Magdalena Fikus

Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, which is a structural homolog of the RAD2 and RAD27 DNA repair genes

Abstract A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.␣cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression.

Quantitative fluorescenct analysis of different conformational forms of DNA bound to the dye, 4′,6-diamidine-2-phenylindole, and sepasated by gel electrophoresis

Abstract A quantitative fluorescent method for estimating the amounts of different conformational forms of the same DNA on agarose gels is described. Supercoiled, open circular, and linear forms of PM2 DNA and fluorescent dye (4′,6-diamidine-2-phenylindole) were used. The results are compared with respective radiometric estimations and are shown to be highly reproducible.

Bioelectrorheological model of the cell. 5. Electrodestruction of cellular membrane in alternating electric field

Recently proposed analysis of the extensil stress developed in a cellular membrane subjected to an alternating electric field (Pawłowski, P., and M. Fikus, 1993. Bioelectrorheological model of the cell. 4. Analysis of the extensil deformation of the membrane in an alternating field. Biophys. J. 65:535-540) was applied in calculations of extensil stress threshold values, sigma eo[d], producing experimentally observed electrodestruction of cells within the frequency range of 7 x 10(1) - 3 x 10(5) Hz. It was shown that the susceptibility (s[d] = 1/sigma eo[d]), of the membrane to this process varies with field frequency and depends on the type of cells. Electrodestruction is facilitated in the 10(5)-Hz field. A rheological hypothesis explaining the experimentally observed dependence of membrane stability on electric field frequency was proposed and successfully tested for two other phenomena: electroporation and electrofusion.

Experimental verification of a theoretical treatment of the mechanism of dielectrophoresis

Abstract Experimental verification of dielectrophoresis models, proposed 20 years ago by Sher and 6 years ago by Sauer, was carried out by a new method allowing the total dielectrophoresis spectrum of a single cell to be obtained. Measurements of the force acting on a cell during positive dielectrophoresis, and for the first time also during negative dielectrophoresis, are reported. Our results include measurements of the dielectrophoretic spectra of Neurospora crassa (slime) cells, and evaluation of the relationship between the critical frequency, f 0 (i.e. the frequency of the electric field which separates the ranges of positive and negative dielectrophoresis) on the one hand, and medium conductivity and cell size on the other. They indicate, that only Sauers model is applicable to dielectrophoresis of living cells modelled as stratified particles of lossy dielectric.

Bioelectrorheological model of the cell. 3. Viscoelastic shear deformation of the membrane

An analytical electromechanical model of a spherical cell exposed to an alternating electric field was used to calculate shear stress generated in the cellular membrane. Shape deformation of Neurospora crassa (slime) spheroplasts was measured. Statistical analysis permitted empirical evaluation of creep of the cellular membrane within the range of infinitesimal stress. Final results were discussed in terms of various rheological models.

(N,N-diethylamino)alkoxy derivatives of phenanthrolines asDNA binding agents

The interaction of the tricyclic angular azaaromatic system having positive charged side chains withDNA, influence of molecular structure of the ligand on the mode of noncovalent binding, and stability of the complex formed were established. Several (N,N-diethylamino)alkoxy derivatives of 1,7-, 1,8-, 1,10- and 4,7-phenanthroline in the protonated form were used as ligand. The electronic and steric factors were shown as responsible for the electrostatic and intercalative interactions of the ligand withDNA. The syntheses of (N,N-diethylamino)alkoxy derivatives from parent phenanthrolines were elaborated.ZusammenfassungDie Wechselwirkung von trizyklischen, azaaromatischen Systemen mit positiver Ladung an Seitenketten mitDNA, der Einfluß der molekularen Struktur des Liganden bei nonkovalenter Bindung und die Stabilität des gebildeten Komplexes wurde bestimmt. Als Liganden wurden mehrere (N,N-diethylamino)alkoxy-Verbindungen von 1,7-, 1,8-, 1,10- und 4,7-Phenanthrolin in protonierter Form benutzt. Es wurde gezeigt, daß die elektronischen und sterischen Faktoren für elektrostatische und intercalative Wechselwirkungen des Ligand undDNA verantwortlich sind. Es wurden Synthesen von (N,N-diethylamino)alkoxy-phenanthrolinen aus entsprechenden Chlorverbindungen ausgearbeitet.

Bioelectrorheological model of the cell. 1. Analysis of stresses and deformations.

An electrorheological model of a cell in alternating electric field is proposed. The model relates changes in the spherical cells shape to the field conditions, electric parameters of cytoplasm, cell membrane and external medium, and to the rheological parameters of the membrane. Stresses were determined using Maxwells stress tensor for isotropic media. Shear stresses in the cell membrane were analyzed. Predictions of the model for variations of shear stress in cellular membranes subjected to an external periodic electric field are presented and related to the conditions prevailing in electrobiological research.

N,N-diethylaminoalkoxy-4,7-phenanthrolines and 1,8-diazafluorene 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.

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.

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.