Attachment of trivaline changes the specificity of binding of netropsin analogs with DNA

In the present communication, synthesis and DNA binding activities of three analogs of the antibiotic netropsin are reported. Each analog contains two N-propylpyrrolecarboxamide units linked covalently to either Dns-Gly-Val-Val-Val-Gly-Gly- (I), Val-Val-Val-Gly-Gly (II) or Gly-Gly (III). It is shown that analogs I and II can self-associate in aqueous solution and methanol as revealed from the fact that UV absorbance and circular dichroism spectra obtained for these analogs are concentration-dependent. By contrast, analogs III exists as a monomer, even at concentration levels of the order of 1.10(-3) M. Determination of the apparent sizes of intramolecular aggregates by gel-filtration shows that analog I in aqueous solution at concentration levels of the order of 1.10(-3) M forms a series of aggregates containing from 2 to 12 monomers. Analog II exhibits a lower tendency to form intermolecular aggregates as compared with that of analog I. Dimerization constants are determined for analogs I and II in aqueous solution and methanol. The binding of N-propylpyrrolecarboxamide units and peptide fragments of analog I to DNA can be independently monitored by circular dichroism and fluorescence methods. If self-associated species of analog I (or II) are present in solution, the ligand exhibits a markedly different order of base pair sequence preferencies as compared with that of analog III. The results obtained are consistent with the inference that analogs I and II in a beta-associated form recognizes base pair sequences containing two runs of 3 AT pairs separated by two GC pairs.     

Physico-chemical basis of the genetic code origin: stereochemical analysis of interactions of amino acids and nucleotides based on the progene hypothesis

A progene hypothesis has been proposed earlier to explain the mechanism of origin of the self-reproducing genetic system. Progenes (precursors of the genetic system) are mixed anhydrides of an amino acid and deoxyribotrinucleotide at the 3'-gamma-terminal phosphate (NpNpNppp-AA); they are produced from dinucleotides (NpNp) and 3'-gamma-aminoacylnucleotidylates (Nppp-AA) as a result of specific interaction between amino acid and dinucleotide. The postulated mechanism of progene formation accounts for the selection of substances, including chirality, the origin of the genetic code as well as for the mechanisms of formation, self-reproduction and evolution of the simpliest genetic system ("gene--polypeptide"). A stereochemical analysis of the progene formation mechanism has allowed us to support the main statements of the hypothesis that relate to the origin of the genetic code and to selection of substances. Atomic groups that could be responsible for the specificity of interaction between dinucleotides and amino acids in progene formation have been revealed. Stereochemical evidence for the physicochemical basis of the origin of the existing genetic code have been produced: 1) a special role of the second nucleotide in the codon is demonstrated in amino acid coding by the progene hypothesis principle; 2) an advantage of T against U in such coding is demonstrated; 3) for 16 amino acids out of 20 an agreement has been obtained between the optimal dinucleotide as revealed by the stereochemical analysis and the codon dinucleotides; 4) an explanation for the third nucleotide selection mechanism is offered. A restoration of the prebiotic code, based on these results, has indicated that the code contains 32 codons, is statistical and group-wise. It encodes 7 groups of isofunctional amino acids: 3 overlapping groups of non-polar amino acids 1) medium-size hydrophobic amino acids (chiefly Val, n-Val and a-But), 2) small and medium-size non-polar amino acids (chiefly Ala Val, n-Val a-But and Gly), 3) small non-polar amino acids (Gly, Ala, a-But) and 4 groups of polar amino acids--1) hydroxy--+dicarbonic (Asp, Glu, Ser and Thr), 2) dicarbonic (Asp and Glu), 3) hydroxy (Ser and Thr) and 4) basic (Arg and Lys). The code includes about 20 amino acids among which are 15-17 canonical and a few common non-canonical. The prebiotic code explains many properties of the existing genetic code and is capable of evolving into the latter by way of a gradual replacement of the physicochemical coding mechanism by the enzymatic coding mechanism.     

