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Biomedical subjects

S G Inge-Vechtomov

Publications and source records attributed to S G Inge-Vechtomov.

At least 19 recordsLinked to original sources

Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+].

The yeast non-Mendelian factor [psi+] has been suggested to be a self-modified protein analogous to mammalian prions. Here it is reported that an intermediate amount of the chaperone protein Hsp104 was required for the propagation of the [psi+] factor. Over-production or inactivation of Hsp104 caused the loss of [psi+]. These results suggest that chaperone proteins play a role in prion-like phenomena, and that a certain level of chaperone expression can cure cells of prions without affecting viability. This may lead to antiprion treatments that involve the alteration of chaperone amounts or activity.

Fungal Proteins

[Ambiguity of translation: a eukaryotic version?].

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.

Chromosome Deletion

Multicopy SUP35 gene induces de-novo appearance of psi-like factors in the yeast Saccharomyces cerevisiae.

Previously, we have shown that plasmid-mediated multiplication of Saccharomyces cerevisiae wild-type SUP35 gene leads to omnipotent suppression and is incompatible with psi-factor, which is an endogenous extrachromosomal suppressor. Here, we describe a frequent de-novo appearance of psi-like factors in mitotic progeny of yeast transformants containing multicopy SUP35 gene.

DNA, Fungal

Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein.

SUP35 is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1 alpha and a unique N-terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF-1 alpha-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi+] strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.

Fungal Proteins

Dosage-dependent translational suppression in yeast Saccharomyces cerevisiae.

The overexpression of SUP35 (SUP2) wild-type gene, caused by increase of its copy number, induces an omnipotent suppression similar to the phenotype of mutants for this gene. The effect of extra-SUP35 was detected for moderate or even low copy number. Moreover, overdosage of the fragment including only the 5'-flanking region and N-terminal 100 bp of protein-coding sequence of SUP35 leads to allosuppression. Multi-SUP35 gene was also incompatible with extrachromosomal suppressor factor psi, presumably because of a high level of mistranslation. The suppressor effect caused by overdosage of another gene, SUP45 (SUP1), is much lower and can be detected only for one construction which is derived from high copy number plasmid. Suppression induced by extra-SUP35 and especially by extra-SUP45 is affected by the cell environment. A model predicting that the balance of gene products is a key for regulation of translational fidelity is discussed.

DNA Replication

Conservative system for dosage-dependent modulation of translational fidelity in eukaryotes.

Variations in dosage of some genes can alter the level of translational fidelity. The Saccharomyces cerevisiae genes that act as dosage-dependent suppressors and/or modulators of suppression, are the following: some tRNA genes (for example, tRNA(Gln)) inducing readthrough by mispairing; genes coding for either translational elongation factor or other proteins taking part in translation; and some genes of unknown function. We suggest that the SUP35 protein is a factor which may play a major role in balance-dependent regulation of translational fidelity. Homologues of this genes have been identified in other yeast genera (Pichia), green algae (Chlamydomonas) and various animals including man. No homologies have been found in the polychaeta (Nereis) or in insects (Drosophila). Rates of evolution differ for two separate parts of the genes; the N-terminal part, which is important for ambiguous translation in Saccharomyces, is markedly variable in the organisms tested. However, the C-terminal part which is required for yeast viability has a common origin but a separate evolution from that of the EF-Tu protein family.

Animals

Structure comparison and evolutionary relations between elongation factors EF-Tu (EF-1 alpha) and SUP 2 proteins.

On the basis of high homology and structural similarity, three genes, SUP2 Saccharomyces cerevisiae, SUP2 Pichia pinus and GST1 Homo sapiens, might be considered as members of one family named SUP2. Comparison of the primary structure of SUP2 proteins and elongation factors EF-Tu(EF-1 alpha) from 19 different species was performed. It was found that SUP2 proteins bear more homology to eukaryotic elongation factor than to procaryotic EF-Tu, though the degree of sequence conservation in SUP2 proteins is smaller than in EF-1 alpha factors. The extensive phylogenetic analysis of SUP2 and EF-Tu(EF-1 alpha) genes was performed by means of 3 methods, 2 phenetic and one cladystic (maximal parsimony). The data support the close relation of SUP2 genes to other elongation factor genes.

