Genome degradation is an ongoing process in Rickettsia.

To study reductive evolutionary processes in bacterial genomes, we examine sequences in the Rickettsia genomes which are unconstrained by selection and evolve as pseudogenes, one of which is the metK gene, which codes for AdoMet synthetase. Here, we sequenced the metK gene and three surrounding genes in eight different species of the genus Rickettsia. The metK gene was found to contain a high incidence of deletions in six lineages, while the three genes in its surroundings were functionally conserved in all eight lineages. A more drastic example of gene degradation was identified in the metK downstream region, which contained an open reading frame in Rickettsia felis. Remnants of this open reading frame could be reconstructed in five additional species by eliminating sites of frameshift mutations and termination codons. A detailed examination of the two reconstructed genes revealed that deletions strongly predominate over insertions and that there is a strong transition bias for point mutations which is coupled to an excess of GC-to-AT substitutions. Since the molecular evolution of these inactive genes should reflect the rates and patterns of neutral mutations, our results strongly suggest that there is a high spontaneous rate of deletions as well as a strong mutation bias toward AT pairs in the Rickettsia genomes. This may explain the low genomic G + C content (29%), the small genome size (1.1 Mb), and the high noncoding content (24%), as well as the presence of several pseudogenes in the Rickettsia prowazekii genome.     

UV-exposure,endogenous DNA damage,and DNA replication errors shape the spectra of genome changes in human skin

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.     

A molecular clock based on the expansion of gene families.

There is evidence to suggest that eukaryotic genomes are subject to frequent insertions and deletions of non-coding DNA. This may lead to a gradual increase or decrease in genome size, or to a dynamic equilibrium in which the overall size remains constant. We argue, however, that there is a bias favouring an accumulation of non-coding DNA in the proximity of genes. Such bias causes a progressive change in genome structure regardless of whether the overall genome size increases, decreases or remains constant. We show that this change may serve as a 'molecular clock', supplementing that provided by nucleotide substitution rates.     

Pseudogene evolution and natural selection for a compact genome

Pseudogenes are nonfunctional copies of protein-coding genes that are presumed to evolve without selective constraints on their coding function. They are of considerable utility in evolutionary genetics because, in the absence of selection, different types of mutations in pseudogenes should have equal probabilities of fixation. This theoretical inference justifies the estimation of patterns of spontaneous mutation from the analysis of patterns of substitutions in pseudogenes. Although it is possible to test whether pseudogene sequences evolve without constraints for their protein-coding function, it is much more difficult to ascertain whether pseudogenes may affect fitness in ways unrelated to their nucleotide sequence. Consider the possibility that a pseudogene affects fitness merely by increasing genome size. If a larger genome is deleterious--for example, because of increased energetic costs associated with genome replication and maintenance--then deletions, which decrease the length of a pseudogene, should be selectively advantageous relative to insertions or nucleotide substitutions. In this article we examine the implications of selection for genome size relative to small (1-400 bp) deletions, in light of empirical evidence pertaining to the size distribution of deletions observed in Drosophila and mammalian pseudogenes. There is a large difference in the deletion spectra between these organisms. We argue that this difference cannot easily be attributed to selection for overall genome size, since the magnitude of selection is unlikely to be strong enough to significantly affect the probability of fixation of small deletions in Drosophila.     