Simple kinetic models explaining critical phenomena in enzymatic reactions with isomerization of the enzyme and substrate

Kinetic models for enzyme reactions are considered which take into account enzyme and substrate isomerization. Application of graph-theoretic methods allows to reveal fragments in schemes which may induce multiple stead-states or concentrational selfoscillations. The role of substrate isomers in the inhibition of enzyme isomers to produce critical phenomena is considered. The boundaries of parameter domains for critical phenomena are estimated. It is shown that the controlled change in concentrations of substrate and enzyme isomers may be important in regulation of enzyme systems, if different enzyme isomers are inhibited mainly by different substrate isomers. The models are used for interpretation of possible critical phenomena in the open reaction catalyzed by lactate dehydrogenase. It is shown that lactate dehydrogenase may act as a trigger in carbohydrate metabolism by changing "critically" its activity in relation to changes in pH and pyruvate fluxes. Slow enzyme inhibition by enolpyruvate is suggested as a possible reason for glycolytic oscillations.     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Comparative study of different variants of Actinomyces roseoflavus producing the polyene antibiotic roseofungin

The respiration chain in the membranes of whole Actinomyces roseoflavus (var. roseofungini) cells from the parent and secondary cultures is sensitive to KCN, non-sensitive to Triton X-100 treatment removing the antibiotic roseofungin from the cells, and has a very high for the bacteria respiration control. When the cells are in contact with atomic tritium at the temperature of liquid nitrogen, roseofungin is tritiated and binds to A. roseoflavus isolated membranes and whole cells, mostly to those of the parent culture as compared to the secondary culture. A fraction of membranes which lost NADH dehydrogenase in the course++ of purification was isolated from the cells disintegrated in the frozen state.     

Effect of ascorbic acid prophylaxis on the frequency of chromosome aberrations, urine mutagenicity and nucleolus test in workers occupationally exposed to cytostatic drugs

The genetic risk of workers occupationally exposed to a series of newly developed cytostatic drugs and the presumed antimutagenic potential of ascorbic acid (AA) were studied in a group of 38 chemical laboratory personnel examined for chromosome aberrations in lymphocytes, urine mutagenicity and nucleolar RNA activity before and after a 6-month prophylactic administration of AA at daily doses of 1 g for 5 days a week. Chromosome aberration tests revealed elevated aberrant cell (AB.C) rates both prior to and after AA supplementation (3.9% and 3.65% of AB.C., respectively). These values were significantly higher than those found in 18 non-exposed matching controls (1.05% of AB.C.). Tests for mutagenic activity in the urine of drug-exposed workers revealed 64% positive urine samples prior to vitaminization and 60% positive urine specimens after it; positive urine samples in the group of controls accounted for 21% of samples. In the nucleolus test, numbers of inactivated micronuclei in the exposed were initially higher than those of controls (33.4% versus 24.3%), but dropped to 20.5% after AA supplementation. These findings show that AA prophylaxis alone cannot substantially reduce the hazards associated with exposure to anti-cancer drugs.     

Predation pressure on an imperfect Batesian micicry complex in the presence of alternative prey

Summary Differential predation pressure and the probability of predation on a Batesian mimicry complex and on alternative prey were estimatedin a field experiment. The mimicry complex was composed of a noxious model (Eleodes obscura (Say)) and a palatable mimic (Stenomorpha marginata (LeConte)). House crickets (Acheta domesticus) (Linn.) were used as alternative prey. The experiment was conducted for 23 nights in August and September to approximate the peak seasonal activity time period during which both models and mimics normally are exposed to predation while foraging and depositing eggs. Each night thirty prey in ratios of 16 models: 7 mimics: 7 crickets were exposed for 2.5 h to a suite of predators consisting of pallid bats (Antrozous pallidus), striped skunks (Mephitis mephitis) and ringtails (Bassariscus astutus) that had free access to the prey. The model-mimic ratio was similar to that found in nature. Predators obtained prey on 11 of the 23 nights and preferred the alternative prey (crickets) in proportions higher than was expected from a predation rate that was equal on all species of prey. Mimics were taken by predators at a rate proportional to their abundance, while models were taken at a rate considerably lower than their relative abundance. This suggests that at least some of the predators could distinguish between models and mimics and were willing to eat the mimics at higher frequencies than they were willing to eat the models. However, although the mimicry is not perfect with respect to the entire predator suite, the mimics still gain an advantage by resembling the models, compared to the predation levels on the alternate prey.