Modelling survival and allele complementation in the evolution of genomes with polymorphic loci.

We have simulated the evolution of sexually reproducing populations composed of individuals represented by diploid genomes. A series of eight bits formed an allele occupying one of 128 loci of one haploid genome (chromosome). The environment required a specific activity of each locus, this being the sum of the activities of both alleles located at the corresponding loci on two chromosomes. This activity is represented by the number of bits set to zero. In a constant environment the best fitted individuals were homozygous with alleles' activities corresponding to half of the environment requirement for a locus (in diploid genome two alleles at corresponding loci produced a proper activity). Changing the environment under a relatively low recombination rate promotes generation of more polymorphic alleles. In the heterozygous loci, alleles of different activities complement each other fulfilling the environment requirements. Nevertheless, the genetic pool of populations evolves in the direction of a very restricted number of complementing haplotypes and a fast changing environment kills the population. If simulations start with all loci heterozygous, they stay heterozygous for a long time.

The differential killing of genes by inversions in prokaryotic genomes.

We have elaborated a method which has allowed us to estimate the direction of translocation of orthologs which have changed, during the phylogeny, their positions on chromosome in respect to the leading or lagging role of DNA strands. We have shown that the relative number of translocations which have switched positions of genes from the leading to the lagging DNA strand is lower than the number of translocations which have transferred genes from the lagging strand to the leading strand of prokaryotic genomes. This paradox could be explained by assuming that the stronger mutation pressure and selection after inversion preferentially eliminate genes transferred from the leading to the lagging DNA strand.

Evolution rates of genes on leading and lagging DNA strands.

One of the main causes of bacterial chromosome asymmetry is replication-associated mutational pressure. Different rates of nucleotide substitution accumulation on leading and lagging strands implicate qualitative and quantitative differences in the accumulation of mutations in protein coding sequences lying on different DNA strands. We show that the divergence rate of orthologs situated on leading strands is lower than the divergence rate of those situated on lagging strands. The ratio of the mutation accumulation rate for sequences lying on lagging strands to that of sequences lying on leading strands is rather stable and time-independent. The divergence rate of sequences which changed their positions, with respect to the direction of replication fork movement, is not stable-sequences which have recently changed their positions are the most prone to mutation accumulation. This effect may influence estimations of evolutionary distances between species and the topology of phylogenetic trees.

Identification of a putative chromosomal replication origin from Helicobacter pylori and its interaction with the initiator protein DnaA.

The key elements of the initiation of Helicobacter pylori chromosome replication, DnaA protein and putative oriC region, have been characterized. The gene arrangement in the H.pylori dnaA region differs from that found in many other eubacterial dnaA regions (rnpA-rmpH-dnaA-dnaN-recF-gyrB). Helicobacter pylori dnaA is flanked by two open reading frames with unknown function, while dnaN-gyrB and rnpA-rmpH loci are separated from the dnaA gene by 600 and 90 kb, respectively. We show that the dnaA gene encoding initiator protein DnaA is expressed in H.pylori cells. The H.pylori DnaA protein, like other DnaA proteins, can be divided into four domains. Here we demonstrate that the C-terminal domain of H.pylori DnaA protein is responsible for DNA binding. Using in silico and in vitro studies, the putative oriC region containing five DnaA boxes has been located upstream of the dnaA gene. DNase I and gel retardation analyses show that the C-terminal domain of H.pylori DnaA protein specifically binds each of five DnaA boxes.

High correlation between the turnover of nucleotides under mutational pressure and the DNA composition.

BACKGROUND: Any DNA sequence is a result of compromise between the selection and mutation pressures exerted on it during evolution. It is difficult to estimate the relative influence of each of these pressures on the rate of accumulation of substitutions. However, it is important to discriminate between the effect of mutations, and the effect of selection, when studying the phylogenic relations between taxa. RESULTS: We have tested in computer simulations, and analytically, the available substitution matrices for many genomes, and we have found that DNA strands in equilibrium under mutational pressure have unique feature: the fraction of each type of nucleotide is linearly dependent on the time needed for substitution of half of nucleotides of a given type, with a correlation coefficient close to 1. Substitution matrices found for sequences under selection pressure do not have this property. A substitution matrix for the leading strand of the Borrelia burgdorferi genome, having reached equilibrium in computer simulation, gives a DNA sequence with nucleotide composition and asymmetry corresponding precisely to the third positions in codons of protein coding genes located on the leading strand. CONCLUSIONS: Parameters of mutational pressure allow us to count DNA composition in equilibrium with this mutational pressure. Comparing any real DNA sequence with the sequence in equilibrium it is possible to estimate the distance between these sequences, which could be used as a measure of the selection pressure. Furthermore, the parameters of the mutational pressure enable direct estimation of the relative mutation rates in any DNA sequence in the studied genome.

