyces cerevisiae cofilin revealed two introns. Another isolated DNA fragment encoded a homolog of the S. cerevisiae V ps10p. Disruption of the corresponding gene resulted in secretion of a vacuolar protein, carboxypeptidase Y, into the culture medium.

[Prion phenomenon in medicine and biology]. / Fenomen prionov v meditsine i biologii.

The data obtained suggest that the fatal changes in brain tissue associated with the prion diseases, are initiated by a conformational rearrangement of constitutively expressed cellular protein PrP. Possible mechanisms of such a conversion of this protein are discussed. Existence of the proteins with the prion properties in low eukaryotes may determine the unusual mechanisms of the "protein" inheritance. A new experimental model for studying the proteins with the prion properties in the yeast Saccharomyces cerevisiae, is described.

[Fusion of glutathione S-transferase with the N-terminus of yeast Sup35p protein inhibits its prion-like properties]. / Sliianie glutation S-transferazy s N-kontsom belka Sup35p drozhzhei ingibiruet ego prionpodobnye svoistva.

The yeast Saccharomyces cerevisiae SUP35 gene that encodes the Sup35p protein homologous to the translation termination eRF3 factor of higher eukaryotes is essential to replication of the nonchromosomally inherited [psi+] determinant. The nonsense suppressor phenotype of this determinant was assumed to be dependent on a specific conformational state of the Sup35p protein; the transition to this state leads to partial inactivation of this protein. In terms of this hypothesis, the Sup35p protein can, like mammalian prions, induce its own specific conformation via protein-protein interactions in the newly synthesized Sup35p molecules; in this way, inheritance of the [psi+] phenotype is ensured in a series of cell generations. In recent years, this hypothesis has been experimentally verified. Allele substitution of the wild-type SUP35 gene by its chimeric GST-SUP35 version, which encodes the glutathione S-transferase sequence fused with the N end of Sup35p, was shown to cause elimination of the [psi+] determinant. The ability to eliminate [psi+] is a recessive trait, because fusions heterozygous for the GST-SUP35 allele did not lose this trait. Elimination of [psi+] seems to be caused by inability of the chimeric protein to bring about oligomerization. The obtained data indicate that the chimeric protein manifests attenuated terminating activity but can interact with the eRF1 translation termination factor encoded by the SUP45 gene.

[Structure and functional similarity of yeast Sup35p and Ure2p proteins to mammalian prions]. / Strukturnoe i funktsional'noe skhodstvo belkov Sup35p i Ure2p drozhzhei s prionami mlekopitaiushchikh.

The results of studies of yeast cytoplasmically-inherited determinants [psi] and [URE3] are summarized. The existence of tandem amino acid repeats in N-terminal regions of yeast Sup35p protein and in prions of higher eukaryotes is shown. The prion-like properties of yeast Sup35p and Ure2p proteins and a role of tandem amino acid repeats localized in N-terminal region of Sup35p protein in inheritance of [psi] determinants is discussed. The suggestion is made that the [psi+] status of yeast cell depends on the specific conformation of the N-terminal domain of Sup35p and that this protein can induce the specific conformational state of its N-terminal domain on newly synthesized Sup35p molecules via protein-protein interaction, thus representing a molecular basis of inheritance of [psi+] determinant. Other proteins containing amino acid repeats of similar type are considered and the suggestion is made that some of these proteins may show prion-like behavior.

[Ambiguity of translation: a eukaryotic version?]. / Neodnoznachnost' transliatsii: versiia éukariot?

In this review, the results of studies on genetic control of translation in the yeasts are considered and analyzed. The studies were performed on the system of the SUP35(SUP2) and SUP45(SUP1) genes. Based on data from genetic analysis, the study of protein synthesis in mutants at these genes, sequencing of both genes, deletion analysis of their functions, and the discovery of homologues of these genes in other eukaryotes, it may be assumed that the gene system under study is a specific evolutionarily conservative system for eukaryotes. This system not only controls translational fidelity, but also ensures integration of translation with other cellular processes.

[Deletion analysis of the SUP2 gene in Saccharomyces cerevisiae]. / Deletsionnyi analiz gena SUP2 drozhzhei Saccharomyces cerevisiae.

The sup2 mutations of the yeast Saccharomyces cerevisiae or plasmid-mediated amplification of the wild type SUP2 gene lead to suppression of different types of nonsense mutations. The Sup2 protein includes a C-terminal region homologous to elongation factor EF-1 alpha and an unique N-terminal region. The SUP2 is an essential gene. The functional role of different regions of the SUP2 gene was investigated, by deleting them without disruption of the reading frame. Such constructs were maintained in yeast on episomal or centromeric plasmids. It was shown that the region, homologous to EF-1 alpha is necessary for viability, while the remaining N-terminal part is nonessential. The region of the first 154 amino acids is necessary and sufficient for the suppressor effect, caused by plasmid-mediated amplification of the SUP2 gene.

[Comparative analysis of the structure of SUP2 genes in Pichia pinus and Saccharomyces cerevisiae]. / Sravnitel'nyi analiz struktury genov SUP2 drozhzhei Pichia pinus i Saccharomyces cerevisiae.

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. A SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of temperature-sensitive sup2 mutation of S. cerevisiae. Nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa exceeding the SUP2 protein of S. cerevisiae for 6 kDa. The SUP2 gene product of P. pinus is similar to the Sup2 protein of S. cerevisiae by its structure and includes a highly conservative (76%) C-terminal region homologus to EF-1 alpha and a lowly conservative N-terminal region. The relation between the evolutionary conservativity of different regions of the Sup2 protein and their functional significance is discussed.

[The relationship of cytoplasmic and mitochondrial protein synthesis in yeasts: the mapping of mitochondrial mutations neutralizing the exhibition of nuclear omnipotent suppressors]. / Sviaz' tsitoplazmaticheskogo i mitopkhondrial'nogo belkovogo sinteza u drozhzhei: kartirovanie mitokhondrial'nykh mutatsii, podavliaiushchikh proiavlenie iadernykh omnipotentnykh supressorov.

The genetic and physical mapping of mitochondrial mutations [CRD] neutralizing respiratory deficiency in sup1 and sup2 mutants was performed. The genetical methods demonstrated improbability of location of these mutations in genes coding for the enzymes of respiratory chain and for 21S rRNA. Southern-blot analysis has shown these mutations to be localized in the Hinc10 fragment of mitochondrial genome. This fact was interpreted as indication that var1 gene is affected by [CRD] mutations. Our results are in agreement with the hypothesis on the participation of sup1(2) proteins in mitochondrial translation.