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Structural and functional characteristics of the yeast red pigment (product of polymerization of N1-(beta-D-ribofuranosyl)-5-aminoimadazole), isolated from adel 1 mutant cells of Saccharomyces cerevisiae, its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N1-methyl-5-aminoimadazole in vitro) has been obtained. Products of in vitro polymerization were identified using mass spectrometry. The ability of these pigments to inhibit amyloid formation using insulin fibrils was compared. The entire compounds studied were able to interact with amyloids and inhibit their growth. Electron and atomic force microscopy revealed a common feature inherent in the insulin fibrils formed in presence of these compounds--they were merged into conglomerates that were more stable and resistant to the effects of ultrasound in comparison with insulin aggregates grown without pigments. We speculate that all these compounds can cause coalescence of fibrils, partially block their loose ends and, thereby, inhibit the attachment of new monomers to growing fibrils.

The effect of the yeast red pigment, the result of polymerization of AIR, and of its low molecular weight derivative (presumably devoid of phosphoribosyl moiety) on the formation of amyloid fibrils in vitro was studied. Both the red pigment and its derivative, the result of acid hydrolysis of the original pigment, were shown to diminish the intensity of amyloid bound Thioflavine T fluorescence. Correlation between the decrease of the intensity of Thioflavine T fluorescence and the concentration of both forms of the red pigment was demonstrated. Both forms were also able to compete with Thioflavine T for amyloid fibrils. Electron microscopy permitted to visualize a drop of fibril size in the case of red pigments presence during their formation.

A new approach: comparative analysis of proteins of the pellets of crude cell lysates of isogenic strains of Saccharomyces cerevisiae differing by their prion composition permitted to identify a large group of prion-associated proteins in yeast cells. 2D-electrophoresis followed by MALDI-analysis of a recipient [psi-] strain and of [PSI+] cytoductant led to identification of 35 proteins whose aggregation state responded to a shift of prion(s) content. Approximately half of these proteins belonged to functional groups of chaperones and enzyme involved in glucose metabolism. Notable were also proteins involved in translation, in oxidative stress response and in protein degradation. The data obtained are compared with the results of other groups who used other approaches to detecting proteins involved in prion aggregates.

Amyloid bound thioflavine T fluorescence was studied in the lysates of yeast strains carrying mutations in genes ADE1 or ADE2 and accumulating red pigment, a result of polymerization of aminoimidazoleribotide (an intermediate of adenine biosynthesis). The fluorescence is drastically enhanced in the case of cells grown in media containing high concentration of adenine (100 mg/l) that blocks accumulation of red pigment. Blocks at first stages of purine biosynthesis de novo also impede red pigment and lead to the same effect on thioflavine fluorescence. At the same time induction of mutations in genes ADE1 or ADE2 in originally white prototrophic strains leads to considerable drop of fluorescence. A fraction of protein polymers was studied by agarose gel electrophoresis and this permitted to conclude that lowering of fluorescence intensity is indeed connected with the decrease of amyloid amount in cells accumulating red pigment. Model experiments with insulin fibers demonstrate that red pigment binds fibrils and blocks their interaction with Thioflavine T. 2D-electrophoretic comparison of pellet proteins of red and white isogenic strains, followed by MALDI, allowed identification of 23 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins, involved in glucose metabolism, closely corresponding to prion-dependent proteins characterized in our previous work. We suppose that, binding amyloid fibrils, red pigment hinders formation of prion aggregates and also, blocking fibril contact with chaperones, impedes prion propagation.

