Zora Šormová

Study of DNA conformation in native, partial and reconstituted nucleohistones, using hydrodynamic methods and optical rotatory dispersion

Abstract 1. 1. The course of the intrinsic viscosity of DNA in native, partial and reconstituted nucleohistones and the correlation of the hydrodynamic parameters s° and [η] support the conclusions reached concerning the compact character of nucleohistone particles, in agreement with the assumed superstructure of DNA. No significant differences in the character of particles of partial and reconstituted nucleohistones were found. The degree of compactness of the particles appears to be determined by the ratio histone DNA rather than by the nature of the histone in the complex. 2. 2. The optical rotatory dispersion (ORD) spectra of native, partial and reconstituted nucleohistones were compared in 0.01 M Tris buffer, where the complex is intact, and in 2 M NaCl where the complex is largely dissociated. An examination of the ORD spectra of DNA in nucleohistones indicated that these spectra were different from the spectrum of free DNA. The difference was the same for DNA in partial and reconstituted nucleohistones and depended only on the ratio of protein to DNA. The deviations of the ORD spectra of DNA can best be explained by a slight change in the parameters of the DNA double helix accompanying supercoil formation.

Plurimodal heterogeneity of base composition of calf thymus DNA

Abstract Calf thymus DNA was fractionated by three cycles of chromatography on methylated serum albumin columns. The separate fractions, as well as the unfractionated DNA, were characterized by the first derivatives of absorption-melting curves, continuous thermal chromatograms on hydroxyapatite columns and isopycnic sedimentation in gradients of CsCl. All these methods indicate a plurimodal distribution of base composition of calf thymus DNA. Fractions with the highest G+C content, rich in satellite Components I, II and III (1.713, 1.720–1.721 and 1.707 g/cm3, respectively) give polyphasic denaturation curves, contrasting with more simple isopycnic-sedimentation profiles; therefore, these molecules seem to contain large cooperatively-melting segments of widely different base composition. Similarly the denaturation profiles of fractions with the lowest average G+C content indicate the presence of independently-melting segments with G+C contents varying between 24 and 40 %.

The effect of 5-azacytidine on the root meristem ofVicia faba

The primary roots ofVicia faba seedlings were placed in a solution of 5-azacytidine and their further growth was observed after being replaced in running tap water. No inhibition of elongation occurred during the action of the 10−5 M solution of 5-azacytidine for 24 hours, but during subsequent cultivation in water in the absence of inhibitor, further growth was blocked. This inhibition could be overcome by cytidine, uridine, sodium azide, 5-azidomethyluracil and simultaneously with the 5-azacytidine solution. Inhibition was accompanied by a high incidence of chromosome stickiness and to a less extent by an incidence of chromosome aberrations. The occurrence of stickiness and chromosome aberrations was prevented by adding excess cytidine to the 5-azacytidine solution.AbstractPrimární kořeny semenáčůVicia faba byly ponořovány do roztoků 5-azacytidinu a po přenesení zpět do protékající vodovodní vody byl sledován jejich další růst. Po působení roztoku 10−5 M 5-azacytidinu 24 h. nedošlo k inhibici dlouživého růstu, ale během další kultivace ve vodě za nepřítomnosti inhibitoru zůstává další růst zablokován. Této inhibici lze předejít cytidinem, uridinem, azidem sodným, 5-azidomethyluracilem a částečně i 2,4-dinitrofenolem, jestliže některá z těchto látek byla přidána v přebytku současně do roztoku 5-azacytidinu. Inhibice je provázena silným výskytem, stickiness chromosomů a v menším měřítku i výskytem chromosomálních aberací. Vzniku stickiness i chromosomálních aberací bylo zabráněno přidáním přebytku cytidinu do roztoku 5-azacytidinu.AbstractПервичные корешки прорастающих семянVicia faba L. погружали в растворы 5-азацитидина и после перенесения их обратно в проточную водопроводную воду следили за дальнейшим ростом. После 24-х часового воздействия 10−5 молярным раствором 5-азацитидина не наблюдалось торможения удлинительного роста, однако, в течение дальнейшего культивирования в воде в отсутствии ингибитора дальнейший рост остается заторможенным. Данное заторможение возможно предупредить цитидином, уридином, азидом натрия, 5-азидометилурацилом и частично 2,4-динитрофенолом, если только данные вещества добавлены в избытке одновременно в раствор 5-азацитидина. Торможение сопровождается значительным появлением слипающихся хромосом (stikiness) и в меньшей мере также и появлением хромосомных абераций. Возникновение слипающихся хромосом и хромосомных абераций предупреждается добавлением избытка цитидина в раствор 5-азацитидина.

