Accounting units in DNA

Chargaff's first parity rule (%A=%T and %G=%C) is explained by the Watson-Crick model for duplex DNA in which complementary base pairs form individual accounting units. Chargaff's second parity rule is that the first rule also applies to single strands of DNA. The limits of accounting units in single strands were examined by moving windows of various sizes along sequences and counting the relative proportions of A and T (the W bases), and of C and G (the S bases). Shuffled sequences account, on average, over shorter regions than the corresponding natural sequence. For an E. coli segment, S base accounting is, on average, contained within a region of 10 kb, whereas W base accounting requires regions in excess of 100 kb. Accounting requires the entire genome (190 kb) in the case of Vaccinia virus, which has an overall "Chargaff difference" of only 0.086% (i.e. only one in 1162 bases does not have a potential pairing partner in the same strand). Among the chromosomes of Saccharomyces cerevisiae, the total Chargaff differences for the W bases and for the S bases are usually correlated. In general, Chargaff differences for a natural sequence and its shuffled counterpart diverge maximally when 1 kb sequence windows are employed. This should be the optimum window size for examining correlations between Chargaff differences and sequence features which have arisen through natural selection. We propose that Chargaff's second parity rule reflects the evolution of genome-wide stem-loop potential as part of short- and long-range accounting processes which work together to sustain the integrity of various levels of information in DNA.     

Relative roles of primary sequence and (G + C)% in determining the hierarchy of frequencies of complementary trinucleotide pairs in DNAs of different species

To an approximation Chargaff's rule (%A = %T; %G = %C) applies to single-stranded DNA. In long sequences, not only complementary bases but also complementary oligonucleotides are present in approximately equal frequencies. This applies to all species studied. However, species usually differ in base composition. With the goal of understanding the evolutionary forces involved, I have compared the frequencies of trinucleotides in long sequences and their shuffled counterparts. Among the 32 complementary trinucleotide pairs there is a hierarchy of frequencies which is influenced both by base composition (not affected by shuffling the order of the bases) and by base order (affected by shuffling). The influence of base order is greatest in DNA of 50% G + C and seems to reflects a more fundamental hierarchy of dinucleotide frequencies. Thus if TpA is at low frequency, all eight TpA-containing trinucleotides are at low frequency. Mammals and their viruses share similar hierarchies, with intra- and intergenomic differences being mainly associated with differences in base composition (percentage G + C). E. coli and, to a lesser extent, Drosophila melanogaster hierarchies differ from mammalian hierarchies; this is associated with differences both in base composition and in base order. It is proposed that Chargaff's rule applies to single-stranded DNA because there has been an evolutionary selection pressure favoring mutations that generate complementary oligonucleotides in close proximity, thus creating a potential to form stem-loops. These are dispersed throughout genomes and are rate-limiting in recombination. Differences in (G + C)% between species would impair interspecies recombination by interfering with stem-loop interactions.     

Complementary principles of bases recoding along one chain of DNA

We have determined the new complementary principles in encoding bases on DNA chain in chromosomes of human genome and some other investigated genomes. The obtained results show that regularity analogues to Chargaff rule (or complementary principle of Watson-Crick) holds not only for two-chain DNA spiral but even for each separate chain. Moreover, we revealed other remarkable regularities concerning repeating sequences of sets of identical letters (bases). On the basis of obtained statistical data one can draw a conclusion that there exist some strict rules of forming DNA structure valid for all species. The obtained results will significantly improve our present view of encoding genetic information and also data on DNA replication process as well as formation of compact shape of chromosome packing.     

A test of Chargaff's second rule

In 1968, Chargaff and his colleagues discovered a rule in Bacillus subtilis: in single stranded DNA, A=T and C=G. This rule has since been confirmed many times in other bacterial and eukaryotic genomes. To the best of our knowledge, this rule has not been tested before in either single stranded DNA or RNA genomes. Over 3400 genomic sequences were examined here and included for the first time both double and single stranded DNA and RNA genomes. We found that: (1) with the exception of the organellar DNA, this parity rule holds for all types of double stranded DNA genomes and (2) that this rule fails to hold for other types of genomes. The parity rule appears to be a selective force on genome evolution and codon use.     