An attempt was made at estimating the overall amyloid content of yeast cells by treating crude cellular lysates with thioflavin T, the agent specifically staining amyloid fibrils. We demonstrated that overproduction of the yeast chaperone Hsp104p, as well as GuHCI treatment of the [PSI+] cells led both to elimination of the [PSI+] factor and to a stable decrease of the overall amyloid content estimated by intensity of fluorescence (IF) of the thioflavin T. At the same time, overexpression of gene SUP35, coding the protein prionizable to [PSI+], led to generation of [PSI+] clones with higher IF of thioflavin T. Cytoduction in the crosses involving PSI factor leads to considerable enhancement of IF; cytoductants with the nucleus of the recipient [psi-] strain not only got [PSI+] factor from the donor strain but also increased their amyloid content. In these model experiments all treatments modifying one of the yeast prions, [PSI+] factor, led to a predictable shift of IF of thioflavin T that behaved like a cytoplasmic hereditary determinant. The data obtained show that IF of thioflavin T staining gives reliable estimates of cellular amyloid content and that mitotically stable shift of IF after a battery of treatments modifying cellular prion set provides quantitative estimate of the input of prionizable protein molecules to the amyloid pool. The combination of thioflavin staining and prionotropic treatments applied here can be possibly used for future attempts of checking yeast strains for cryptic prions.

A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.

In this work, studies on the phenomenon of concealed heterokaryosis that we previously detected in the saccharomycetes yeast strains were continued. New approaches to high effectiveness of isolation of cytoductants carrying the concealed nucleus were implemented, and the composition of individual concealed heterokaryons, zygotic clones, and the first zygotic buds was analyzed by a micromanipulator. The relationship between a delay in the growth of the parental strain (a potential donor of the concealed nucleus) and a decline in the frequency of the appearance of concealed heterokaryons (HKC) was observed. It is assumed that different replication rates of two nuclei of the heterokaryon probably underlie the appearance of HKC. A drastically decreased level of replication of one of the parental nuclei may be connected with the fact that binuclear buds appear extremely rarely and give rise to the rapidly "purified" progeny consisting of cells carrying the second nucleus with normal replication. A lack of the phenotype allows rare binuclear cells to persist as concealed heterokaryons. HKC may be detected only when cells of either parental type are isolated on the corresponding selective media.

In this work, the studies on the previously detected phenomenon of concealed heterokaryosis in Saccharomyces cerevisiae were continued. In genetic and Southern blotting experiments, one of the nuclei in the heterokaryon was shown to be active (capable of division and ensuring the corresponding cell phenotype), whereas the other was not expressed until the heterokaryotic clone was transferred to the medium selective for this concealed nucleus. Moreover, the concealed nucleus was able to assume the active state after fusion with the second parental nucleus. It was analyzed whether the nuclei with new marker combinations occurring in meiosis can behave as exceptional nuclei. Tetrad analysis of hybrids carrying the kar1 mutation in their nuclei revealed the relatively high percentage of exceptional tetrads (more than 10%). One spore in these tetrads usually formed diploid cells capable of sporulation. The presented data of genetic and molecular biological studies testify in favor of the assumption that abnormal spores contain two nuclei, which form an "illegitimate" hybrid after fusion. An extraneous spore (termed x) has usually a genotype close to that of one of the spores in this tetrad. Thus, it was assumed that the additional DNA replication round occurs in the absence of cell division during one of meiotic divisions. Results of cytological analysis conducted by the method of specific DNA staining confirmed the existence of exceptional tetrads, one spore of which contains two nuclei.

A phenomenon discovered earlier, cryptic heterokaryosis in Saccharomyces yeast, has been further investigated. A phenotypically silent nucleus in a yeast cell may resume its expression after fusion with another parental cell. The resulting hybrid is capable of sporulation. By the growth of a cytoductant with an expressing nucleus from one parent and a silent nucleus from the other parent on suitable selective media, the silent nucleus can be activated. The presence of deletion or insertion mutations in several genes in YPH strains allows nuclei of the YPH type to be traced not only genetically but also by blotting.

