Protein surface shielding agents in protein crystallization.

The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds.

Biophysical aspects of cancer--electromagnetic mechanism.

Hypothesis of coherent vibration states in biological systems based on nonlinear interaction between longitudinal elastic and electric polarization fields with metabolic energy supply was formulated by Frohlich. Conditions for excitation of coherent states and generation of electromagnetic fields are satisfied in microtubules which form electrical polar structures. Numerical models are used for analysis of Frohlich's vibration states in cells. Reduction of activity and of energy production in mitochondria, and disintegration of cytoskeleton structures by phosphorylation on the pathway of cancer trasformation can diminish excitation of the Frohlich's vibration states and of the generated electromagnetic field, which results in disturbances of the interaction forces between cells. Interaction forces between cancer cells may be smaller than interaction forces between healthy cells and cancer cells as follows from numerical models. Mechanism of malignity, i.e. local invasion, detachment of cancer cells, and metastasis, is assumed to depend on the electromagnetic field.

The fission yeast ortholog of eIF3a subunit is not functional in Saccharomyces cerevisiae.

The Schizosaccharomyces pombe eIF3a ortholog (SpeIF3a) was shown to be unable to substitute for S. cerevisiae eIF3a (SceIF3a) in its essential function in the initiation of translation. Overproduction of SpeIF3a altered the distribution of SceIF3a but formation of the endogenous eIF3 complex was not affected. SpeIF3a was found to be more tightly bound to S. cerevisiae ribosomes than SceIF3a and other eIF3 subunits (eIF3g, eIF3i, eIF3j). The host cells displayed aberrant morphology and altered chitin deposition. SpeIF3a probably competes with SceIF3a for binding to either ribosomes or yet to be identified substrates.

Preparation of human recombinant kinesin heavy chain and epitope mapping of its structural domains.

The isolation of the cDNA sequence encoding the human neuronal kinesin (a force-generating motor protein which transports various membrane organelles along microtubules in an ATP-dependent manner) heavy chain (nKHC) and the construction of expression vectors to produce the full-length nKHC and its domains in Escherichia coli is described. By tuning up the conditions for the expression of nKHC, a sufficient amount of the soluble protein intragenously tagged with 6xHis tag was obtained and purified by nickel chromatography. The recombinant structural domains of nKHC, including the motor domain (FKHC1--amino acids 1-330), the microtubule binding domain (FKHC2--amino acids 174-315) and the coiled-coil stalk domain (FKHC3--amino acids 331-906) were used to determine the epitope location for monoclonal antibodies KN-01, KN-02, and IB II raised against different kinesin heavy chains. The antibodies were shown to recognize epitopes located in the stalk domain of nKHC and represent thus useful probes for this domain.

Colocalization of cortical microtubules and F-actin in Dipodascus magnusii using confocal laser scanning microscopy.

Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin. A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei. In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex. In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites. Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network. The actin system of D. magnusii consisted of patches and filaments. Patches were found to accumulate at the tips of growing cells. Bands of fine actin filaments were usually observed before F-actin rings were established. A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells. Cells with developing septa showed medial F-actin discs associated at both sides with microtubules. Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D. magnusii.

The W303 genetic background affects the isw2 delta mutant phenotype in Saccharomyces cerevisiae.

We performed detailed phenotypic analysis of the isw2 delta strains of the W303 genetic background and compared its results with those obtained previously in BY-derived genetic background. Shmoolike morphology was observed in the isw2 delta strain of alpha-mating type of the BY strains, but not in its W303-derived counterpart. On the other hand, derepression of a-specific genes in the isw2 delta (MAT alpha) strain was observed in both genetic backgrounds, although to a different extent. Unlike in BY-derived strain hyperactivation of the Ras2/cAMP pathway reduced invasiveness of the isw2 delta strain (MAT alpha) of the W303 background. Sensitivity to Calcofluor White indicating a cell wall-integrity defect was significantly increased in the isw2 delta strains of the W303 background in contrast to BY-derived strains. Our data indicate that the effects of the isw2 deletion strongly depend on the background in which the deletion, is made.