Amino Acid Sequence

Interactions between chromosomal omnipotent suppressors and extrachromosomal effectors in Saccharomyces cerevisiae.

Chromosomal omnipotent suppressor mutations recovered in psi+ strains of Saccharomyces cerevisiae were brought into psi- cytoplasm. SUP46, SUP138 and SUP139 acted as dominant omnipotent suppressors in the psi- cytoplasm though their suppressor activity was substantially reduced. SUP46 and SUP138 conferred recessive thermosensitivity and antibiotic sensitivity in psi- cytoplasm as in psi+ cytoplasm. On the other hand, sup111 through sup115, which acted as recessive omnipotent suppressors in the psi+ cytoplasm, manifested no, or very low, suppressor activity in the psi- cytoplasm. They, however, still enhanced the efficiency of the SUP29 tRNA suppressor in psi- cytoplasm. A multicopy plasmid carrying the wild-type SUP35 gene enhanced the efficiency of sup111 in psi- cytoplasm.

Extrachromosomal Inheritance

[The effect of sterol metabolism in a model ecological system Drosophila-yeasts on the crossing-over in Drosophila].

The absence of sterols available for metabolism causes the death of Drosophila larva. Addition of suboptimal cholesterol doses to this medium allows the portion of larvae to survive. Sterol-deficient diet at the preimaginal stages leads to suppression of both spontaneous and high-temperature induced crossingover in Drosophila females. Two possible explanations for dependence of recombination process on sterol metabolism are suggested: 1) the shortage of precursor for ecdisons biosynthesis was the cause of discordance of meiotic events; 2) suppression of crossingover occurs, due to alteration of cell membrans' structure.

Animals

Phenotypic expression of primary lesions of genetic material in Saccharomyces yeasts.

"Illegitimate" mating of yeasts (alpha x alpha), either spontaneous or induced by uv light or ethyl methanesulfanate, in a selective system for "cytoduction" revealed that about 95% of cytoductants expressed their original (alpha) mating type. Inducing the mating by treating the recipient of cytoplasm with uv light reached two orders of magnitude. An additional copy of MAT alpha in the alpha recipient almost completely eliminated the effect, which means that nonheritable mating type changes observed are formally recessive and are localized within MAT alpha complex. About 1% of cytoductants obtained were nonmating types and some of them were identified as mat alpha l mutants. Radl8 mutant as a recipient showed a considerably elevated spontaneous frequency of illegitimate hybridization and cytoduction. The cytoductants also preserved the original mating type. These facts suggest that nonheritable changes of mating type are due to repairable primary (premutational) lesions in MAT alpha genetic material. The significance of these results for understanding the mechanism of nonheritable variability is discussed.

DNA Damage

[Modification changes of the genetic material in Saccharomyces yeasts].

The problem of mating-type switches in heterothallic yeast cells was investigated. In selective system for cytoduction in alpha x alpha crosses alpha-cytoductants were predominantly obtained. Thus matings in alpha x alpha crosses can proceed through non-heritable changes (modifications) of the mating type alpha----a. The frequency of alpha-cytoductants after UV-irradiation of the recipient cells exceeded the control value 50-90 times. The extra copy of MAT alpha dramatically decreased the frequency of cytoductants in alpha x alpha crosses, either spontaneously or after UV-irradiation. The rad18 recipient defective in postreplication repair had 70-times increased level of mating-type modifications, as compared with isogenic Rad+ strain. An explanation consistent with these data is that mating-type modifications are due to phenotypic expression of primary lesions of MAT alpha locus. Such lesions might be expressed as transient a-mating type. After the mating event, these lesions can be repaired or turned to true mutations within the MAT locus. In fact, approximately half of non-mating cytoductants from alpha x alpha crosses had the phenotype of mat alpha 2 mutants.

Chromosome Mapping

Nucleotide sequence of the SUP2 (SUP35) gene of Saccharomyces cerevisiae.