The yeast strain YPH857 carrying multiple genetic markers was shown to segregate clones that had the pleiotropic suppression phenotype. The phenotype was designated Ppsu+. This suppression involves deletion alleles of the TRP1 and HIS3 genes and an insertion in the URA3 gene. Unlike the original YPH857 culture that carries an unidentified mutation of resistance to cycloheximide, the Ppsu+ clones exhibited a decreased level of resistance to this inhibitor of protein synthesis. In addition, they have a lower mating ability and can produce asci on a standard medium for sporulation. A comparative analysis of total DNA from the YPH857 strain and Ppsu+ segregants by Southern blotting provided evidence for the presence of an extraneous nucleus in these segregants. Ppsu+ strains were shown to contain wild-type alleles, apart from deletion and insertional alleles typical for the YPH857 strain. Moreover, they contain the 2 microns DNA of the Scp3 type with deletion of one of the two EcoRI and HpaI recognition sites (whereas the 2 microns DNA of YPH857 belongs to the Scp1 type) and exhibit heterogeneity with respect to the presence of one EcoR1 recognition site in the gene of 5S ribosomal RNA. It was supposed that the "cryptic" nucleus belongs to a strain of low viability and can survive as an unexpressed DNA in a small fraction of cells. Nuclei from the cryptic and YPH857 strains can be fused at a low rate to yield Ppsu+ cells capable of sporulation. In certain cases, a Ppsu+ clone may be heterogeneous: some of its cells contain nuclei in an unfused heterokaryotic state. This assumption has been confirmed by selecting Cyhr colonies with the original YPH857 genome among Ppsu+ clones on the cycloheximide-containing medium.

Heterokaryons capable of segregating clones of different phenotypes were obtained in saccharomycetes yeast. Clones expressing phenotypic traits encoded by the nuclear genome of one parent and the mitochondrial genome of another were shown to contain cells that carried the second phenotypically silent nucleus. The cryptic nucleus can be uncovered by growing these "cytoductants" on the corresponding medium. The use of strains carrying an insertion in the URA3 gene and the endogenic plasmid, 2 microns DNA, made it possible to detect this nucleus by blot hybridization.

Recent data concerning cloning and sequencing of mdr genes involved in multiple drug resistance in higher eukaryotes are reviewed. Structures of ABC-superfamily members, including the mdr products as well as mechanisms of their superproduction at various levels are considered. The possible role of MDR-transporter in normal tissues and various approaches to overcoming the MDR phenotype are discussed. Non-P-glycoprotein mechanisms of drug resistance capable to modify MDR phenotype and applications of mdr in biotechnology are provided.

The review of yeast endogenous 2 micron plasmid and of some 2 micron plasmid based vectors is devoted to structure--function relationships with a particular emphasis on the stable replication control mechanisms. Some original data on possible novel plasmid encoded proteins are provided.

Inheritance of the mutant phenotype of respiratory deficience in RD-50 strain was studied. The deficience can be restored, giving respiratory sufficience, in crosses with rho0 testers. The "restorable" phenotype of the mutant, named RDc+, was shown to be determined by a nuclear pet-like mutation (pet50). The restorable RDc+ phenotype is stabilized in the presence of the pet50 allele, but can remain as such in the presence of the wild-type allele PET50. Restoration also takes place in cytoductants with the nucleus of kar partner. In order to explain the behaviour of the mitochondrial mutation mit50, we suppose it to be a microdeletion, capable of reversion, due to integration of a putative mt episome. Some features of the nuclear mutation pet50, particularly, its segregation in mitotic progeny of some revertants to respiratory competence point to its peculiarity. We suppose the mutation pet50 to be an insertion into the chromosomal PET50 gene. This insertion may be excised, remaining within the cell in the free state for some time, and then either eliminate or reintegrate into the chromosome.

A number of manifestations of mitochondrial DNA instability have been reviewed. Differences in organization of mitochondrial genomes of different origin have been regarded as well as variability concerning the genetic code. Examples of molecular heterogeneity of mtDNA and among them insertions and optional introns in Saccharomyces cerevisiae are given. Specific mutations in ascomycets and higher plants have been discussed as an aspect of instability since they cause the appearance of mitochondrial plasmids and episomes. One can regard the rate of mtDNA evolution particularly the high frequency of molecular rearrangement as connected with the fact that some of its regions behave as "egoistic" DNA. According to the Doolittle-Crick concept phenotypical selection always supports any useful function of that DNA, emerging by chance. Therefore we admit that some of the optional insertions into mt genes in S. cerevisiae have the adaptive function. It is also possible that in the course of evolution some higher plants "have learned" to use the DNA's ability to generate plasmids and episomes in order to create new means of gene activity regulation.

