Evolution of DNA structure: Direction,mechanism, rate

Summary On the basis of the results of an analysis of frequencies of pyrimidine oligonucleotides, the degree of pyrimidine clustering of DNA in species from different taxa has been determined. A tendency for an increase in the index of clustering of DNA was revealed in the sequence: invertebrates, fishes, amphibians, reptiles, birds, mammals. A mechanism is postulated, according to which the increase in the degree of clustering of DNA during the evolution may be associated with the accumulation of mutations, Purine Pyrimidine transversions, resulting in a selective enrichment of one of the chains of DNA with pyrimidines and the otherwith purines, i.e. in an increase in the degree of purine-pyrimidine imbalance (asymmetry) of DNA complementary chains. This mechanism of DNA evolution is supported by the presence of positive correlation between the degree of clustering and the degree of the chain asymmetry of natural DNAs, as well as the character of the amino acid substitutions in cytochromes c in different species. The progressive evolution of different groups of organisms on the whole may have been accompanied by an acceleration of the rates of evolution of the DNA structure.On the basis of the amino acid sequence of cytochromes c in different species the degree of clustering and the degree of the chain asymmetry of the corresponding structural genes of DNA was found to have a general tendency towards an increase in the following order: invertebrates, fishes, amphibians, reptiles, birds, mammals. Thus, evolution of cytochrome c cistron is a vector process based on a selection of mutations which, on the one hand, are neutral to protein, and, on the other hand, result in the sense chain of DNA being enriched with pyrimidines and the nonsense one (and the corresponding mRNA) - with purines. Hence, it is the polynucleotide template rather than protein, that must have been the object of selection. The frequency of substitutions in cytochromes c cistron for vertebrates is 1.56×10^–9 per nucleotide per year. It is believed that the evolutionary modification of the DNA structure may be associated with an increase in the interference resistance of the translation, i.e. with selection for codons of highest readout stability.Abbreviations Used Py pyrimidine - Pu purine - H heavy, i.e. the pyrimidine rich strand of DNA - L light, i.e. the purine rich strand of DNA     

Content and localisation of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants.

5-Methylcytosine has been found in all pyrimidine isopliths isolated from the DNA of cotton plants, but it localizes predominantly in tri- (about 52%) and dipyrimidine (about 22%) clusters. The 5-methylcytosine distribution by pyrimidine isopliths in DNA of cotton plants is specific and quite different from that in other plant and animal DNA studied. The total 5-methylcytosine content in DNA from wilt-infected cotton plants (2.3 mol %) is less than half that found in DNA from non-infected cotton plants (4.9 mol %). No other visible differences (G+C content, Tm, deltaT, s20,w, frequencies of pyrimidine clusters and others) in these DNA have been found. This suggests that in wilt-infected plants, no essential alteration in DNA sequence or molecular population takes place. As a result of wilt infection 5-methylcytosine completely disappears from dipyrimidine oligonucleotides of cotton plant DNA; its content decreases markedly in long pyrimidine clusters (heptaoligonucleotides and longer) and in C3, C2 T, CT2 fragments. Thus, DNA in wilt-infected plant cells is specifically undermethylated (demethylated). The induced alteration in DNA methylation may be considered one of the possible mechanisms for the specific distortion of gene activity of host cells and primary fungal pathogenic action on plants.     

The structure of animal mitochondrial DNA (base composition,pyrimidine clusters,character of methylation)

