Standard deviations and correlations of GC levels in DNA sequences

In a DNA sequence that exhibits long-range correlations, standard deviations among the GC levels of its segments can be up to an order of magnitude higher than in a sequence consisting of independent, identically distributed nucleotides. Conversely, plots of inter-segment standard deviations vs. segment length reveal quantitative information about the correlations present in a sequence. We present and discuss formulae that relate long-range (power-law) correlations between the nucleotides of a sequence to the expected standard deviations of the GC levels of its segments, and to the correlations between them.     

Progressive structural transitions within Mu transpositional complexes

Assembly of the Mu transpososome is dependent on specific binding sites for the MuA transposase near the ends of the phage genome. MuA also contacts terminal nucleotides but only upon transpososome assembly, and base-specific recognition of the terminal nucleotides is critical for assembly. We show that Mu ends lacking the terminal 5 bp can form transpososomes, while longer DNA substrates with mutated terminal nucleotides cannot. The impact of the mutations can be suppressed by base mismatches near the end of Mu. Deletion of the flanking strands or mutation of the terminal nucleotides has differential effects on the cleavage and strand transfer reactions. These results show that the terminal nucleotides control the assembly and activation of transpososomes by influencing conformational changes around the active site.     

The phage Mu transpososome core: DNA requirements for assembly and function.

The two chemical steps of phage Mu transpositional recombination, donor DNA cleavage and strand transfer, take place within higher order protein-DNA complexes called transpososomes. At the core of these complexes is a tetramer of MuA (the transposase), bound to the two ends of the Mu genome. While transpososome assembly normally requires a number of cofactors, under certain conditions only MuA and a short DNA fragment are required. DNA requirements for this process, as well as the stability and activity of the ensuing complexes, were established. The divalent cation normally required for assembly of the stable complex could be omitted if the substrate was prenicked, if the flanking DNA was very short or if the two flanking strands were non-complementary. The presence of a single nucleotide beyond the Mu genome end on the non-cut strand was critical for transpososome stability. Donor cleavage additionally required at least two flanking nucleotides on the strand to be cleaved. The flanking DNA double helix was destabilized, implying distortion of the DNA near the active site. Although donor cleavage required Mg2+, strand transfer took place in the presence of Ca2+ as well, suggesting a conformational difference in the active site for the two chemical steps.     

Bacteriophage Mu DNA replication in vitro

An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Mu replication was monitored by DNA hybridization. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Mu DNA synthesis is semidiscontinuous. In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA. The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand. Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. However, the host Okazaki pieces hybridize to both Mu strands symmetrically. Most, if not all, host sequences were represented in mature Mu viral DNA. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right.     

Strong asymmetric mutation bias in endosymbiont genomes coincide with loss of genes for replication restart pathways

A large majority of bacterial genomes show strand asymmetry, such that G and T preferentially accumulate on the leading strand. The mechanisms are unknown, but cytosine deaminations are thought to play an important role. Here, we have examined DNA strand asymmetry in three strains of the aphid endosymbiont Buchnera aphidicola. These are phylogenetically related, have similar genomic GC contents, and conserved gene order structures, yet B. aphidicola (Bp) shows a fourfold higher replication-induced strand bias than B. aphidicola (Sg) and (Ap). We rule out an increase in the overall substitution frequency as the major cause of the stronger strand bias in B. aphidicola (Bp). Instead, the results suggest that the higher GC skew in this species is caused by a different spectrum of mutations, including a relatively higher frequency of C to T mutations on the leading strand and/or of G to A mutations on the lagging strand. A comparative analysis of 20 gamma-proteobacterial genomes revealed that endosymbiont genomes lacking recA and other genes involved in replication restart processes, such as priA, which codes for primosomal helicase PriA, displayed the strongest strand bias. We hypothesize that cytosine deaminations accumulate during single-strand exposure at arrested replication forks and that inefficient restart mechanisms may lead to high DNA strand asymmetry in bacterial genomes.     

Is GC bias in the nuclear genome of the carnivorous plant Utricularia?driven by ROS-based mutation and biased gene conversion?

At less than 90 Mbp, the tiny nuclear genome of the carnivorous bladderwort plant Utricularia is an attractive model system for studying molecular evolutionary processes leading to genome miniaturization. Recently, we reported that expression of genes encoding DNA repair and reactive oxygen species (ROS) detoxification enzymes is highest in Utricularia traps, and we argued that ROS mutagenic action correlates with the high nucleotide substitution rates observed in the Utricularia plastid, mitochondrial, and nuclear genomes. Here, we extend our analysis of 100 nuclear genes from Utricularia and related asterid eudicots to examine nucleotide substitution biases and their potential correlation with ROS-induced DNA lesions. We discovered an unusual bias toward GC nucleotides, most prominently in transition substitutions at the third position of codons, which are presumably silent with respect to adaptation. Given the general tendency of biased gene conversion to drive GC bias, and of ROS to induce double strand breaks requiring recombinational repair, we propose that some of the unusual features of the bladderwort and its genome may be more reflective of these nonadaptive processes than of natural selection.     