Analysis of bilateral inverse symmetry in whole bacterial chromosomes

The positions of the 64 DNA tri-nucleotides (triplets) along the Borrelia burgdorferi chromosome were determined and cumulative position plots (CPP) were obtained. Analysis of CPP for complementary triplets revealed close correlations in complementary triplet frequencies (CTF) between opposing leading and lagging strands. Such bilateral inverse symmetry (BIS) applied also to complementary mono- and di-nucleotides and to some >3 n-tuples. At the level of individual bases BIS explains Chargaff's second parity rule for whole bacterial chromosomes. Using shuffled control sequences we show that single-base BIS was not the source of higher-order BIS. Analysis of CTF in 45 other chromosomes suggests that BIS is a general property of eubacteria. BIS at the various levels may be due to the very similar numbers of codons used in chromosomal halves. Evolutionarily, BIS could have resulted from asymmetric substitution of bases combined with genetic rearrangements. However, the provocative theoretical alternative of whole-genome inverse duplication is here considered.     

Historical opinion: Erwin Chargaff and his 'rules' for the base composition of DNA: why did he fail to see the possibility of complementarity?

Erwin Chargaff was one of the more interesting and colourful figures of the historic decade that heralded the proposal of the double helical structure of DNA by Watson and Crick in 1953. In describing Chargaff's important contribution to the study of DNA, particularly its base composition, this article seeks to suggest why, despite his substantial achievements, he failed to anticipate some of the key features of the Watson-Crick model, particularly complementarity between bases--a failure that left him deeply embittered for the rest of his life.     

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle‐scale barcodes. Next‐generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high‐quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long‐range PCR and sequenced using next‐generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early‐diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome‐scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.     

War and world of Erwin Chargaff (Dedicated to 110th anniversary of birth)

The article shortly describes the life path of Erwin Chargaff, one of the most famous figures in the history of molecular biology and genetics. Chargaff was born in Chernivtsi (Austria-Hungary, now Ukraine) but during the First World War his family was forced to move to Vienna. After graduating from the University of Vienna, Chargaff worked in Berlin, where he studied bacterial lipids. Due to Nazis coming to power in Germany, Chargaff moved to Paris and later (1935) emigrated to the USA and obtained a position at the Columbia University, where he initially invastigated the role of phospholipids in blood clotting. In year 1944, applying novel methods Chargaff initiated intensive investigation of the chemical composition of nucleic acids from taxonomically distant species and established two rules, which were later named after him. The first Chargaff's rule provided a significant support to Watson and Crick in construction of their double helical DNA model. The explosion of atomic bombs over Hiroshima and Nagasaki forced Chargaff to think about the moral responsibility of researchers and science to mankind. He began to raise these issues in the press and manifested himself as a talented journalist, who criticized the bureaucratization of science and its transformation into a way of earning money. Despite decades of life in America, spiritually Erwin Chargaff always remained a European, who never forgot his roots and always remembered his native land.     

Codon usage and bias in mitochondrial genomes of parasitic platyhelminthes

Sequences of the complete protein-coding portions of the mitochondrial (mt) genome were analysed for 6 species of cestodes (including hydatid tapeworms and the pork tapeworm) and 5 species of trematodes (blood flukes and liver- and lung-flukes). A near-complete sequence was also available for an additional trematode (the blood fluke Schistosoma malayensis). All of these parasites belong to a large flatworm taxon named the Neodermata. Considerable variation was found in the base composition of the protein-coding genes among these neodermatans. This variation was reflected in statistically-significant differences in numbers of each inferred amino acid between many pairs of species. Both convergence and divergence in nucleotide, and hence amino acid, composition was noted among groups within the Neodermata. Considerable variation in skew (unequal representation of complementary bases on the same strand) was found among the species studied. A pattern is thus emerging of diversity in the mt genome in neodermatans that may cast light on evolution of mt genomes generally.     

Simultaneous Transmission of Three Stable Genotypes in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

REPLICATION of the double stranded DNA genomes of bacteria takes place by a semi-conservative mechanism¹, but although autoradiographic studies have confirmed that eukaryotic DNA is replicated semi-conservatively^2,3, our lack of knowledge about the structure of the eukaryotic chromosome means that this finding does not prove that the conserved unit is a single polynucleotide strand of DNA. Indirect information supporting the hypothesis that the conserved unit consisted of a single polydeoxyribonucleotide strand has accumulated from transmission studies in chemical mutation experiments. Two phenotypic classes of mutants are readily distinguishable as a result of chemical mutagenesis; mosaic (fractional) mutants and complete (whole body) mutants. Mutation studies assume that the treated gametes contain one DNA polymer per chromosome and that the polymer is made up of two complementary nucleotide strands. With chemical mutagens (excluding acridine dyes) it is probable that only one of the two complementary strands would be chemically altered⁴. Following fertilization, DNA replication occurs, fixing both the mutational event and its complementary wild type site. The zygote will possess a mutation which will be phenotypically expressed in the adult fly depending on the early morphogenic movements in the fly's development. Assuming that only one strand is altered, the two cell lines would contain a mutant genotype and a wild type genotype. Two genotypically mutant cell lines would arise if two mutational events occur in the opposing polydeoxyribonucleotide strands within the same genie region. In this case, there would be no genotypically wild type cells in the embryo.     