A phylogenetic analysis of indel dynamics in the cotton genus

Genome size evolution is a dynamic process involving counterbalancing mechanisms whose actions vary across lineages and over time. Whereas the primary mechanism of expansion, transposable element (TE) amplification, has been widely documented, the evolutionary dynamics of genome contraction have been less thoroughly explored. To evaluate the relative impact and evolutionary stability of the mechanisms that affect genome size, we conducted a phylogenetic analysis of indel rates for 2 genomic regions in 4 Gossypium genomes: the 2 coresident genomes (A(T) and D(T)) of tetraploid cotton and its model diploid progenitors, Gossypium arboreum (A) and Gossypium raimondii (D). We determined the rates of sequence gain or loss along each branch, partitioned by mechanism, and how these changed during species divergence. In general, there has been a propensity toward growth of the diploid genomes and contraction in the polyploid. Most of the size difference between the diploid species occurred prior to polyploid divergence and was largely attributable to TE amplification in the A/A(T) genome. After separating from the true parents of the polyploid genomes, both diploid genomes experienced slower sequence gain than in the ancestor, due to fewer TE insertions in the A genome and a combination of increased deletions and decreased TE insertions in the D genome. Both genomes of the polyploid displayed increased rates of deletion and decreased rates of insertion, leading to a rate of near stasis in D(T) and overall contraction in A(T) resulting in polyploid genome contraction. As expected, TE insertions contributed significantly to the genome size differences; however, intrastrand homologous recombination, although rare, had the most significant impact on the rate of deletion. Small indel data for the diploids suggest the possibility of a bias as the smaller genomes add less or delete more sequence through small indels than do the larger genomes, whereas data for the polyploid suggest increased sequence turnover in general (both as small deletions and small insertions). Illegitimate recombination, although not demonstrated to be a dominant mechanism of genome size change, was biased in the polyploid toward deletions, which may provide a partial explanation of polyploid genomic downsizing.     

Deletional bias and the evolution of bacterial genomes

Although bacteria increase their DNA content through horizontal transfer and gene duplication, their genomes remain small and, in particular, lack nonfunctional sequences. This pattern is most readily explained by a pervasive bias towards higher numbers of deletions than insertions. When selection is not strong enough to maintain them, genes are lost in large deletions or inactivated and subsequently eroded. Gene inactivation and loss are particularly apparent in obligate parasites and symbionts, in which dramatic reductions in genome size can result not from selection to lose DNA, but from decreased selection to maintain gene functionality. Here we discuss the evidence showing that deletional bias is a major force that shapes bacterial genomes.     

Genomic heterogeneity of background substitutional patterns in Drosophila melanogaster

Mutation is the underlying force that provides the variation upon which evolutionary forces can act. It is important to understand how mutation rates vary within genomes and how the probabilities of fixation of new mutations vary as well. If substitutional processes across the genome are heterogeneous, then examining patterns of coding sequence evolution without taking these underlying variations into account may be misleading. Here we present the first rigorous test of substitution rate heterogeneity in the Drosophila melanogaster genome using almost 1500 nonfunctional fragments of the transposable element DNAREP1_DM. Not only do our analyses suggest that substitutional patterns in heterochromatic and euchromatic sequences are different, but also they provide support in favor of a recombination-associated substitutional bias toward G and C in this species. The magnitude of this bias is entirely sufficient to explain recombination-associated patterns of codon usage on the autosomes of the D. melanogaster genome. We also document a bias toward lower GC content in the pattern of small insertions and deletions (indels). In addition, the GC content of noncoding DNA in Drosophila is higher than would be predicted on the basis of the pattern of nucleotide substitutions and small indels. However, we argue that the fast turnover of noncoding sequences in Drosophila makes it difficult to assess the importance of the GC biases in nucleotide substitutions and small indels in shaping the base composition of noncoding sequences.     

Recombination drives vertebrate genome contraction

Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Molecular nature of 11 spontaneous de novo mutations in Drosophila melanogaster

To investigate the molecular nature and rate of spontaneous mutation in Drosophila melanogaster, we screened 887,000 individuals for de novo recessive loss-of-function mutations at eight loci that affect eye color. In total, 28 mutants were found in 16 independent events (13 singletons and three clusters). The molecular nature of the 13 events was analyzed. Coding exons of the locus were affected by insertions or deletions >100 nucleotides long (6 events), short frameshift insertions or deletions (4 events), and replacement nucleotide substitutions (1 event). In the case of 2 mutant alleles, coding regions were not affected. Because approximately 70% of spontaneous de novo loss-of-function mutations in Homo sapiens are due to nucleotide substitutions within coding regions, insertions and deletions appear to play a much larger role in spontaneous mutation in D. melanogaster than in H. sapiens. If so, the per nucleotide mutation rate in D. melanogaster may be lower than in H. sapiens, even if their per locus mutation rates are similar.     

