The product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, specifically functions in mitochondria.

We reported previously that the product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, belongs to a family of proteins that are involved in DNA repair and replication. The family includes S. cerevisiae proteins Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Here, we report that Din7p specifically affects metabolism of mitochondrial DNA (mtDNA). We have found that dun1 strains, defective in the transcriptional activation of the DNA damage-inducible genes RNR1, RNR2, and RNR3, exhibit an increased frequency in the formation of the mitochondrial petite (rho(-)) mutants. This high frequency of petites arising in the dun1 strains is significantly reduced by the din7::URA3 allele. On the other hand, overproduction of Din7p from the DIN7 gene placed under control of the GAL1 promoter dramatically increases the frequency of petite formation and the frequency of mitochondrial mutations conferring resistance to erythromycin (E(r)). The frequencies of chromosomal mutations conferring resistance to canavanine (Can(r)) or adenine prototrophy (Ade(+)) are not affected by enhanced synthesis of Din7p. Experiments using Din7p fused to the green fluorescent protein (GFP) and cell fractionation experiments indicate that the protein is located in mitochondria. A possible mechanism that may be responsible for the decreased stability of the mitochondrial genome in S. cerevisiae cells with elevated levels of Din7p is discussed.

Choline and halogen derivatives of CMA (9-oxo-10-acridine acetic acids) as tools for monitoring the interaction of the interferon inducers via a specific receptor.

New choline and halogen derivatives of CMA (9-oxo-10-acridine acetic acid) were investigated as interferon (IFN) inducers in mice and in the mouse bone marrow-derived macrophage cultures. Two of the choline derivatives, DMCMA and CSCMA, were active IFN inducers presumably because they were hydrolyzed so as to release CMA. The halogen analogues of CMA were inactive or weak IFN inducers in vivo and in vitro. On the contrary, the Br and I derivatives of CMA were potent inhibitors of IFN induction by CMA in vitro. The behavior of the agonists and antagonists of CMA suggests that the induction of interferon may occur indirectly via a specific CMA-receptor complex.

Restriction of PM2 supercoiled DNA by Hap II endonuclease.

Restriction of the covalently closed circular DNA from phage PM2 (CCC PM2 DNA) by Hap II endonuclease was studied under varying enzyme concentration. At low Hap II concentration, accumulation of the intermediate product, OC DNA, was observed at the early stages of the reaction. The resulting final mixture of restriction products consists of OC and L DNA, and their relative content depends on the concentration of the enzyme used. The affinity of the enzyme for the intact recognition site of the substrate in different conformational forms does not seem to be affected. Basically identical results were obtained with the two different CCC DNA used: PM2 and SV40 DNA.

Effect of temperature and 4,6'-diamidine-2-phenylindole on restriction of supercoiled Col E1 DNA by Eco RI endonuclease.

Supercoiled Col E1 DNA is split by Eco RI endonuclease at 37 degrees C without intermediate formation of open circular DNA. Accumulation of this restriction product is observed at low temperature. The fluorescent dye, 4,6'-diamidine-2-phenylindole (DAPI) inhibits restriction by Eco RI endonuclease. This effect is due to the DAPI:DNA rather than to the DAPI:Eco RI interactions.

Expression of small synthetic genes coding for hEGF, human epidermal growth factor, and CPTI II, serine proteinase inhibitor from Cucurbitacea, cloned in a novel expression/secretion vector in Saccharomyces cerevisiae.

Efficient synthesis of two small eukaryotic polypeptides of human and plant origin was carried out using a novel expression/secretion yeast vector, pYET. The yield was optimized in respect of the yeast strain, expression cassette construction, promoter regulation and culture conditions. Both cloned genes code for biotechnologically important proteins: human epidermal growth factor and a serine proteinase inhibitor from Cucurbitacea.

3' Noncoding sequences of the CTA 1 gene enhance expression of the recombinant serine protease inhibitor, CPTI II, in Saccharomyces cerevisiae.