TUCAN/CARDINAL and DRAL participate in a common pathway for modulation of NF-kappaB activation

Prior to genome sequencing, information on base composition (GC level) and its variation in mammalian genomes could be obtained using density gradient ultracentrifugation. Analyses using this approach led to the conclusion that mammalian genomes are organized into mosaics of fairly homogeneous regions, called isochores. We present an initial compositional overview of the chromosomes of the recently available draft human genome sequence, in the form of color-coded moving window plots and corresponding GC level histograms. Results obtained from the draft human genome sequence agree well with those obtained or deduced earlier from CsCl experiments. The draft sequence now permits the visualization of the mosaic organization of the human genome at the DNA sequence level.     

Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.     

Correlation approach to identify coding regions in DNA sequences.

Recently, it was observed that noncoding regions of DNA sequences possess long-range power-law correlations, whereas coding regions typically display only short-range correlations. We develop an algorithm based on this finding that enables investigators to perform a statistical analysis on long DNA sequences to locate possible coding regions. The algorithm is particularly successful in predicting the location of lengthy coding regions. For example, for the complete genome of yeast chromosome III (315,344 nucleotides), at least 82% of the predictions correspond to putative coding regions; the algorithm correctly identified all coding regions larger than 3000 nucleotides, 92% of coding regions between 2000 and 3000 nucleotides long, and 79% of coding regions between 1000 and 2000 nucleotides. The predictive ability of this new algorithm supports the claim that there is a fundamental difference in the correlation property between coding and noncoding sequences. This algorithm, which is not species-dependent, can be implemented with other techniques for rapidly and accurately locating relatively long coding regions in genomic sequences.     

The orientation bias of Chi sequences is a general tendency of G-rich oligomers

The Chi sequences are specific oligomers that stimulate DNA repair by homologous recombination, and are different sequences in each organism. Approximately 75% of the copies of the Chi sequence (5'-GCTGGTGG-3') of Escherichia coli reside on the leading strand, and this orientation bias is often believed to be a consequence of the biological role of Chi sequences as the signal sequence of RecBCD pathway in DNA replication. However, our computer analysis found that many G-rich oligomers also show this asymmetric orientation pattern. The shift in the Chi orientation bias appears around the replication origin and terminus, but these locations are also coincident with the shift points in GC content or GC skew. We conducted the same analysis with the genome of Bacillus subtilis, and found that in addition to Chi, other G-rich oligomers show similar asymmetric orientation patterns, whose shift points were coincident with those of the GC skew. However, the genome of Haemophilus influenzae Rd, whose GC skew is not so pronounced, does not clearly show asymmetric orientation patterns of Chi or other G-rich oligomers. These results lead us to suggest that the uneven distribution of the Chi orientation between the two strands of the double helix is mostly due to the uneven distribution of G content (GC skew) and that the replication-related function of Chi sequences is not the primary factor responsible for the evolutionary pressure causing the orientation bias.     

Cre induces an asymmetric DNA bend in its target loxP site

Cre initiates recombination by preferentially exchanging the bottom strands of the loxP site to form a Holliday intermediate, which is then resolved on the top strands. We previously found that the scissile AT and GC base pairs immediately 5' to the scissile phosphodiester bonds are critical in determining this order of strand exchange. We report here that the scissile base pairs also influence the Cre-induced DNA bends, the position of which correlates with the initial site of strand exchange. The binding of one Cre molecule to a loxP site induces a approximately 35 degrees asymmetric bend adjacent to the scissile GC base pair. The binding of two Cre molecules to a loxP site induces a approximately 55 degrees asymmetric bend near the center of the spacer region with a slight bias toward the scissile A. Lys-86, which contacts the scissile nucleotides, is important for establishing the bend near the scissile GC base pair when one Cre molecule is bound but has little role in positioning the bend when two Cre molecules are bound to a loxP site. We present a model relating the position of the Cre-induced bends to the order of strand exchange in the Cre-catalyzed recombination reaction.     

DNA asymmetric strand bias affects the amino acid composition of mitochondrial proteins.

Variations in GC content between genomes have been extensively documented. Genomes with comparable GC contents can, however, still differ in the apportionment of the G and C nucleotides between the two DNA strands. This asymmetric strand bias is known as GC skew. Here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. We compared orthologous genes between animal mitochondrial genomes that show large differences in GC and AT skews. Specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative GC skew and a positive AT skew, to those of flatworms, which show the opposite skews for both GC and AT base pairs. We found that the mammalian proteins are highly enriched in amino acids encoded by CA-rich codons (as predicted by their negative GC and positive AT skews), whereas their flatworm orthologs were enriched in amino acids encoded by GT-rich codons (also as predicted from their skews). We found that these differences in mitochondrial strand asymmetry (measured as GC and AT skews) can have very large, predictable effects on the composition of the encoded proteins.     