Rates and Genomic Consequences of Spontaneous Mutational Events in Drosophila melanogaster

Because spontaneous mutation is the source of all genetic diversity, measuring mutation rates can reveal how natural selection drives patterns of variation within and between species. We sequenced eight genomes produced by a mutation-accumulation experiment in Drosophila melanogaster. Our analysis reveals that point mutation and small indel rates vary significantly between the two different genetic backgrounds examined. We also find evidence that ∼2% of mutational events affect multiple closely spaced nucleotides. Unlike previous similar experiments, we were able to estimate genome-wide rates of large deletions and tandem duplications. These results suggest that, at least in inbred lines like those examined here, mutational pressures may result in net growth rather than contraction of the Drosophila genome. By comparing our mutation rate estimates to polymorphism data, we are able to estimate the fraction of new mutations that are eliminated by purifying selection. These results suggest that ∼99% of duplications and deletions are deleterious—making them 10 times more likely to be removed by selection than nonsynonymous mutations. Our results illuminate not only the rates of new small- and large-scale mutations, but also the selective forces that they encounter once they arise.     

Understanding the Differences between Genome Sequences of Escherichia coli B Strains REL606 and BL21(DE3) and Comparison of the E. coli B and K-12 Genomes

Each difference between the genome sequences of Escherichia coli B strains REL606 and BL21(DE3) can be interpreted in light of known laboratory manipulations plus a gene conversion between ribosomal RNA operons. Two treatments with 1-methyl-3-nitro-1-nitrosoguanidine in the REL606 lineage produced at least 93 single-base-pair mutations (∼ 90% GC-to-AT transitions) and 3 single-base-pair GC deletions. Two UV treatments in the BL21(DE3) lineage produced only 4 single-base-pair mutations but 16 large deletions. P1 transductions from K-12 into the two B lineages produced 317 single-base-pair differences and 9 insertions or deletions, reflecting differences between B DNA in BL21(DE3) and integrated restriction fragments of K-12 DNA inherited by REL606. Two sites showed selective enrichment of spontaneous mutations. No unselected spontaneous single-base-pair mutations were evident. The genome sequences revealed that a progenitor of REL606 had been misidentified, explaining initially perplexing differences. Limited sequencing of other B strains defined characteristic properties of B and allowed assembly of the inferred genome of the ancestral B of Delbrück and Luria. Comparison of the B and K-12 genomes shows that more than half of the 3793 proteins of their basic genomes are predicted to be identical, although ∼ 310 appear to be functional in either B or K-12 but not in both. The ancestral basic genome appears to have had ∼ 4039 coding sequences occupying ∼ 4.0 Mbp. Repeated horizontal transfer from diverged Escherichia coli genomes and homologous recombination may explain the observed variable distribution of single-base-pair differences. Fifteen sites are occupied by phage-related elements, but only six by comparable elements at the same site. More than 50 sites are occupied by IS elements in both B and K, 16 in common, and likely founding IS elements are identified. A signature of widespread cryptic phage P4-type mobile elements was identified. Complex deletions (dense clusters of small deletions and substitutions) apparently removed nonessential genes from ∼ 30 sites in the basic genomes.     

Patterns of Insertion and Deletion in Mammalian Genomes

Nucleotide insertions and deletions (indels) are responsible for gaps in the sequence alignments. Indel is one of the major sources of evolutionary change at the molecular level. We have examined the patterns of insertions and deletions in the 19 mammalian genomes, and found that deletion events are more common than insertions in the mammalian genomes. Both the number of insertions and deletions decrease rapidly when the gap length increases and single nucleotide indel is the most frequent in all indel events. The frequencies of both insertions and deletions can be described well by power law.Key Words: Insertion, deletion, gap, indel, mammalian genome.     