DNA asymmetry and the replicational mutational pressure.

The mode of replication and organisation of bacterial genomes impose asymmetry on their nucleotide composition. The asymmetry is seen in coding and non-coding sequences and is reflected in the amino acid composition of proteins. The mechanisms generating asymmetry include: unequal mutation rates connected with replication and transcription, selection forces positioning genes and signal sequences nonrandomly in the genome, and protein coding constraints on coding sequences. There are different methods of visualising and measuring the asymmetry. Some of them can assess the contribution of individual mechanisms to the observed asymmetry and those have been described in greater detail. Asymmetric mutational and selection pressures differentiate the rates of evolution of genes on leading and lagging strands. The genes relocated to the opposite strand have to adapt to a different mutational pressure or are eliminated. Translocations from leading to lagging strands are more often selected against than from lagging to leading strands. Comparison of intergenic sequences that have lost the coding function to the original genes enables finding the frequencies of the twelve substitution rates in sequences free from selection. In the absence of selection, the half-time of substitution of a given type of nucleotide is linearly correlated with the fraction of that nucleotide in the sequence.

Is there replication-associated mutational pressure in the Saccharomyces cerevisiae genome?

Compositional bias of yeast chromosomes was analysed using detrended DNA walks. Unlike eubacterial chromosomes, the yeast chromosomes did not show the specific asymmetry correlated with origin and terminus of replication. It is probably a result of a relative excess of autonomously replicating sequences (ARS) and of random choice of these sequences in each replication cycle. Nevertheless, the last ARS from both ends of chromosomes are responsible for unidirectional replication of subtelomeric sequences with pre-established leading/lagging roles of DNA strands. In these sequences a specific asymmetry is observed, resembling the asymmetry introduced by replication-associated mutational pressure into eubacterial chromosomes.

Total number of coding open reading frames in the yeast genome.

At the end of 1996 we approximated the total number of protein coding ORFs in the Saccharomyces cerevisiae genome, based on their properties, as 4700-4800. The number is much smaller than the 5800 which is widely accepted. According to our calculations, there remain about 200-300 orphans-ORFs without known function or homology to already discovered genes, which is only about 5% of the total number of genes. Our results would be questionable if the analysed set of known genes was not a statistically representative sample of the whole set of protein coding genes in the S. cerevisiae genome. Therefore, we repeated our estimation using recently updated databases. In the course of the last 18 months, previously unknown functions of about 500 genes have been found. We have used these to check our method, former results and conclusions. Our previous estimation of the total number of coding ORFs was confirmed.

Origin and properties of non-coding ORFs in the yeast genome.

In a recent paper we have estimated the total number of protein coding open reading frames (ORFs) in the Saccharomyces cerevisiae genome, based on their properties, at about 4800. This number is much smaller than the 5800-6000 which is widely accepted. In this paper we analyse differences between the set of ORFs with known phenotypes annotated in the Munich Information Centre for Protein Sequences (MIPS) database and ORFs for which the probability of coding, counted by us, is very low. We have found that many of the latter ORFs have properties of antisense sequences of coding ORFs, which suggests that they could have been generated by duplication of coding sequences. Since coding sequences generate ORFs inside themselves, with especially high frequency in the antisense sequences, we have looked for homology between known proteins and hypothetical polypeptides generated by ORFs under consideration in all the six phases. For many ORFs we have found paralogues and orthologues in phases different than the phase which had been assumed in the MIPS database as coding.

How does replication-associated mutational pressure influence amino acid composition of proteins?

We have performed detrended DNA walks on whole prokaryotic genomes, on noncoding sequences and, separately, on each position in codons of coding sequences. Our method enables us to distinguish between the mutational pressure associated with replication and the mutational pressure associated with transcription and other mechanisms that introduce asymmetry into prokaryotic chromosomes. In many prokaryotic genomes, each component of mutational pressure affects coding sequences not only in silent positions but also in positions in which changes cause amino acid substitutions in coded proteins. Asymmetry in the silent positions of codons differentiates the rate of translation of mRNA produced from leading and lagging strands. Asymmetry in the amino acid composition of proteins resulting from replication-associated mutational pressure also corresponds to leading and lagging roles of DNA strands, whereas asymmetry connected with transcription and coding function corresponds to the distance of genes from the origin or terminus of chromosome replication.

