Modification of hydrophobic sorbents by cobalt chloride in order to concentrate low molecular polar organic substances from the air for subsequent gas chromatographic determination.

The article presents a new method of modification of hydrophobic sorbents. To improve sorption pre-concentration of polar organic compounds in the air analysis, these sorbents are coated with cobalt chloride. This modification increases retention volume of lower alcohols by 5-10 fold as compared to that of unmodified sorbents and solves the problem of gas-chromatographic determination at 1-2 ppb (micrograms/m(3)) by using the most common flame ionization detector. It should be noted that the modification of hydrophobic sorbents by cobalt chloride has little influence on their porosimetry parameters (specific surface area, proportions of meso- and micropores) and modified sorbents are capable of retaining hydrophobic nonpolar and weakly polar analytes as well as original unmodified sorbents. Thus, a fairly simple procedure leads to a large positive effect.

[The birth and death of genes].

Duplications of DNA regions with subsequent divergence of the duplicated copies by mutations is traditionally considered to be the major mechanism of the new genes appearance. After duplication, only a small fraction of the paralogs remains unchanged, while most copies are converted into pseudogenes or acquire new functions as a consequence of either subfunctionalization or neofunctionalization events. In some cases, certain regions of duplicated copies can combine with each other, giving rise to functionally new genes. Analysis of the primate genomes revealed a burst of segmental duplications in apes. It was demonstrated that some of these duplications include genes that are specifically duplicated only in human. Genome sequencing, followed by transcriptome analysis, enabled the identification of transcribed pseudogenes in mammalian genomes, that contradicts with the traditional view of pseudogenes as inactive copies of functioning genes.

[Modification of [PSI+] prion properties by the combination of amino acid changes within Sup35 protein N-domain].

[PSI+] prion is an amyloid isoform of a release factor Sup35p (eRF3). The structure of these protein aggregates remains unclear despite a long term history of prion amyloids investigations. The N-terminal domain of Sup35p (which is responsible for a propagation of prion) shapes superpleated beta-structure, according to modern concepts. Recently we constructed five double mutations within SUP35 sequence encoding the N-terminal prion-forming domain and investigated properties of mutant proteins. Mutations sup35-M1 (YQ46-47KK) and sup35-M2 (QQ61-62KK) lead to [PSI+] prion loss, while other mutant alleles (sup35-M3 QQ70-71KK; sup35-M4 QQ80-81KK; sup35-M5 QQ89-90KK) maintained prion. For the detail analysis of effects of mutant alleles on Sup35p aggregation we characterized propagation and properties of [PSI] prion in yeast strains bearing different mutant allele combinations. The data obtained have refined a supposed organization of beta-sheets forming by different regions of Sup35p prion-forming domain within amyloid. Also we obtained evidences that mutant sup35-M2 and sup35-M4 alleles change structure of prion aggregates. The prion destabilization by these mutations possibly is connected with decrease of heteroaggregate fragmentation by chaperones.

[The identification of Saccharomyces cerevisiae genes leading to synthetic lethality of prion [PSI+] with Sup45 mutations].

Previously, we proposed a test system allowing to perform search for genes that influence the properties of the Sup35 and Sup45 protein. This test is based on the phenomenon of lethality of diploids that combine mutations in SUP45 gene with [PSI+] prion. Lethality of this combination depends on the type of sup45 mutation, and the properties of the prion. [PSI+] variant, which is a strong suppressor ([PSI+]s), showing synthetic lethality with all the nonsense mutations and some missense sup45 mutations in the heterozygote state. The presence of extra copies of a gene under test that affects the phenotypic manifestation of prion [PSI+] or translation termination factors properties, leads to the increase or decrease in diploid lethality. Gene library screening using this test system allowed us to establish the effect of ten fragments of genomic DNA of yeast on synthetic lethality. Deletion analysis of these regions has led to the identification of the HLJ1 and TEF2 genes, as affecting Sup35 protein prionization and/or the efficiency of translation termination.

[The influence of UPF genes on the severity of SUP45 mutations].

Eukaryotic cells possess special mechanism of the degradation of mRNAs containing premature termination codons (PTCs)--nonsense-mediated mRNA decay (NMD) pathway. In yeast Saccharomyces cerevisiae, the activity of this pathway depends on the recognition of the PTC by the translational machinery and interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins. Previously we have shown that decreasing of eRF1 amount causes an impairment of NMD. Here we show that deletion of either UPF1 or UPF2 increased viability of sup45 mutants, while effect of UPF3 deletion is allele-specific. Two-hybrid data have shown that aa 1-555 of eRF1 participate in interaction with Upf1. Deletion of each UPF gene leads to allosuppresson of ade1-14 mutation without changing eRF1 amount. Depletion of Upf1 does not influence synthetic lethality of sup45 and prion [PSI+]. It is possible that the absence of Upf1 (or its activator Upf2) leads to more effective formation of the translation termination complex and, consequently, increased viability of cells containing mutant termination factors.