Plant genome evolution: lessons from comparative genomics at the DNA level

Angiosperm genomes show tremendous variability in genome size and chromosome number. Nevertheless, comparative genetic mapping has revealed genome collinearity of closely related species. Sequence-based comparisons were used to assess the conservation of gene arrangements. Numerous small rearrangements, insertions/deletions, duplications, inversions and translocations have been detected. Importantly, comparative sequence analyses have unambiguously shown micro-collinearity of distantly related plant species. Duplications and subsequent gene loss have been identified as a particular important factor in the evolution of plant genomes.     

Pseudogenes, junk DNA, and the dynamics of Rickettsia genomes

Studies of neutrally evolving sequences suggest that differences in eukaryotic genome sizes result from different rates of DNA loss. However, very few pseudogenes have been identified in microbial species, and the processes whereby genes and genomes deteriorate in bacteria remain largely unresolved. The typhus-causing agent, Rickettsia prowazekii, is exceptional in that as much as 24% of its 1.1-Mb genome consists of noncoding DNA and pseudogenes. To test the hypothesis that the noncoding DNA in the R. prowazekii genome represents degraded remnants of ancestral genes, we systematically examined all of the identified pseudogenes and their flanking sequences in three additional Rickettsia species. Consistent with the hypothesis, we observe sequence similarities between genes and pseudogenes in one species and intergenic DNA in another species. We show that the frequencies and average sizes of deletions are larger than insertions in neutrally evolving pseudogene sequences. Our results suggest that inactivated genetic material in the Rickettsia genomes deteriorates spontaneously due to a mutation bias for deletions and that the noncoding sequences represent DNA in the final stages of this degenerative process.     

Genomic rearrangements by LINE-1 insertion-mediated deletion in the human and chimpanzee lineages

Long INterspersed Elements (LINE-1s or L1s) are abundant non-LTR retrotransposons in mammalian genomes that are capable of insertional mutagenesis. They have been associated with target site deletions upon insertion in cell culture studies of retrotransposition. Here, we report 50 deletion events in the human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ~18 kb of sequence from the human genome and ~15 kb from the chimpanzee genome. Our data suggest that during the primate radiation, L1 insertions may have deleted up to 7.5 Mb of target genomic sequences. While the results of our in vivo analysis differ from those of previous cell culture assays of L1 insertion-mediated deletions in terms of the size and rate of sequence deletion, evolutionary factors can reconcile the differences. We report a pattern of genomic deletion sizes similar to those created during the retrotransposition of Alu elements. Our study provides support for the existence of different mechanisms for small and large L1-mediated deletions, and we present a model for the correlation of L1 element size and the corresponding deletion size. In addition, we show that internal rearrangements can modify L1 structure during retrotransposition events associated with large deletions.     

Reductive genome evolution in chemoautotrophic intracellular symbionts of deep-sea <Emphasis Type="Italic">Calyptogena</Emphasis> clams

To understand reductive genome evolution (RGE), we comparatively analyzed the recently reported small genomes of two chemoautotrophic, intracellular symbionts of deep-sea clams, Calyptogena okutanii and C. magnifica. Both genomes lack most genes for DNA recombination and repair such as recA and mutY. Their genome architectures were highly conserved except one inversion. Many deletions from small (<100 bp) to large (1–11 kbp) sizes were detected and the deletion numbers decreased exponentially with size. Densities of deletions and short-repeats, as well as A+T content were higher in non-coding regions than in coding regions. Because Calyptogena symbiont genomes lack recA, we propose that deletions and the single inversion occurred by RecA-independent recombination (RIR) at short-repeats with simultaneous consumption of repeats, and that short-repeats were regenerated by accelerated mutations with enhanced A+T bias due to the absence of mutY. We further propose that extant Calyptogena symbiont genomes are in an actively reducing stage of RGE consisting of small and large deletions, and the deletions are caused by short-repeat dependent RIR along with regeneration of short-repeats. In future, the RGE rate will slowdown when the gene repertoires approach the minimum gene set necessary for intracellular symbiotic life. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. Kuwahara and Y. Takaki equally contributed to this work.     