Isolation procedure for bacterial DNA based on gel permeation chromatography on a sepharose column.

Abstract Gel permeation chromatography of bacterial lysates, no matter what type of lysis is used, on a Sepharose 4B column equilibrated with buffered 2 M NaCl was demonstrated to be generally useful as a simple method for the quantitative and reproducible isolation of high-molecular-weight DNA (average molecular weight 2.2 × 10 7 daltons). Conventional analyses of this DNA and its re-chromatography on a MAK column and electrophoretic fractionations in polyacrylamide and agarose gels show less heterogeneity and a lower polysaccharide content in comparison with DNA preparations isolated by other complex processes, no RNA contamination and 2–3% of residual proteins. The method is also recommended for bacteria affected by different factors, where classical methods for the quantitative isolation of DNA fail.

Fractionation of DNA from Bacillus subtilis and its transforming activity for various markers

The physical, chemical, and functional heterogeneity of tranforming DNA was studied by preparative fractionation techniques providing resolution with respect to differences in molecular weight (gel filtration, sucrose density gradient), base composition (CsCl density gradient), or both these parameters simultaneously (methylalbumin-coated celite 545 MAK column). A comparison of the basic characteristics of the obtained fractions (melting temperature, Tm; density, ρ; sedimentation coefficient, S20,w; and transforming activity for ade, leu, and met markers) showed that the factor decisive for functional activity represents, in addition to the sequential arrangement of nucleotides in the chain, the average base composition. Hence, using the methylalbumin column or CsCl density gradient centrifugation, DNA fractions can be isolated which show a several times higher transforming activity for any of the markers examined. By contrast, the remaining fractionation methods, even though considerably decreasing the heterogeneity of the fractions as regards their molecular weight (such as zonal centrifugation), do not offer a possibility of fractionation of the activity for individual markers. This indicates a statistically random degradation of transforming DNA during its isolation. The order of the investigated markers according to their guanine-cytosine content is ade leu met and corresponds also to the order of their positions on the genetic map of Bacillus subtilis.

Mechanism of resistance to 5-azacytidine inBacillus Subtilis

Spontaneous mutants resistant to 5-azacytidine (5-AzCyd-r) and to 5-aza-2′-deoxycytidine (5-dAzCyd-r) were isolated inBacillus subtilis. There is no cross resistance between the two markers. Genetic transfer by a transformation process was possible with a low efficiency only with the 5-AzCyd-r marker. Independently isolated 5-AzCyd-r mutants did not show any differences in the level of resistance.

Sea urchin satellite DNA. Its isolation, some properties and relative amount at different stages of sea urchin development

It has been shown recently [ 1 3 ] that DNA isolated from sea urchin cells contains a certain amount of satellite DNA with buoyant density in CsC1 density gradient considerably higher than that of the main band DNA. Although different buoyant density values were obtained for the main band using DNA preparations isolated from various species o f sea urchins, a small amount of a heavy satellite DNA with relatively invariable density was found in all samples studied (table 1). Recently Patterson and Stafford [2] described preparation of sea urchin DNA samples highly enriched in satellite DNA, and reported hybridization of this DNA with ribosomal RNA. In our present paper resuits are presented concerning heavy satellite DNA isolation using a different method, and some physicochemical characterization o f this DNA. Preliminary data are also included on the variation of the satellite DNA content in DNA preparations isolated from sea urchin cells in different developmental stages.

The influence of thymine and 5-bromouracil on the sensitivity ofEscherichia coli to alkylation, UV and X-irradiation

The influence is followed of an alkylating agent (triethylene-melamine) UV and X-irradiation on the survival ofEscherichia coli 15 T∋ells grown in a minimal medium containing enzymatic hydrolysate of caseine. Thymine-less death of a considerable number of cells was observed in a culture grown in this medium. A conclusive difference in the sensitivity to the lethal agents used was found between a culture grown in a thymine-less medium and bacteria grown in a medium containing excess (20 μg/ml) of thymine. The culture grown with a sufficient thymine concentration was more sensitive to alkylation and X-rays, whereas bacteria surviving conditions of thymine-less death were more resistant to the above agents. However, such cells were more sensitive to UV-irradiation. The differences found are discussed from the point of view of DNA concentrations found in the individual cultures.