[Overexpression of genes encoding tRNA(Tyr) AND tRNA(Gln) improves viability of nonsense mutants in SUP45 gene in yeast Saccharomyces cerevisiae].

The variety of mechanisms providing viability of organisms bearing nonsense-mutations in the essential genes is unknown at present. In yeast Saccharomyces cerevisiae nonsense-mutants containing premature stop-codon in mRNA of the essential SUP45 gene were obtained. These strains are viable in the absence of mutant suppressor tRNA, therefore it is supposed that there are alternative mechanisms providing nonsense-suppression and mutants viability. Analysis of transformants obtained by transformation of strain bearing nonsense-mutant allele of SUP45 gene with multicopy yeast genomic library revealed three genes encoding wild type tRNA(Tyr) and four genes encoding wild type tRNA(Gln) that improve nonsense-mutants viability. Moreover, overexpression of these genes leads to the increase in the amount of full-length eRF1 protein in cell and compensates nonsense-mutants sensitivity to high temperature. Probable mechanisms of tRNA(Tyr) and tRNA(Gln) influence on the increase of viability of nonsense-mutants in SUP45 gene are discussed in this work.

[Mutations in the Sup35 gene impairs degradation of mRNA containing premature stop codons].

Eukaryotic cells possess special mechanism of the degradation of mRNAs containing premature termination codons (PTCs)--nonsense-mediated mRNA decay (NMD) pathway. In yeast Saccharomyces cerevisiae the activity of this pathway depends on the recognition of the PTC by the translational machinery and interaction of translation termination factors eRF1 (Sup45) and eRF3 (Sup35) with Upfl, Upf2 and Upf3 proteins. Previously we have shown that decreasing of eRF1 amount causes an impairment of NMD. Here we show that sup35 nonsense and missense mutations lead to accumulation of PTC-containing transcripts such as his7-1 mRNA and CYH2 pre-mRNA. Thus sup35 mutations do not only decrease translation fidelity but also influence mRNA stability. Remarkably, deletion of either UPF1 or UPF2 increased viability of sup35 mutants, while UPF3 deletion leads to decreased viability of sup35 mutants.

[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.

[Evaluation of the gene encoding translation termination factor eRF3 as a possible phylogenetic marker].

We have tested if a part of nuclear gene GSPT2 encoding N and M domains of translation termination factor eRF3b could be applied as a new molecular marker, using order Rodentia as a model group. The proposed fragment cannot be used as a phylogenetic marker at intrageneric level because of low variability within families and impossibility to resolve relationships in family Cricetidae. However, this part of GSPT2 gene allows to divide higher taxa reliably. Phylogenetic relationships between families established using the proposed molecular marker mainly correspond with contemporary conceptions. The new marker indicates close relationship of genus Acomys with family Gerbillidae in agreement with other molecular data but opposing to morphological ones. Thus the part of gene GSPT2 encoding N and M domains of eRF3b protein can be applied as an adequate phylogenetic marker in placental mammals at family and higher taxonomic levels. Also it can be used while solving controversial questions of phylogeny and taxonomy.

[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.

[Increased tRNA concentration in yeast containing mutant termination translation factors eRF1 and eRF3].

Earlier we have characterized strains bearing mutations in essential genes SUP45 and SUP35 of yeast S. cerevisiae, encoding translation termination factors eRF1 and eRF3 respectively. In the present work nonsense-mutants on genes SUP45 and SUP35 have been compared by a level of eight tRNA: tRNATyr, tRNAGln, tRNATrp, tRNALeu and tRNAArg (previously described as potentially suppressor tRNA), and also tRNAPro, tRNAHis and tRNAGly. We have not revealed preferable increase in amount of natural suppressor tRNA. The majority of the investigated mutations leads to increase in a level of all investigated tRNA. The mechanisms providing viability of nonsense-mutants on essential genes SUP45 and SUP35 are discussed.

[Characterization of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1]. / Kharakteristika missens-mutatsii v gene SUP45 drozhzhei Saccharomyces cerevisiae, kodiruiushchem faktor terminatsii translatsii eRF1.

Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1-eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.