The plasmid mutation AntR determining multiple resistance to antibiotics--tetracycline and cycloheximide in Saccharomyces cerevisiae was earlier obtained and genetically characterized. In this work we describe experiments on cytoduction and transformation, proving the localization of this mutation in the yeast 2 mu DNA. As a result of cotransformation of the sensitive cells carrying a double mutation in the gene LEU2 with the yeast vector marked by LEU2 and 2 mu DNA obtained from the yeast AntR mutant, the Leu+ AntR clones were selected. Though the primary co-transformans contain both plasmids in an unlinked state, we managed to get clones in which the markers AntR and LEU2 were linked. The putative recombinant molecules were cloned in Escherichia coli and then introduced into the yeast recipient cells, differing by the presence of the endogenous 2 mu DNA. Retransformation of cir0 cells results in the appearance of the clones in which LEU2 and AntR markers segregate together. Thus, the result of cotransformation and selection in vivo is that the mutation of multiple resistance was included into the yeast vector plasmid, presumably, in its 2 mu part.

The Bam HI fragment of rat liver mtDNA was used for construction and analysis of the cloning vehicles for prokaryota and eukaryota. Transformation of mutant bacterial cells deficient in DNA polymerase I polA with recombinant pBR-mtBA DNA was shown previously. Now the transformation of bacterial cells with recombinant pmt Tn9 DNA was observed. The Bam HI-A fragment of animal mtDNA may be used as vehicle replicon not only in bacterial cells but also in yeasts. The recombinant molecule was constructed from plasmide DNA YJpI which contained yeast chromosomal LEU2 gene and Bam HI-A mtDNA fragment.

The mitochondrial mutation of the resistance to tetracycline (Tr) is shown to have one-parent inheritance. The polarity of recombination between the gene Tr and other mitochondrial mutations also depends on the alleles controlling the mating type as well as in another nuclear gene i, suppressing Tr-resistance. The "arrest" of Tr-transmission in alpha-haploid or in a haploid with gene-suppressor induces the process of mitochondrial recombination.

We propose a method for isolation of enhancer-like sequences from a yeast genomic library. The method was used to identify DNA inserts capable of increasing bacterial bla gene expression when located downstream from the transcription initiation site. Plasmids carrying different fragments of one such insert were used to localize the enhancer-like function on a 1.2 kb fragment.

Most Leu- clones of yeast transformants (cir0, pJDB219) can stabilize the replication of 2 micron-vectors with REP3, the stability obtained being comparable to the one for the standard cir0 strain. One of the Leu- clones was used to isolate a plasmid with Rep 1.2 functions ("Rep-helper plasmid"). The plasmid was shown to carry a partially active LEU2 gene by transforming both E. coli and S. cerevisiae to Leu+ phenotype. A restriction analysis performed demonstrated that the Rep-helper plasmid has lost approximately 1.9 kb compared to the parent pJDB219, deletion and rearrangement having taken place at the bacterial and 2 mem components boundary. The Rep-helper plasmid carrying host strains allows to quantify the REP3 function on different 2 microns vectors. Some but not all cir+ stabilized vectors show greater stability in Rep-helper strains compared to the standard cir0 ones. Manipulating the Rep-helper plasmid level, by selecting for Leu+ phenotype, stabilized REP3 +/- plasmid p3030, but mostly destabilizes REP3+ plasmid YEp13HIS3.

Nuclear suppression of the mitochondrial mutation of tetracycline resistance (Tr) is found. The suppressor mutation (i) is monogenous and recessive. Besides phenotypical suppression of mitochondrial resistance to tetracycline, it causes "the arrest" of the Tr plasmagene transmission. The effect of the suppressor is specific.