Summary Base composition, content of pyrimidine isopliths and the degree of methylation of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from various vertebrates and protozoonCrithidia oncopelti have been studied. MtDNAs from mammals (ox, rat) do not differ in fact in the GC content from the respective nDNA. The GC content in mtDNA from fishes (sheat fish) and birds (duck, chicken) is 1.5–2.5 mole % higher than in the respective nDNA. Kinetoplast DNA (kDNA) fromCrithidia oncopelti (GC = 42.9 mole %) differs significantly in base composition from nDNA (GC = 51.3 mole %). All the mtDNA and kDNA studied differ from the respective nDNA by a lower degree of pyrimidine clustering. Th amount of mono and dipyrimidine fragments in mtDNA is more than 30 mole %, whereas in nDNA it does not exceed 23 mole %. The quantity of long pyrimidine clusters (hexa and others) is 2–4 times lower in mtDNA than in nDNA. The lower degree of clustering of pyrimidine nucleotides seems to be a specific feature of all the mtDNA studied. This may be indicative of common traits in the organization and origin of mtDNA. All mtDNA of vertebrates contain 5-methylcytosine as a minor base (1.5–3.15 mole %) and surpass by 1.5–2 times the respective nDNA in the methylation degree. It has been found that in animals mtDNA is species specific as far as the 5-methyl-cytosine content is concerned. In mitochondria and nuclei of rat liver certain DNA methylase activity has been detected, which providesin vitro the methylation of cytosine residues both in homologous DNA and various heterologous DNAs. The specificity of methylationin vitro of cytosine residues in the same heterologous DNA fromE. coli B varies with the source of enzymes. The mitochondrial enzyme methylates cytosine as the lone monopyrimidine residue, whereas the nuclear enzyme methylates cytosine in the di- and tripyrimidine fragments.     

Distribution of pyrimidine sequences of different length and base composition in bacteriophage T5 DNA]

Distribution of pyrimidine tracts of different length (isopliths) with general formula PynPn+1 in bacteriophage T5 DNA was studied. The first seven isoplith fractions were subfractionated by the chain length and the quantity of the resulting non-isomeric oligonucleotides was determined. The pattern of distribution of pyrimidine tracts of various length and base composition in bacteriophage T5 DNA is different from that previously observed in the DNAs of bacteriophages T3 and T7. The observed differences in distribution of pyrimidine nucleotides are in accordance with the other peculiarities of bacteriophage T5 genome.     

Determination of pyrimidine nucleotide sequences of DNA]

A modification of the Burton method for determination of pyrimidine nucleotide blocks (isopliths) of DNA, providing a higher yield of large-sized nucleotide isopliths, is described. The amount of side products (interisopliths) does not exceed their amount upon DNA hydrolysis according to the Burton method. Another advantage of the technique recommended is a considerable shortening of hudrolysis time (20 min instead of 18 hours). The modification described has been successfully used to determine the pyrimidine nucleotide blocks of some warm-blooded animals DNAs. It has been found that the DNA of animals with higher sensitivity to ionised irradiation contains more oligothymidylic sequences as compared to the DNA of animals, less sensitive to irradiation.     

Radiosensitivity of coding and noncoding DNA sequences of various organisms]

Relationship between pyrimidine distribution patterns and radiosensitivity (Z) of DNA molecules of different species was derived by computer analysis of recurrence frequency of pyrimidine clusters. Blocking factors (beta) and Z for coding and non-coding DNA sequences of species from different taxonomic classes have been calculated within a new model. The radiosensitivity of coding DNA sequences practically does not vary whereas Z values were increased during evolution from simplest to higher organisms. The beta and Z values calculated for several groups of individual genes were shown to vary considerably.     

Sequence signatures of direct complementarity between mRNAs and cognate proteins on multiple levels

A potential connection between physico-chemical properties of mRNAs and cognate proteins, with implications concerning both the origin of the genetic code and mRNA–protein interactions, is unexplored. We compare pyrimidine content of naturally occurring mRNA coding sequences with the propensity of cognate protein sequences to interact with pyrimidines. The latter is captured by polar requirement, a measure of solubility of amino acids in aqueous solutions of pyridines, heterocycles closely related to pyrimidines. We find that the higher the pyrimidine content of an mRNA, the stronger the average propensity of its cognate protein’s amino acids to interact with pyridines. Moreover, window-averaged pyrimidine profiles of individual mRNAs strongly mirror polar-requirement profiles of cognate protein sequences. For example, 4953 human proteins exhibit a correlation between the two with |R| > 0.8. In other words, pyrimidine-rich mRNA regions quantitatively correspond to regions in cognate proteins containing residues soluble in pyrimidine mimetics and vice versa. Finally, by studying randomized genetic code variants we show that the universal genetic code is highly optimized to preserve these correlations. Overall, our findings redefine the stereo-chemical hypothesis concerning code’s origin and provide evidence of direct complementary interactions between mRNAs and cognate proteins before development of ribosomal decoding, but also presently, especially if both are unstructured.     