A nucleotide sequence of the yeast Saccharomyces cerevisiae omnipotent suppressor SUP2 (SUP35) gene is presented. The sequence contains a single open reading frame (ORF) of 2055 bp, which may encode a 76.5-kDa protein. A single transcript of 2.3 kb corresponding to a complete ORF is found. Analysis of codon bias suggests that the SUP2 gene is not highly expressed. The C-terminal part of the deduced amino acid sequence shows a high homology to yeast elongation factor EF-1 alpha, whereas the N-terminal part is unique for the SUP2 protein. The N terminus contains a number of short repeating elements and possesses an unusual amino acid composition. Analysis of the nucleotide and deduced amino acid sequences indicates that three additional proteins could possibly be expressed, two of which might be initiated on internal ATG codons and a third might be formed by alternative splicing. One of these proteins is supposed to be imported into mitochondria. Possible functions of the SUP2 gene product(s), especially its putative activity as a soluble factor controlling the fidelity of translation, are discussed.

Amino Acid Sequence

[Selective systems for obtaining recessive ribosomal suppressors in saccharomycete yeasts].

Recessive mutations only occurring in two genes (ribosomal suppressors sup1 and sup2) can be obtained using special selective system. We demonstrate that the absolute selectivity of the system is based on selection for simultaneous reversions to prototrophy in mutants requiring adenine and histidine in haploids marked by two different nonsense mutations--his7-1 (UAA) and ade1-14 (UGA, this being identified in the present study). In support to this conclusion, we developed an analogous system utilising his7-1 (UAA) and lys2-87 (UGA). The selectivity of the system is shown to be influenced both by the choice of nonsense alleles and by genotypic background.

Culture Media

Localization of possible functional domains in sup2 gene product of the yeast Saccharomyces cerevisiae.

Primary structures of yeast sup2 gene and polypeptide product coded by the gene are compared with the current nucleotide and amino acid sequence data base. The amino acid sequence of the sup2 product shows homology to elongation factors from different sources. Especially high homology is found in the regions, corresponding to conservative aminoacyl-tRNA- and GTP-binding domains, described in elongation factors and other proteins. The data obtained are discussed in relation to the functions of sup2 polypeptide product in protein synthesis.

Amino Acid Sequence

[Hybridization of cells of the same mating type in Saccharomyces yeasts].

The problem of mating-type switches in heterothallic yeast cells was investigated. 93% of non-mating hybrids were obtained in a X a crosses. The hybrids obtained in alpha X alpha crosses expressed alpha-mating type predominantly. Hybrids with no major rearrangements or loss of chromosome III were detected among these hybrids. In the selective system for cytoduction in a X a crosses the significant part of all cytoductants were alpha-maters, i.e. those originated through a----alpha switches. In alpha X alpha crosses alpha cytoductants were predominantly obtained either spontaneously or after UV-irradiation, though the frequency of cytoductants after UV-irradiation exceeded the control value several times. So, we developed the method for selection of mating-type "switchers" (a in equilibrium alpha), avoiding the diploid stage, and demonstrated the possibility of hybridization among the alpha-cells without hereditary changes at the MAT locus.

Alleles

[Effect of sterol metabolism in the yeast-Drosophila system on the frequency of radiation-induced aneuploidy in Drosophila melanogaster oocytes].

The influence of sterol metabolism upon mutagenesis in Drosophila melanogaster was investigated using ecological-genetic yeast - drosophila system. Sterol deficiency in the organism of Drosophila was caused by using the strain of Saccharomyces cerevisiae 9-2P712 with a mutation in the nysr1 locus which blocks synthesis of ergosterol as a nutrition substrate for flies. It was concluded that maintenance of females on the mutant yeast strain causes an increase of radiation-induced X-chromosome loss in mature oocytes. Resistance of oocytes to X-ray irradiation is restored, reaching the control level, when 0,1% cholesterol solution in 10% ethanol is added to the yeast biomass. The possible membrane and hormonal mechanisms of elevation of induced aneuploidy and the role of sterol metabolism in ensuring resistance of insects to damaging factors are discussed.

Aneuploidy