Expression of the gene coding for the recombinant trypsin inhibitor, CPTI II, was enhanced tenfold when yeast transcription terminating sequences were added to the expression cassette of the pJK6 yeast vector. The yield was further increased about 20% in the BJ5464 yeast strain, defective in vacuolar proteases.

Synthesis, cloning and expression in Escherichia coli of the gene coding for the trypsin inhibitor from Cucurbita pepo.

A chemically synthesized gene coding for the serine proteinase inhibitor CPTI II was cloned in E. coli and its expression was investigated in cytoplasmic and secretion systems. Under all conditions investigated the biologically active form of the inhibitor was found only in the latter system, although the yield was rather low.

N,N-diethylaminoalkoxy-4,7-phenanthrolines and 1,8-diazaflourene derivatives, a novel class of potential interferon inducers and antiviral agents: interactions with nucleic acids in vitro and cellular activities.

Tilorone aza-analogues, derivatives of 4,7-phenanthroline and 1,8-diazafluorene, were examined as DNA-complexing agents by spectral and electrophoretic methods. The binding process includes at least two types of interactions: electrostatic and, possibly, intercalation. Complex formation with the denatured DNA was also observed, but its nature remained unsolved. Binding and thermodynamic parameters were determined. All ligands studied showed weak antiviral activity and essentially no interferon induction when assayed in vitro and in vivo. It was concluded that interferon induction by tilorone may involve specific cell receptors or intermediaries.

Genotoxicity assessment of low-molecular weight interferon inducers by the SOS Chromotest.

Tilorone and its aza-analogs, as well as CMA and its butyric analog (CNPA) were investigated as potential genotoxic agents by the SOS Chromotest. The SOS-inducing potency values (SOSIP) were 0.0033 and 0.0009 for SAF and vivakorfen, respectively, after activation with S9 fraction of mouse liver only. In contrast, an SOSIP value for tilorone of 0.0011 was observed in a non-activated assay. The SOSIPs of investigated compounds were low and comparable to the lowest values determined for other genotoxins. CMA and CNPA were not SOS inducers in any test system.

Bioelectrorheological model of the cell. 8. Cellular deformation under prolonged and recurrent shear stress.

The influence of a prolonged and recurrent shear stress created by a periodic electric field on the mechanical properties of Neurospora crassa cells was investigated. Conditions were found under which modifications of cellular structures responding to stress become irreversible, and plastic flow of the viscoelastic structural elements is observed. The symmetry of the response of the cell under stress application and relaxation was lost, when compared to the reference conditions. To interpret the results a general rheological model was proposed. As previously described (Pawlowski et al., 1997), the existence of the three hypothetical supramolecular regions of the membrane (F, S and O) was suggested. Rheological parameters for the above regions were calculated. Theoretical functions were satisfactorily fitted to the experimental results.

Bioelectrorheological model of the cell. 7. Cellular deformation in the presence of cytochalasin B.

The influence of cytochalasin B on the mechanical properties of Neurospora crassa cells subjected to a periodic electric field was investigated. Shear and extensional deformations were considered and studied separately. Conditions were found under which shear deformations become irreversible. Rheological models helped in the interpretation of the results in terms of the different response to the shear stress of the three hypothetical supramolecular regions of the membrane-skeleton network (F, S and 0). Rheological parameters for the above regions related to the proposed models were calculated. The models were satisfactorily fitted to the experimental results. The influence of cytochalasin B on the course of extensional deformation of cells was investigated and characterized semiquantitatively.

A new method for the investigation of cellular dielectrophoresis.

A new method and a new type of measuring-microchamber for the investigation of cellular dielectrophoresis is presented. The method is based on the observation of the trajectory of a single cell in gravitational and electric fields being crossed and orientated perpendicularly to the observation direction. By means of this method the whole dielectrophoretic spectrum ranging from negative to positive dielectrophoresis may be easily and quickly obtained. The experiments carried out on different cell types showed that the dielectrophoretic spectra, as well as the dependence of the critical frequency upon medium conductivity and cell size agree well with the predictions of a new model of the dielectrophoretic mechanism proposed by Sauer.