Action of a Transposable Element in Coding Sequence Fusions

The original Casadaban technique for isolating fused cistrons encoding hybrid beta-galactosidase proteins used a Mucts62 prophage to align the upstream coding sequence and lacZ prior to selection. Kinetic analysis of araB-lacZ fusion colony emergence indicated that the required DNA rearrangements were regulated and responsive to conditions on selection plates. This has been cited as an example of "directed mutation." Here we show genetically that the MuA and integration host factor (IHF) transposition functions are involved in the formation of hybrid araB-lacZ cistrons and propose a molecular model for how fusions can form from the initial strand-transfer complex. These results confirm earlier indications of direct Mu involvement in the fusion process. The proposed model explains how rearranged Mu sequences come to be found as interdomain linkers in certain hybrid cistrons and indicates that the fusion process involves a spatially and temporally coordinated sequence of biochemical reactions.     

Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

Endonuclease VIII (Nei), which recognizes and repairs oxidized pyrimidines in the base excision repair (BER) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. Recently, we and others identified three homologs of Escherichia coli endonuclease VIII-like (NEIL) proteins in humans. Here, we report identification of human NEIL homologs in Mimivirus, a giant DNA virus that infects Acanthamoeba. Characterization of the two mimiviral homologs, MvNei1 and MvNei2, showed that they share not only sequence homology but also substrate specificity with the human NEIL proteins, that is, they recognize oxidized pyrimidines in duplex DNA and in bubble substrates and as well show 5'2-deoxyribose-5-phosphate lyase (dRP lyase) activity. However, unlike MvNei1 and the human NEIL proteins, MvNei2 preferentially cleaves oxidized pyrimidines in single stranded DNA forming products with a different end chemistry. Interestingly, opposite base specificity of MvNei1 resembles human NEIL proteins for pyrimidine base damages whereas it resembles E. coli formamidopyrimidine DNA glycosylase (Fpg) for guanidinohydantoin (Gh), an oxidation product of 8-oxoguanine. Finally, a conserved arginine residue in the "zincless finger" motif, previously identified in human NEIL1, is required for the DNA glycosylase activity of MvNei1. Thus, Mimivirus represents the first example of a virus to carry oxidative DNA glycosylases with substrate specificities that resemble human NEIL proteins. Based on the sequence homology to the human NEIL homologs and novel bacterial NEIL homologs identified here, we predict that Mimivirus may have acquired the DNA glycosylases through the host-mediated lateral transfer from either a bacterium or from vertebrates.     

Clustering of identical oligomers in coding and noncoding DNA sequences.

We develop a quantitative method for analyzing repetitions of identical short oligomers in coding and noncoding DNA sequences. We analyze sequences presently available in the GenBank separately for primate, mammal, vertebrate, rodent, invertebrate and plant taxonomic partitions. We find that some oligomers "cluster" more than they would if randomly distributed, while other oligomers "repel" each other. To quantify this degree of clustering, we define clustering measures. We find that (i) clustering significantly differs in coding and noncoding DNA; (ii) in most cases, monomers, dimers and tetramers cluster in noncoding DNA but appear to repel each other in coding DNA. (iii) The degree of clustering for different sources (primates, invertebrates, and plants) is more conserved among these sources in the case of coding DNA than in the case of noncoding DNA. (iv) In contrast to other oligomers, we find that trimers always prefer to cluster. (v) Clustering of each particular oligomer is conserved within the same organism.     

