The consequences of political dictatorship for Russian science.

The Soviet communist regime had devastating consequences on the state of Russian twentieth century science. Country Communist leaders promoted Trofim Lysenko--an agronomist and keen supporter of the inheritance of acquired characters--and the Soviet government imposed a complete ban on the practice and teaching of genetics, which it condemned as a "bourgeois perversion". Russian science, which had previously flourished, rapidly declined, and many valuable scientific discoveries made by leading Russian geneticists were forgotten.

Oligonucleotide model with non-identical complementary strands for chromatographic studies of structure-dependent photosusceptibility.

In a previous work, we used a quantitative chromatographic analysis of two self-complementary oligonucleotides to correlate the conformational differences between the oligonucleotide duplexes and photochemical susceptibilities of constituent oligomers. In this work we describe a new double-stranded oligonucleotide model with non-identical complementary strands. To separately analyze photoproducts in two strands, one of them is used in a partially protected form (the hydrophobic 5'-dimethoxytrityl group uncleaved). Using a reversed-phase column, the oligomers and products of their UV photomodification are separated into two groups of peaks. This facilitates the quantitation of photoproducts in each of the complementary strands. Three 15-mer oligonucleotides, 5'-TTTTTAT-TAAATATA-3' (F5), 5'-AAAAATAATTTATAT-3' (F6) and 5'-TATATTTAATAAAAA-3' (F7) form the parallel-stranded (ps) F5.F6 and the ordinary antiparallel-stranded (aps) F5.F7 duplexes. For these particular sequences, the rate of cyclobutane thymine dimer formation in the ps DNA has been estimated as ca. 1.5-2 times that in the ordinary aps DNA.

Divalent metal cations upon coordination to the N7 of purines differentially stabilize the PyPuPu DNA triplex due to unequal Hoogsteen-type hydrogen bond enhancement.

The PyPuPu triplexes consisting of CG*G triads are stabilized by alkaline earth cations (Ca2+, Mg2+) and transition metal cations (Mn2+, Co2+, Ni2+, Zn2+, Cd2+), while similar triplexes including TA*A triads are stabilized only by transition metal cations. We hypothesize that such a differential triplex stabilization by divalent metal cations can be the consequence of their coordination to the N7 of the third strand purines with concomitant polarization effects on the bases resulting in unequal Hoogsteen-type hydrogen bond enhancement.

Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex.

A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.

Protonated pyrimidine-purine-purine triplex.

We have studied a protonated pyrimidine-purine-purine (Py-Pu-Pu) triplex, which is formed between the d(C)nd(G)n duplex and the d(AG)m oligonucleotide as the third strand and carries the CG*A+ protonated base-triads. We have observed such an intermolecular complex between a plasmid carrying the d(C)18 d(G)18 insert and the d(AG)5 oligonucleotide without bivalent cations in 200 mM of Na+ at pH4.0. Bivalent cations additionally stabilize the complex. We propose the structures for nearly isomorphous base-triads TA*A, CG*G and CG*A+. To identify the H-DNA-like structure, which includes the triplex between d(C)n d(G)n duplex and the AG-strand, we have cloned in a superhelical plasmid the insert: G10TTAA(AG)5. The data on photofootprinting and chemical modification with diethyl pyrocarbonate, potassium permanganate and dimethyl sulfate demonstrate that the H-like structure with triplex carrying CG*G and CG*A+ base triads is actually formed under acid conditions. In the course of this study we have come across unexpected results on probing of Py-Pu-Pu triplexes by dimethyl sulfate (DMS): the protection effect is observed not only for guanines entering the duplex but also for guanines in the third strand lying in the major groove. We have demonstrated this effect not only for the case the novel protonated Py-Pu-Pu triplex but also for the traditional non-protonated Py-Pu-Pu intramolecular triplex (H*-DNA) formed by the d(C)37 d(G)37 insert in supercoiled plasmid in the presence of Mg2+ ions.

Effect of intermolecular triplex formation on the yield of cyclobutane photodimers in DNA.

