V. Fučík

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.

Restriction and modification in Bacillus subtilis 168

SummaryGene hsrM (nonB) of Bacillus subtilis 168, causing non-permissiveness to phage SP10 (Saito et al. 1979) and reduced plating efficiency of unmodified phage ϕ105, is responsible for non-permissiveness of B. subtilis 168 for phages π15 and PZA. Upon transformation to sporulation deficiency (allele spoOA) B. subtilis 168 becomes permissive for ϕ15 and PZA and loses the ability to restrict ϕ105. spoOA str-1 double transformants of B. subtilis 168, however, retain the restriction 168 and non-permissiveness for ϕ15 and PZA phages, in spite of their Spo− phenotype. Therefore it appears that a functional product of the spoOA gene is required for expression of gene hsrM in wild-type bacteria, but is not essential in streptomycin-resistant bacteria. Phage genomes (PZA) were trapped in spores of the restriction deficient strain with much higher efficiency than in the wild-type.

Tolerated variations in a genome: the case of closely related Bacillus phages PZA, phi 29 and phi 15--a review.

Restriction-site analysis was used to estimate the relationship of bacteriophages PZA, phi 29 and phi 15. Complete nucleotide sequences of PZA and luminal diameter 29 genomes were compared and tolerated variations were assessed. Most of the base-pair changes are silent nucleotide substitutions in the third position of codons but amino acid changing substitutions are also observed. The terminal portions of the phage genomes diverged faster than their central parts. Gene mutations in phage PZA were induced by hydroxylamine and their frequency was compared with the evolutionary mutability.

NEW MEMBERS OF BACILLUS SUBTILIS PHAGE GROUP CONTAINING A PROTEIN LINK IN THEIR CIRCULAR DNA

ABSTRACT Newly described phages (PZE and PZA) are morphologically similar to o29. Comparison of these three phages has revealed that they have the following characteristics in common: the contour length of DNA molecules corresponds to 11-12 Md, mol.% of G+C amounts to about 40%, as established by Tm, buoyant densities and by enzymatic digestion of 32 P-labelled DNA to mononucleotides. Circular DNA from the particles has transfecting activity and can be linearized by proteases. While o29 and PZE seem to be nearly identical organisms, the phage PZA differs in host range properties, antigenic activity DNA restriction patterns and efficiency of DNA-DNA hybridization.

Enzymatic synthesis of 5-bromo-2’-deoxyuridine-2-14C and of 5-iodo-2’-deoxyuridine-2-14C and their incorporation into deoxyribonucleic acid (Allium cepa)

Abstract5-Bromo-2’-deoxyuridine-2-14C was prepared from 5-bromouracil-2-14C and 2’-de-oxyguanosine using trans-N-deoxyribosylase fromLactobacillus helveticus and incorporated into DNA ofAllium cepa roots. After isolating the DNA and hydrolyzing it enzymatically to deoxynucleoside-5’-phosphates a radioactive nucleotide was detected which yielded 5-bromo-2’-deoxyuridine-2-14C on enzymatic dephosphorylation. The incorporation of 5-iodo-2’-deoxy-uridine-2-14C was followed only by microautoradiography.Abstract5-Brom-2’-desoxyuridin-2-14C byl připraven z 5-bromuracilu-2-14C a 2’-desoxyguanosinu za použití trans-N-desoxyribosylázy zLactobacillus helveticus a inkorporován do DNK v kořenechAllium cepa. Po izolaci DNK a její enzymové hydrolýze na desoxynukleosid-5’-fosfáty byl zjištěn radioaktivní nukleotid, jehož enzymovou defosforylací byl získán 5-bromo-2’-desoxy-uridin-2-14C. Inkorporace 5-jodo-2’-desoxyuridinu-2-14C byla sledována pouze mikroautoradio-grafií.Abstract5-бром-2’-дезоксиуриди н-2-14С приготовлен из 5-бромурадиила-2-14С и 2’-дезокеи гуанозина с использованием транс-N-дезоксирибози лазы изLactobacillus helveticus и инкорпорировац в ДНК в корпяхAllium cepa. После изоляции ДНК и ее энзимати-ческого гидролиза на дезоксинуклеозид-5’-ϕ осϕаты установлен радиоактивный нуклеотид, эизиматичсской деϕосϕориляцией которого получен 5-бром-2’-дезоксиуриди н-2-14С. лнкорпорация 5-йод-2’-дезокеиуридин а-2-14С изучалась путем микроавто-радиогчаϕ ии.