Lipid tail chain asymmetry and the strength of membrane-induced interactions between membrane proteins

Many lipids are composed of asymmetric tail chains that differ by their molecular weight (MW) and/or degree of saturation. Previous studies found that membrane moduli vary with the degree of lipid tail asymmetry. However, to date little is known regarding the effect (if any) of tail asymmetry on the membrane-induced interactions between embedded proteins. In this paper we use a self-consistent field model to examine the effect of lipid tail asymmetry on membrane proteins. We first examine the case where the overall tail length (sum of both chains) is held constant, which implies that the membrane thickness remains constant as well, independent of tail asymmetry. We find that, in these systems, the membrane area stretch and bending moduli decrease with increasing chain asymmetry, thereby reducing the magnitude of the membrane-induced barrier to protein aggregation. Since in symmetric lipid bilayers the energy barrier is typically of order ∼ 1-2 times the thermal energy kT, the asymmetry-induced reduction in barrier height may increase the probability of protein aggregation significantly. In systems where one tail chain is held constant, increasing asymmetry involves changes in the bilayer thickness which are found to dominate any effect arising from the asymmetry.     

Lipid tail chain asymmetry and the strength of membrane-induced interactions between membrane proteins

Many lipids are composed of asymmetric tail chains that differ by their molecular weight (MW) and/or degree of saturation. Previous studies found that membrane moduli vary with the degree of lipid tail asymmetry. However, to date little is known regarding the effect (if any) of tail asymmetry on the membrane-induced interactions between embedded proteins. In this paper we use a self-consistent field model to examine the effect of lipid tail asymmetry on membrane proteins. We first examine the case where the overall tail length (sum of both chains) is held constant, which implies that the membrane thickness remains constant as well, independent of tail asymmetry. We find that, in these systems, the membrane area stretch and bending moduli decrease with increasing chain asymmetry, thereby reducing the magnitude of the membrane-induced barrier to protein aggregation. Since in symmetric lipid bilayers the energy barrier is typically of order approximately 1-2 times the thermal energy kT, the asymmetry-induced reduction in barrier height may increase the probability of protein aggregation significantly. In systems where one tail chain is held constant, increasing asymmetry involves changes in the bilayer thickness which are found to dominate any effect arising from the asymmetry.     

Isolation of mitochondrial DNA, purified of nuclear DNA, from animal tissues (degree of methylation and level of pyrimidine nucleotide clustering--criteria of purity)]

A method of preparation of mitochondria free of nuclear DNA and its fragments by treatment of mitochondria with DEAE-cellulose has been developed. This method is based on binding nuclear nucleic acids and nucleoproteins to DEAE-cellulose particles in the media used for isolation of mitochondria. Treatment with DEAE-cellulose under the conditions described does not induce any visible degradation of mitochondria and mitochondrial DNA. The mitochondrial DNA preparations obtained from beef and rat liver are represented with closed circular molecules of contour length about 5.5 mu. The 5-methylcytosine content in beef and rat mitochondrial DNA (3.03 and 2.0 mole %, respectively) is twice as much as in corresponding nuclear DNA. Besides, mitochondrial DNA strongly differs from nuclear ones by a lower degree of pyrimidine clustering: the amount of mono- and dipyrimidine fragments (about 32 mole %) in mitochondrial DNA is 1.5 times as large and the content of long pyrimidine clusters (hexa- and others) is 2--4 times as low as those in nuclear DNA. The methylation level and the pyrimidine clustering degree may be used as criteria for the purity of mitochondrial DNA from nuclear DNA.     

k Chain variable regions from three galactan binding myeloma proteins.

A series of seven BALB/c myeloma proteins has been identified with binding specificity for antigens containing beta(1 leads to 6)-D-galactopyranosyl moieties. We have determined the primary amino acid sequence of the first 108 residues from the light chains of three of these proteins. The framework portions of the variable regions of these three light chains are identical with residue 100 at which position three different amino acids are found in the three chains. An additional interchange was found at position 106 in one of the proteins. Based on recent DNA sequence studies suggesting that the variable region ends at residue 97, these substitutions indicate the possible existance of multiple genes coding for the region beginning at residue 98 and continuing toward the carboxy terminus. A single amino acid interchange was observed in complementarity determining regions occurring in L3. This substitution (Ile-Trp) would require changes in all three codon bases to produce the respective amino acids if one were derived from the other. Two of these chains are thus indistinguishable for their first 100 amino acids and are the first pair of k chains to exhibit complete identity over their variable regions.     