Patterns of nucleotide substitution,insertion and deletion in the human genome inferred from pseudogenes

Nucleotide substitution, insertion and deletion (indel) events are the major driving forces that have shaped genomes. Using the recently identified human ribosomal protein (RP) pseudogene sequences, we have thoroughly studied DNA mutation patterns in the human genome. We analyzed a total of 1726 processed RP pseudogene sequences, comprising more than 700 000 bases. To be sure to differentiate the sequence changes occurring in the functional genes during evolution from those occurring in pseudogenes after they were fixed in the genome, we used only pseudogene sequences originating from parts of RP genes that are identical in human and mouse. Overall, we found that nucleotide transitions are more common than transversions, by roughly a factor of two. Moreover, the substitution rates amongst the 12 possible nucleotide pairs are not homogeneous as they are affected by the type of immediately neighboring nucleotides and the overall local G+C content. Finally, our dataset is large enough that it has many indels, thus allowing for the first time statistically robust analysis of these events. Overall, we found that deletions are about three times more common than insertions (3740 versus 1291). The frequencies of both these events follow characteristic power–law behavior associated with the size of the indel. However, unexpectedly, the frequency of 3 bp deletions (in contrast to 3 bp insertions) violates this trend, being considerably higher than that of 2 bp deletions. The possible biological implications of such a 3 bp bias are discussed.     

Substitution, Insertion, Deletion, Suppression, and Altered Substrate Specificity in Functional Protocatechuate 3,4-Dioxygenases

Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for Acinetobacter strains containing spontaneous mutations blocking expression of pcaH or -G, genes encoding the alpha and beta subunits of protocatechuate 3, 4-dioxygenase. The crystal structure of the Acinetobacter oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivating Acinetobacter protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate Acinetobacter strains in which mutations within pcaH or -G cause a heat-sensitive phenotype. These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in pcaH or -G were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into the Acinetobacter chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.     

Rapid sequence turnover at an intergenic locus in Drosophila

Closely related species of Drosophila tend to have similar genome sizes. The strong imbalance in favor of small deletions relative to insertions implies that the unconstrained DNA in Drosophila is unlikely to be passively inherited from even closely related ancestors, and yet most DNA in Drosophila genomes is intergenic and potentially unconstrained. In an attempt to investigate the maintenance of this intergenic DNA, we studied the evolution of an intergenic locus on the fourth chromosome of the Drosophila melanogaster genome. This 1.2-kb locus is marked by two distinct, large insertion events: a nuclear transposition of a mitochondrial sequence and a transposition of a nonautonomous DNA transposon DNAREP1_DM. Because we could trace the evolutionary histories of these sequences, we were able to reconstruct the length evolution of this region in some detail. We sequenced this locus in all four species of the D. melanogaster species complex: D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. Although this locus is similar in size in these four species, less than 10% of the sequence from the most recent common ancestor remains in D. melanogaster and all of its sister species. This region appears to have increased in size through several distinct insertions in the ancestor of the D. melanogaster species complex and has been shrinking since the split of these lineages. In addition, we found no evidence suggesting that the size of this locus has been maintained over evolutionary time; these results are consistent with the model of a dynamic equilibrium between persistent DNA loss through small deletions and more sporadic DNA gain through less frequent but longer insertions. The apparent stability of genome size in Drosophila may belie very rapid sequence turnover at intergenic loci.