We have studied the effect of intermolecular triplexes formation on the yield of cyclobutane photodimers in DNA. DNA duplex within the pyrimidine-purine-pyrimidine triplex d(TC)nd(GA)nd(CT)n is protected from the formation of cyclobutane photodimers in the case of the stabilization of this triplex by acid pH, and in the case of supplementary stabilization by Mg2+ or Zn2+. We have studied pH-independent pyrimidine-purine-purine triplexes stabilized by bivalent cations. In such triplexes, the protection from the formation of [6-4] photodimers is observed, whereas the protection from cyclobutane dimer formation does not take place. The formation of the d(TC)nd(GA)nd(GA)n triplex leads to an inversion of the intensities of cyclobutane CT and TC photodimers. We observed a sharp decrease in photoreactivity with respect to cyclobutane dimers in the duplex tract d(C)18d(G)18 in the presence of Ba2+, Cd2+, Co2+, Mn2+, Zn2+ and Ni2+. The formation of the d(C)nd(G)nd(G)n triplex leads to 'antifootprinting', i.e. an increase in the yield of cyclobutane photodimers.

Photofootprinting of DNA triplexes.

We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6-4) pyrimidine photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined conditions. At acid pH, d(C)n.d(G)n.d(C)n and d(CT)n.d(GA)n.d(CT)n triplexes are detected by this method. The d(CT)n.d(GA)n.d(CT)n triplexes are additionally stabilized by divalent cations and spermidine. PyPuPu triplexes are pH-independent and are stabilized by divalent cations, such as Mg++ and Zn++. The effect depends on the type of cation and on the DNA sequence. The d(CT)n.d(GA)n.d(GA)n triplex is stabilized by Zn++, but not by Mg++, whereas the d(C)n.d(G)n.d(G)n triplex is stabilized by Mg++. In H-DNA, virtually the entire pyrimidine chain is protected against photodimerization, whereas only half of the pyrimidine chain participating in a triplex is protected in the CGG intramolecular triplex.

Stabilization of PyPuPu triplexes with bivalent cations.

We studied the formation of stable PyPuPu intermolecular triplexes under neutral pH in the presence of bivalent cations (Mg, Ca, Mn, Co, Ni, Cu, Zn, Cd, and Ba) with the help of the photo- and DMS footprinting assays. The cations which stabilize d(C)n.d(G)n.d(G)n and d(TC)n.d(GA)n.d(AG)n triplexes were determined. Among them, Zn++ ions stabilized both triplexes, whereas Mg++ ions stabilize CGG triplexes, but do not stabilize TC.GA.AG triplexes. We have shown that an arbitrary purine sequence forms the PyPuPu triplex in the presence of Zn++ ions, and that the purine third strand is antiparallel with respect to the purine strand within the duplex.

Protection against UV-induced pyrimidine dimerization in DNA by triplex formation.

Cyclobutane and [6-4]-pyrimidine dimers are major photoproducts of ultraviolet-irradiated DNA. The yield of these photoproducts is dependent on the sequence and structure of the DNA. By analysing the photofootprints of fragments produced by cleavage of the DNA chain near [6-4]-pyrimidine dimers, we show here that a homopurine-homopyrimidine insert (with either d(TC)x or d(C)n) in plasmid pUC19 is, as expected, a good target for UV-induced pyrimidine-dimer formation. But we find that dimerization is virtually completely suppressed when the pyrimidine oligonucleotides d(TC)y or d(C)m are added to DNA carrying d(TC)x- or d(C)n-containing inserts, respectively. This effect is dependent on the type of oligonucleotide used and is site-specific. The protection occurs under acidic conditions that favour the formation of intermolecular triplexes between the homopurine-homopyrimidine inserts and homologous oligopyrimidines. We therefore conclude that triplex formation effectively protects the DNA duplex from UV-induced damage (pyrimidine dimerization). This observation makes the photofootprinting assay a very promising method for studying intermolecular and intramolecular triplexes (H-form DNA) both in vitro and in vivo.

Hereditary variability of plants under the action of exogenous DNA.

