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.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

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.     

Effect of CO2 enrichment and nitrogen availability on resource acquisition and resource allocation in a grass,Bromus mollis

Summary The effects of CO₂ enrichment on the growth, biomass partitioning, photosynthetic rates, and leaf nitrogen concentration of a grass, Bromus mollis (C₃), were investigated at a favorable and a low level of nitrogen availability. Despite increases in root: shoot ratios, leaf nitrogen concentrations were decreased under CO₂ enrichment at both nitrogen levels. For the low-nitrogen treatment, this resulted in lower photosynthetic rates measured at 650 l/l for the CO₂-enriched plants, compared to photosynthetic rates measured at 350 l/l for the non-enriched plants. At higher nitrogen availability, photosynthetic rates of plants grown and measured at 650 l/l were greater than photosynthetic rates of the non-enriched plants measured at 350 l/l. Water use efficiency, however, was increased in enriched plants at both nitrogen levels. CO₂ enrichment stimulated vegetative growth at both high and low nitrogen during most of the vegetative growth phase but, at the end of the experiment, total biomass of the high and low CO₂ treatments did not differ for plants grown at low nitrogen availability. While not statistically significant, CO₂ tended to stimulate seed production at high nitrogen and to decrease it at low nitrogen.