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.     

A prevalent persistent global nonrandomness that distinguishes coding and non-coding eucaryotic nuclear DNA sequences

Summary Coding sequences of eucaryotic nuclear DNA were characterized by an excess of short runs and a deficit of long runs of weak and of strong hydrogen bonding bases; non-coding sequences by a deficit of short runs and an excess of long runs, in the same of purines and of pyrimidines. The conservation of these attributes across DNA sequences coding for proteins of widely different function, across widely different eucaryotic species for the same protein and across related genes that diverged a long time ago and that now show large differences in base and, if coding, amino acid sequence suggested that these attributes have survival value. It was concluded that these attributes constitute probalistic constraints on th primary structure (base sequence) of both coding and non-coding DNA.     

Efficient production of the industrial biocatalysts hydantoinase and N-carbamyl amino acid amidohydrolase: Novel non-metabolizable inducers

Abstract The biosynthesis of the hydantoin-hydrolysing enzymes hydantoinase and N -carbamyl amino acid amidohydrolase from Agrobacterium sp. IP I-671, a Gram-negative bacterium used as a biocatalyst for the production of enantiomerically pure ( R ) amino acids, was found to be highly inducible by the addition to the cultivation medium of different non-metabolizable thiolated hydantoins or pyrimidines. Among these inducers the hexacyclic pyrimidine thioderivatives were more potent than all the pentacyclic thiohydantoin compounds. Addition of 2,4-thiouracil to the cultures, at a rate of 0.1 g (g cell dry mass)⁻¹, led to no appreciable growth inhibition and yielded a biocatalyst exhibiting a 40-fold higher hydantoinase and a 15-fold higher N -carbamyl amino acid amidohydrolase activity than the corresponding inducer-free cultures.     

Determination of structural differences in DNA by pyrimidine isopliths ratios]

Using a modified Burton procedure, twelve pyrimidine nucleotide isopliths of DNA from five mammalian species (human, rabbit, guinea pig, rat and cattle) were determined. A method is proposed for mathematical estimation of DNA block analysis data, revealing a correlation between the specific DNA primary structure, the systemic status of the organism under investigation and the organism's radiosensitivity. In some cases DNA structural differences as determined by pyrimidine isoplith ratios help to distinguish between families of the same mammalian order. Quantitative isoplith ratios demonstrate that ionizing radiation treatment brings about certain changes in DNA primary structure. Their direction is quite the opposite to the main trend in the changes of DNA structure in the course of biological evolution.     

tra cistrons and proteins encoded by the Escherichia coli antibiotic resistance plasmid R6-5

Summary Hybrid plasmids obtained by cloning individual EcoRI and HindIII fragments of the conjugative plasmid, R6-5, were analyzed for their ability to complement transfer-deficient point mutations of Flac. As a result, the locations of 10 tra cistrons were defined on the physical map of R6-5. Two cistrons, traE and traG, are interrupted by EcoRI restriction sites and one cistron, traC, probably contains a HindIII restriction site. The origin of DNA transfer, oriT, was also localized. Surprisingly the hybrid plasmid carrying oriT is mobilized by the F factor as well as by R6-5. The surface exclusion cistrons, traS and traT, were mapped and their biological expression analyzed. A total of 18 proteins encoded by cistrons within the tra region were detected by SDS polyacrylamide gel electrophoresis of proteins synthesized in minicells; they represent about 53% of the coding capacity of the cloned DNA. R6-5 DNA fragments containing the cistrons traC, traE, and traT directed the synthesis of proteins which comigrated during SDS gel electrophoresis with the F-coded proteins previously characterized as TraCp, TraEp, and TraTp. A further two proteins encoded by R6-5 comigrated with F-encoded (but genetically unidentified) proteins whose cistrons map in the corresponding part of the tra region. In contrast, no R6-5 proteins corresponding to F proteins TraAp, TraDp, TraJp, TraMp, 6a or 6c were detected. These results are discussed in relation to known DNA sequence homologies between the F and R6-5 plasmids. A preliminary physical map of the tra region of R6-5 is presented and compared with that of F.