DNA denatures upon drying after ethanol precipitation.

We have observed that ethanol precipitation and subsequent drying of small (less than 400 bp) radiolabelled DNA fragments is able to induce a transition to a form that migrates aberrantly on acrylamide gels. This unusual form has increased sensitivity to S1 nuclease, decreased sensitivity to restriction enzymes, and a concentration dependence for the reversion to the duplex form. Apparently, DNA denatures upon dehydration so that redissolving at low dilution will allow the collapse of DNA fragments into single-stranded hairpin structures. These structures are stable enough at low dilution to prevent complete reannealing of single stranded species. These single stranded species show strong binding to unidentified proteins present in nuclear extracts. This may give rise to misleading interpretations of mobility shift assays, especially if the single-stranded conformers have a similar mobility to the duplex fragment, which can occur in fragments that are 50-100 bp long. Evidence is presented that DNA, in general, denatures upon dehydration, but that hindrances to rotation in the solid state may prevent long fragments from dissociating.     

Construction of a 42 base pair double stranded DNA microcircle.

A procedure for the construction of double stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. Starting with two synthetic oligonucleotides which are able to form dumbbell shaped structures, two subsequent ligation reactions yield a microcircle of double stranded DNA of 42 base pairs. This is by far the smallest circle of double stranded DNA yet described. These microcircles can be constructed in quantities required for high resolution structural analyses such as X-ray crystallography and NMR spectroscopy.     

Structure and organization of the hisA gene of the thermophilic archaebacterium Methanococcus thermolithotrophicus.

A restriction fragment of Methanococcus thermolithotrophicus genomic DNA was cloned into pUC8 to produce plasmid pET9301, which complements mutations in the hisA gene of Escherichia coli. Sequencing the DNA (2,155 base pairs) cloned from this thermophilic methanogen demonstrated that the M. thermolithotrophicus hisA gene is located within a cluster of open reading frames (ORFs) and is 68 and 69% homologous at the nucleotide level to the hisA genes of the mesophilic methanococci M. voltae and M. vannielii, respectively. The ORF (ORF 206) immediately 5' to the hisA gene of M. thermolithotrophicus is partially deleted in the genomes of the two mesophilic species, whereas ORF 114, which is 5' to ORF 206, is conserved in all three species.     

Quantitative hybridization-arrest of mRNA in Xenopus oocytes using single-stranded complementary DNA or oligonucleotide probes.

The expression of heterologous mRNA in Xenopus oocytes was quantitatively inhibited by coinjection of single-stranded complementary DNA or synthetic complementary oligonucleotides. The lymphokines Interleukin-2 (IL-2) and Interleukin-3 (IL-3) were used as model systems to test the effectiveness of this procedure. Messenger RNA samples were hybridized to single stranded complementary DNA or oligonucleotides, injected into oocytes and the oocyte incubation medium assayed for the presence or absence of specific translation products 48 hours later. When IL-2 mRNA was hybridized to a large excess of long (490 bases) single stranded complementary DNA, the expression of IL-2 was effectively blocked (greater than 98%). Complementary oligonucleotides (18-23 bases) were almost as effective as the polynucleotide in inhibiting IL-2 activity (greater than 95%). Oligonucleotides derived from the 5' end, middle or 3' end of the coding sequence were all effective in arresting IL-2 mRNA translation. Oligonucleotide hybrid-arrest was effective even when no NaCl was present in the hybridization buffer, indicating that the annealing reaction could occur within the oocyte after injection. Definite proof that hybrid-arrest could occur in vivo was shown by the fact that oligonucleotides injected before or after mRNA injection, while not as effective as co-injection, still showed substantial inhibition of specific mRNA translation. The oligonucleotide hybrid-arrest method was equally effective in the case of IL-3, demonstrating its general applicability.     

Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences

DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.     

Compensatory nature of Chargaff’s second parity rule

The second parity rule of Chargaff (A≈T and G≈C within one strand) holds all over the living world with minor exceptions. It is maintained with higher accuracy for long sequences. The question addressed in the article is how different sequence types, with different biases from the parity, contribute to the general effect. It appears that the sequence segments with biases of opposite sign are intermingled, so that with sufficient sequence lengths the parity is established. The parity rule seems to be a cumulative result of a number of independent processes in the genome evolution, with the parity as their intrinsic property. Symmetrical appearance of simple repeats and of Alu sequences in the human DNA strands, and other contributions to the Chargaff parity II rule are discussed.