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.

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.

Reversion of the antimitotic effect of N-formylbiuret by ureidosuccinic acid and uracil inAllium cepa L.

AbstraetA study was made of the influence of N-formylbiuret, a degradation product of 5-azauracil, on the course of mitosis in the meristem of the roots ofAllium cepa. In the presence of N-formylbiuret in a concentration of 4-10-3 M in the nutrient fluid there is a decrease of 20 to 30% in the mitotic index of the meristem of the roots. This decrease in the mitotic index can be prevented by the addition of ureidosuccinic acid or uracil to the medium. N-formylbiuret which, as a result of interaction with dihydroorotase, inhibits the biosynthesis of the pyrimidine precursors of nucleic acids, does not display irreversible toxicity when observing its cytological effect. When decreasing the mitotic index with N-formylbiuret no chromosomal aberrations were observed in the anaphase or telophase.Under the same conditions, the structurally similar halogenacetyl derivatives of urea in decimally smaller concentrations show marked cytotoxicity with the agglutination of chromosomes. The reversion of the antimitotic effect of these substances by the pyrimidine precursors of nucleic acids was not demonstrated.AbstractByl sledován vliv N-formylbiuretu, degradačního produktu 5-azauracilu, na průběh mitóz v kořenových meristémechAllium cepa. V přítomnosti 4. 10-3 M koncentrace N-formylbiuretu v živném roztoku dochází v kořenových meristémech k 20–30% snížení mitotického indexu. Toto snížení mitotického indexu lze odstranit přidáním kyseliny ureidojantarovéa uracilu do média. N-Formylbiuret, který v důsledku interakce s dihydroorotázou inhibuje biosyntézu pyrimidinových prekursorů nukleových kyselin, nejeví tak při sledování cytologických účinků irreversibilní toxicitu. Při snížení mitotického indexu N-formylbiuretem nebylyv anafázi a v telofázi pozorovány žádné chromozomální aberace.Strukturálně příbuzné halogenacetylderiváty močoviny se za stejných podmínek v řádově nižších koncentracích vyznačují značnou cytotoxicitou spojenou s aglutinací chromozómů. Zvrat antimitotického účinku těchto látek pyrimidinovými prekursory nukleových kyselin nebyl prokázán.AbstractИзуралось влиянис N-ϕормилбиурета, деградационного продукта 5-азаурацла, натечение митоза в меристемах корнейAllium cepa L.В происутствии 4.10-3 Мконцентрацин N-ϕормилбиурета в пительном растворе имеет место 20-% снижение митотического индекса в меристемах корней. Данное снижение снижение митотического индекса возможно устранить добавлением уреидоянтарной кислоты и урацила в среду.N-ϕормилбиурет, который вследствие взаимодействия с дегидрооротазой тормозит биосинтез пиримидиновых предшественников нуклеиновых кислот, не оказывает при митотичении цитолотических эϕϕектов необратимой токсичности. При снижении митоическото индекса N-ϕормилбиуретом не наблюдалось в анаϕазе и в телоϕазе никаких хромосомных абберраций. Структурой родственные талотенацетилдерив аты мочевины обладаю тв одинаковых условиях при в десятку раз пониженных концентрациях значиельной цитотоксичностью, сязанной с агглютинацией хром.осом.Обратимост ь антимитотического действия этих веществ пиримидиновыми предшественниками нуклеиновы кислот не доказана.