Injection of exogenous barley donor DNA into grains of barley recipient plants at the milk maturity stage, with a specially designed syringe, led to the appearance of transformed plants. The transformation (in rare cases) was caused by the unsheared DNA since the DNA passing through the syringe needle remained relatively stable (10(6) to 10(7) daltons) as was confirmed by DNA sedimentation analysis.14 plants grown from seeds injected with highly polymeric DNA containing close to 30 per cent protein had transformed pollen grains. In the 2nd generation only 2 plants from the 8 studied preserved these changes. In the progeny of these two plants, i.e., in the 3rd seed generation after injection, 82.1 per cent of plants preserved the transformed characters. The next, 4th generation, preserved a transformed phenotype in 89.6 per cent of plants.It was also shown that reversion to a recipient-like state was not always constant. We found the reversion of transformed properties (i.e., normal starch and two-rowed spikes) in 40 per cent of the 4th generation descendants of one of the plants which had lost the phenotypical expression of these properties in the 3rd generation but had them in the 2nd generation.The study of the morphological properties of transformed plants showed that with respect to phenotypic expression some characters were changed towards the donor type, some remained as in the recipients and some were of the intermediate type.

Participation of the intracellular enzymes in the control of mutational processes : Part 4: the role of UV-specific endonuclease and medium composition in the induction of genic mutations in Escherichia coli.

The influence of UV-specific endonuclease and medium composition on the frequency and spectrum of genic mutations in Escherichia coli KI2 uvr (+) (with normal repair enzymes) and urv A6 (defective in UV-specific endonuclease) was studied. Mutations at the locus glu (gene controlling assimilation of glucose) were induced by ultra-violet irradiation and hydroxylamine treatment. To identify mutant colonies, triphenyl tetrazolium chloride (TTC) was added to the medium since it coloured the mutant colonies bright crimson and readily permitted distinction between pure mutant clones (complete mutations) and mixed clones (mosaic or sector mutations).A maximum mutation frequency after UV-irradiation was observed in E. coli uvr (+) cells but not in the E. coli uvr A6 strain. The curve of mutagenesis with a maximum was found in both studied strains after treatment by hydroxylamine which did not cause DNA damage recognized by UV-specific endonuclease.The highest frequency of mutations (at the point of maximum) in the series of experiments with enriched growth medium was almost 10 times higher than in the series of the experiments with poor medium.It was established that in bacteria with normal repair enzymes the frequency of complete mutations was higher than the frequency of mosaic mutations. It was also observed that the rate of UV-mutagenesis was higher in the case of E. coli uvr (+).The study of the distribution of mosaic mutant sectors in experiments with bacteria suspended in either a nutrient broth or a buffer during UV-irradiation revealed that the size of mutant sectors was rather variable and that the differences in the number of nucleoids per cell did not always determine the distribution of mutant sector sizes.

Participation of the intracellular enzymes in the control of the mutation process : III. Influence of the inhibition of repair and replication on γ-ray induced chromosomal aberrations in barley.

The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive (3)H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10-11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication - hydroxyurea - had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.

Repair of single-strand DNA breaks and recovery of chromosomal and chromatid aberrations after treatment of plant seeds with propyl methanesulfonate in vivo.

Repair of single-strand breaks of DNA and simultaneous recovery of chromosomal aberrations were studied after treatment of barley seeds with the monofunctional alkylating chemical mutagen, propyl methanesulfonate in vivo. In soaked seeds the diminution of single-strand breaks of DNA induced by PMS was correlated with the decrease of chromosomal aberrations, whereas in dried seeds the repair of DNA breaks was depressed and, in accord with this, the frequency of chromosomal aberrations increased. The prolonged storage of seeds led to a more delayed repair of chromosomal aberrations in dry seeds and a more delayed accelerated repair in soaked seeds.

Molecular mechanisms of the origin of chromosome aberrations and the structural organisation of eukaryotic DNA.

A new hypothesis on the appearance of exchange chromosomal aberrations has been suggested. According to this hypothesis, temporal duplex polynucleotide structure should arise during G1 and G2 phases during the correction of DNA. The size of the duplex, as a rule, should be restricted to the size of complementary nucleotide sequences in the regions of repetitions. Any polynucleotide break in a duplex zone would result in chromosome breakage and if complementary broken ends interact with each other, then exchange chromosome aberrations may be formed. This hypothesis would explain such previously obscure phenomena as extremely high frequencies of exchanges after mutagen treatment, the nature of mitotic crossing-over, negative interference, change of aberration types before replication, the low frequency of damaged structural genes during aberration formation, etc.