Dual function of eIF3j/Hcr1p in processing 20 S pre-rRNA and translation initiation.

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.

Rpg1p/Tif32p, a subunit of translation initiation factor 3, interacts with actin-associated protein Sla2p.

The yeast two-hybrid system was used to screen for proteins that interact in vivo with Saccharomyces cerevisiae Rpg1p/Tif32p, the large subunit of the translation initiation factor 3 core complex (eIF3). Eight positive clones encoding portions of the SLA2/END4/MOP2 gene were isolated. They overlapped in the region of amino acids 318-550. Subsequent deletion analysis of Sla2p showed that amino acids 318-373 were essential for the two-hybrid protein-protein interaction. The N-terminal part of Rpg1p (aa 1-615) was essential and sufficient for the Rpg1p-Sla2p interaction. A coimmunoprecipitation assay provided additional evidence for the physical interaction of Rpg1p/Tif32p with Sla2p in vivo. Using immunofluorescence microscopy, Rpg1p and Sla2p proteins were colocalized at the patch associated with the tip of emerging bud. Considering the essential role of Rpg1p as the large subunit of the eIF3 core complex and the association of Sla2p with the actin cytoskeleton, a putative role of the Rpg1p-Sla2p interaction in localized translation is discussed.

Phospholipase C is involved in kinetochore function in Saccharomyces cerevisiae.

The budding yeast PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C. Here, we present evidence that Plc1p associates with the kinetochore complex CBF3. This association is mediated through interactions with two established kinetochore proteins, Ndc10p and Cep3p. We show by chromatin immunoprecipitation experiments that Plc1p resides at centromeric loci in vivo. Deletion of PLC1, as well as plc1 mutations which abrogate the interaction of Plc1p with the CBF3 complex, results in a higher frequency of minichromosome loss, nocodazole sensitivity, and mitotic delay. Overexpression of Ndc10p suppresses the nocodazole sensitivity of plc1 mutants, implying that the association of Plc1p with CBF3 is important for optimal kinetochore function. Chromatin extracts from plc1Delta cells exhibit reduced microtubule binding to minichromosomes. These results suggest that Plc1p associates with kinetochores and regulates some aspect of kinetochore function and demonstrate an intranuclear function of phospholipase C in eukaryotic cells.

Rpg1p, the subunit of the Saccharomyces cerevisiae eIF3 core complex, is a microtubule-interacting protein.

The essential gene RPG1/TIF32 of Saccharomyces cerevisiae encodes the 110-kDa subunit of the translation initiation factor 3 (eIF3) core complex. In this study, the Rpg1p-specific monoclonal antibody PK1/1 was used to analyse the cellular distribution of Rpg1p by epifluorescence and confocal laser scanning microscopy (CLSM). In budded cells, a portion of Rpg1p was obviously co-localised with microtubules. In addition, CLSM revealed an accumulation of Rpg1p in a patch at the very end of cytoplasmic microtubules reaching the bud tip. A punctate fluorescence pattern was typical for separated unbudded cells. Distribution of Rpg1p was confirmed using a strain expressing exclusively a hemaglutinin-tagged version of Rpg1p. In nocodazole-treated cells, the pattern of the PK1/1 staining was disturbed. No staining was observed in Rpg1p-depleted cells. In vitro experiments revealed that Rpg1p was specifically co-immunoprecipitated with alpha-tubulin from the yeast cell free extract and this observation was further supported by showing that Rpg1p co-sedimented with hog brain microtubules. We conclude that Rpg1p is a microtubule-interacting protein that indicates an interesting connection between the translation initiation machinery and cytoskeleton in yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae gene ISW2 encodes a microtubule-interacting protein required for premeiotic DNA replication.