The role of the genetic code in generating new coding sequences inside existing genes.

The genetic code has a very interesting property--it generates an open reading frame (ORF) inside a coding sequence, in a specific phase of the antisense strand with much higher probability than in the random DNA sequences. Furthermore, these antisense ORFs (A-ORFs) possess the same features as real genes--the asymmetry in the nucleotide composition at the first and second positions in codons. About two thirds of the 2997 overlapping ORFs in the yeast genome possess this feature. Thus, the question arises: has this feature of the genetic code been exploited in the evolution of genes? We have searched the FASTA data bases for homologies with the antisense translation products of a specific class of genes and we have found some sequences with relatively high homology. Many of them have scores which could be randomly found in the searched data bases with a probability lower than 10(-6). We conclude that some genes could arise by positioning a copy of the original gene under a promoter in the opposite direction in such a way that both, the original gene and its copy initially use the same nucleotides in the third, degenerated positions in codons.

Asymmetry of coding versus noncoding strand in coding sequences of different genomes.

We have used the asymmetry between the coding and noncoding strands in different codon positions of coding sequences of DNA as a parameter to evaluate the coding probability for open reading frames (ORFs). The method enables an approximation of the total number of coding ORFs in the set of analyzed sequences as well as an estimation of the coding probability for the ORFs. The asymmetry observed in the nucleotide composition of codons in coding sequences has been used successfully for analysis of the genomes completed at the time of this analysis.

Conjugational cotransfer of IncFI and IncI conjugative plasmids forming aggregate in a pathogenic Citrobacter freundii strain.

Two plasmids, pEM4 (IncFI) determining virulence and pEM6 (IncI) determining colicine I production and resistance to tetracycline, have been found in a pathogenic strain of Citrobacter freundii. Even though pEM4 and pEM6 plasmids are conjugative and transfer themselves very efficiently during conjugation, a high cotransfer of both plasmids is observed--an unusually high fraction of transconjugants acquiring pEM6 acquires pEM4 and vice versa. The observed cotransfer of these plasmids is connected with their ability to complement their conjugational functions. An insertion mutant of pEM4 with decreased frequency of conjugational transfer has been isolated. This mutant (pEM44) lost its ability to mobilize nonconjugative plasmids. Its transfer is stimulated by pEM6 and it is transferred from (pEM44, pEM6) donors almost exclusively with pEM6 plasmid. The role of cotransfer and stimulation phenomena in spreading of plasmid aggregates in bacterial population is discussed.

Mobilization of R387 Inc K conjugative plasmid by the conjugative plasmid R64 Inc I.

Plasmid aggregate (R387, R64) was constructed in E. coli K12 strain. Plasmid R387 Inc K was stimulated to conjugational transfer by plasmid R64 Inc I. This stimulation was caused neither by recombination between both plasmids nor by trans-complementation of R387 conjugational systems by gene(s) product(s) of R64 plasmid. The observed phenomenon resembled rather mobilization of nonconjugative plasmids by conjugative ones. As in mobilization, the observed increase in R387 transfer frequency could take place only when both interacting plasmids were present in donor cells. Moreover, the entry exclusion system functioning in recipient cells, toward stimulating R64 plasmid affected strongly the conjugational transfer of stimulated R387 plasmid. Analogous phenomenon was observed during mobilization of nonconjugative plasmids by conjugative ones.

Genetic properties of plasmids isolated from pathogenic strain of Citrobacter freundii.

It was found that Citrobacter freundii SLJ10 strain was an etiological factor of swine diarrhoea in North Poland. This strain harboured a plasmid aggregate consisting of two plasmids-pEM4 responsible for virulence and pEM6 for selective advantage of the strain, determining resistance to tetracycline and production of bacteriocin. These two plasmids cooperated in conjugal transfer and formed an infective block of genetic information, which could be transferred to other strains of Enterobacteriaceae and cause their virulence.

R factors of pathogenic strains of Escherichia coli isolated from infant diarrhoea.

Several drug resistance patterns were determined in 170 pathogenic strains of E. coli isolated in 6 Polish towns from infant diarrhoea. The most frequent were strains resistant to 5 different drugs: ampicillin, tetracycline, chloramphenicol, streptomycin and sulfonamide. Conjugative R factors of 30 strains of the same resistance pattern (Ap Tc Cm Sm Su) were characterised by determining their Fi(F) character, incompatibility and molecular weight.