Two ancient bacterial endosymbionts have coevolved with the planthoppers (Insecta: Hemiptera: Fulgoroidea)

Background

Pseudoscorpions are chelicerates and have historically been viewed as being most closely related to solifuges, harvestmen, and scorpions. No mitochondrial genomes of pseudoscorpions have been published, but the mitochondrial genomes of some lineages of Chelicerata possess unusual features, including short rRNA genes and tRNA genes that lack sequence to encode arms of the canonical cloverleaf-shaped tRNA. Additionally, some chelicerates possess an atypical guanine-thymine nucleotide bias on the major coding strand of their mitochondrial genomes.

Results

We sequenced the mitochondrial genomes of two divergent taxa from the chelicerate order Pseudoscorpiones. We find that these genomes possess unusually short tRNA genes that do not encode cloverleaf-shaped tRNA structures. Indeed, in one genome, all 22 tRNA genes lack sequence to encode canonical cloverleaf structures. We also find that the large ribosomal RNA genes are substantially shorter than those of most arthropods. We inferred secondary structures of the LSU rRNAs from both pseudoscorpions, and find that they have lost multiple helices. Based on comparisons with the crystal structure of the bacterial ribosome, two of these helices were likely contact points with tRNA T-arms or D-arms as they pass through the ribosome during protein synthesis. The mitochondrial gene arrangements of both pseudoscorpions differ from the ancestral chelicerate gene arrangement. One genome is rearranged with respect to the location of protein-coding genes, the small rRNA gene, and at least 8 tRNA genes. The other genome contains 6 tRNA genes in novel locations. Most chelicerates with rearranged mitochondrial genes show a genome-wide reversal of the CA nucleotide bias typical for arthropods on their major coding strand, and instead possess a GT bias. Yet despite their extensive rearrangement, these pseudoscorpion mitochondrial genomes possess a CA bias on the major coding strand. Phylogenetic analyses of all 13 mitochondrial protein-coding gene sequences consistently yield trees that place pseudoscorpions as sister to acariform mites.

Conclusion

The well-supported phylogenetic placement of pseudoscorpions as sister to Acariformes differs from some previous analyses based on morphology. However, these two lineages share multiple molecular evolutionary traits, including substantial mitochondrial genome rearrangements, extensive nucleotide substitution, and loss of helices in their inferred tRNA and rRNA structures.     

Genome DNA Sequence Variation, Evolution, and Function in Bacteria and Archaea

Comparative genomics has revealed that variations in bacterial and archaeal genome DNA sequences cannot be explained by only neutral mutations. Virus resistance and plasmid distribution systems have resulted in changes in bacterial and archaeal genome sequences during evolution. The restriction-modification system, a virus resistance system, leads to avoidance of palindromic DNA sequences in genomes. Clustered, regularly interspaced, short palindromic repeats (CRISPRs) found in genomes represent yet another virus resistance system. Comparative genomics has shown that bacteria and archaea have failed to gain any DNA with GC content higher than the GC content of their chromosomes. Thus, horizontally transferred DNA regions have lower GC content than the host chromosomal DNA does. Some nucleoid-associated proteins bind DNA regions with low GC content and inhibit the expression of genes contained in those regions. This form of gene repression is another type of virus resistance system. On the other hand, bacteria and archaea have used plasmids to gain additional genes. Virus resistance systems influence plasmid distribution. Interestingly, the restriction-modification system and nucleoid-associated protein genes have been distributed via plasmids. Thus, GC content and genomic signatures do not reflect bacterial and archaeal evolutionary relationships.     

Genome phylogeny based on short-range correlations in DNA sequences.

The surprising fact that global statistical properties computed on a genomewide scale may reveal species information has first been observed in studies of dinucleotide frequencies. Here we will look at the same phenomenon with a totally different statistical approach. We show that patterns in the short-range statistical correlations in DNA sequences serve as evolutionary fingerprints of eukaryotes. All chromosomes of a species display the same characteristic pattern, markedly different from those of other species. The chromosomes of a species are sorted onto the same branch of a phylogenetic tree due to this correlation pattern. The average correlation between nucleotides at a distance k is quantified in two independent ways: (i) by estimating it from a higher-order Markov process and (ii) by computing the mutual information function at a distance k. We show how the quality of phylogenetic reconstruction depends on the range of correlation strengths and on the length of the underlying sequence segment. This concept of the correlation pattern as a phylogenetic signature of eukaryote species combines two rather distant domains of research, namely phylogenetic analysis based on molecular observation and the study of the correlation structure of DNA sequences.     

Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10(4) to 10(6) CFU/microg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Efficient identification and regional positioning of YAC and cosmid clones to human Chromosome 21 by radiation fusion hybrids

The use of integrated mapping strategies involving bacterial, yeast, and rodent cells as hosts simplifies the construction of maps, which combine long-range order, high resolution, and easy access to the cloned DNA. Radiation-fusion hybrids offer a specially powerful long-range mapping system for human chromosomes. We describe here techniques for establishing a radiation-fusion hybrid map of Chromsome (Chr) 21 q and its integration with local information on YAC and cosmid positions.