A molecular genetic characterization of the ORF YOR304W (ISW2), identified in a screen of a yeast lambdagt11 library using a monoclonal antibody that reacts with a 210 kDa mammalian microtubule-interacting protein, is presented in this paper. The protein encoded by the ORF YOR304W is 50% identical to the Drosophila nucleosome remodelling factor ISWI and is therefore a new member of the SNF2 protein family and has been recently entered into SDG as ISW2. Although not essential for vegetative growth, we found that the ISW2 gene is required for early stages in sporulation. The isw2 homozygous deletant diploid strain was blocked in the G(1) phase of the cell cycle, unable to execute the premeiotic DNA replication and progress through the nuclear meiotic division cycle. ISW2 expression from a multicopy plasmid had the same effect as deletion, but ISW2 expression from a centromeric plasmid rescued the deletion phenotype. In vegetatively growing diploid cells, the Isw2 protein was preferentially found in the cytoplasm, co-localizing with microtubules. An accumulation of the Isw2 protein within the nucleus was observed in cells entering sporulation. Together with data published very recently by Tsukiyama et al. (1999), we propose a role for the Isw2 protein in facilitating chromatin accessibility for transcriptional factor(s) that positively regulate meiosis/sporulation-specific genes.

The Saccharomyces cerevisiae HCR1 gene encoding a homologue of the p35 subunit of human translation initiation factor 3 (eIF3) is a high copy suppressor of a temperature-sensitive mutation in the Rpg1p subunit of yeast eIF3.

The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast. In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain. Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis. The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature. In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found. We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex.

Microelectronic sensors for measurement of electromagnetic fields of living cells and experimental results.

Microelectronic sensors are used for measurements of electromagnetic fields generated by synchronized cultures of yeast cells. Cold sensitive mutant tub2-401 of Saccharomyces cerevisiae is used. The measured electromagnetic signals in the frequency range from 8 to 9 MHz are compared with evolution of the reassembled microtubules. The detected signals peak in the time interval 25-30 min and 45-60 min after the release of the cells from the restrictive to the permissive temperature. The first maximum corresponds to the stage when the mitotic spindle is formed and binds chromatids. The second maximum is measured when the processes of anaphase A and of anaphase B take place.

Rpg1, the Saccharomyces cerevisiae homologue of the largest subunit of mammalian translation initiation factor 3, is required for translational activity.

Eukaryotic initiation factor 3 (eIF3) consists of at least eight subunits and plays a key role in the formation of the 43 S preinitiation complex by dissociating 40 and 60 S ribosomal subunits, stabilizing the ternary complex, and promoting mRNA binding to 40 S ribosomal subunits. The product of the Saccharomyces cerevisiae RPG1 gene has been described as encoding a protein required for passage through the G1 phase of the cell cycle and exhibiting significant sequence similarity to the largest subunit of human eIF3. Here we show that under nondenaturing conditions, Rpg1p copurifies with a known yeast eIF3 subunit, Prt1p. An anti-Rpg1p antibody co-immunoprecipitates Prt1p, and an antibody directed against the Myc tag of a tagged version of Prt1p co-immunoprecipitates Rpg1p, demonstrating that both proteins are present in the same complex. A cell-free translation system derived from the temperature-sensitive rpg1-1 mutant strain becomes inactivated by incubation at 37 degreesC, and its activity can be restored by the addition of the Rpg1-containing protein complex. Finally, the rpg1-1 temperature-sensitive mutant strain shows a dramatic reduction of the polysome/monosome ratio upon shift to the restrictive temperature. These data show that Rpg1p is an authentic eIF3 subunit and plays an important role in the initiation step of translation.

RPG1: an essential gene of saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase.

In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G1 phase. We characterized a novel gene, RPG1, which is also involved in this phase. RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein. Elutriated or alpha-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature. The cells remained unbudded and neither grew nor duplicated DNA. rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G1 cells after a shift to a non-permissive temperature. Similarly, the asynchronous rpg1-1 cells accumulated in G1 at the non-permissive temperature, but mother and daughter cells did not separate. A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum. Our data show that Rpg1p is required for passage through the G1 phase and may be involved in growth control. Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3.