Conservation and divergence in multigene families: alternatives to selection and drift

It is generally assumed that conservation and divergence of DNA signify function (selection) and no function (drift), respectively. This assumption is based on the view that a mutation is a unique event on a single chromosome, the fate of which depends on selection or drift. Knowledge of the rates, units and biases of widespread mechanisms of non-reciprocal DNA exchange, in particular within multigene families, provides alternative explanations for conservation and divergence, notwithstanding biological function. Such mechanisms of DNA turnover cause continual fluctuations in the copy-number of variant genes in an individual and, hence, promote the gradual and cohesive spread of a variant gene throughout a family (homogenization) and throughout a population (fixation). The dual processes (molecular drive) of homogenization and fixation are inextricably linked. Data are presented of the expected stages of transition in the spread of variant repeats by molecular drive in some non-genic families of DNA, seemingly not under the influence of selection. When a molecularly driven change in a given gene family is accompanied by the coevolution (mediated by selection) of other DNA, RNA or protein molecules that interact with the gene family then biological function is observed to be maintained despite sequence divergence. Conversely, the mechanics of DNA turnover and a turnover bias in favour of ancestral sequences can dramatically retard the rate of sequence change, in the absence of function. Examples of the maintenance of function by molecular coevolution and conservation of sequences in the absence of function, are drawn mainly from the rDNA multigene family.     

High-throughput analysis of satellite DNA in the grasshopper <Emphasis Type="Italic">Pyrgomorpha conica</Emphasis> reveals abundance of homologous and heterologous higher-order repeats

Satellite DNA (satDNA) constitutes an important fraction of repetitive DNA in eukaryotic genomes, but it is barely known in most species. The high-throughput analysis of satDNA in the grasshopper Pyrgomorpha conica revealed 87 satDNA variants grouped into 76 different families, representing 9.4% of the genome. Fluorescent in situ hybridization (FISH) analysis of the 38 most abundant satDNA families revealed four different patterns of chromosome distribution. Homology search between the 76 satDNA families showed the existence of 15 superfamilies, each including two or more families, with the most abundant superfamily representing more than 80% of all satDNA found in this species. This also revealed the presence of two types of higher-order repeats (HORs), one showing internal homologous subrepeats, as conventional HORs, and an additional type showing non-homologous internal subrepeats, the latter arising by the combination of a given satDNA family with a non-annotated sequence, or with telomeric DNA. Interestingly, the heterologous subrepeats included in these HORs showed higher divergence within the HOR than outside it, suggesting that heterologous HORs show poor homogenization, in high contrast with conventional (homologous) HORs. Finally, heterologous HORs can show high differences in divergence between their constituent subrepeats, suggesting the possibility of regional homogenization.     

Aspects of nonrandom turnover involved in the concerted evolution of intergenic spacers within the ribosomal DNA of Drosophila melanogaster

Polymerase chain reaction (PCR)-amplified, sequenced, and digitally typed intergenic spacers (IGSs) of the ribosomal (r)DNA in D. melanogaster reveal unexpected features of the mechanisms of turnover involved with the concerted evolution of the gene family. Characterization of the structure of three isolated IGS length variants reveals breakage hot spots within the 330-base-pair (bp) subrepeat array found in the spacers. Internal mapping of variant repeats within the 240-bp subrepeat array using a novel digital DNA typing procedure (minisatellite variant repeat [MVR]-PCR) shows an unexpected pattern of clustering of variant repeats. Each 240-bp subrepeat array consists of essentially two halves with the repeats in each half identified by specific mutations. This bipartite structure, observed in a cloned IGS unit, in the majority of genomic DNA of laboratory and wild flies and in PCR-amplified products, has been widely homogenized yet is not predicted by a model of unequal crossing over with randomly placed recombination breakpoints. Furthermore, wild populations contain large numbers of length variants in contrast to uniformly shared length variants in laboratory stocks. High numbers of length variants coupled to the observation of a homogenized bipartite structure of the 240-bp subrepeat array suggest that the unit of turnover and homogenization is smaller than the IGS and might involve gene conversion. The use of PCR for the structural analysis of members of the rDNA gene family coupled to digital DNA typing provides powerful new inroads into the mechanisms of DNA turnover affecting the course of molecular evolution in this family. Correspondence to: G. A. Dover     

Interhomologue sequence variation of alpha satellite DNA from human chromosome 17: Evidence for concerted evolution along haplotypic lineages

Alpha satellite DNA is a family of tandemly repeated DNA found at the centromeres of all primate chromosomes. Different human chromosomes 17 in the population are characterized by distinct alpha satellite haplotypes, distinguished by the presence of variant repeat forms that have precise monomeric deletions. Pairwise comparisons of sequence diversity between variant repeat units from each haplotype show that they are closely related in sequence. Direct sequencing of PCR-amplified alpha satellite reveals heterogeneous positions between the repeat units on a chromosome as two bands at the same position on a sequencing ladder. No variation was detected in the sequence and location of these heterogeneous positions between chromosomes 17 from the same haplotype, but distinct patterns of variation were detected between chromosomes from different haplotypes. Subsequent sequence analysis of individual repeats from each haplotype confirmed the presence of extensive haplotype-specific sequence variation. Phylogenetic inference yielded a tree that suggests these chromosome 17 repeat units evolve principally along haplotypic lineages. These studies allow insight into the relative rates and/or timing of genetic turnover processes that lead to the homogenization of tandem DNA families. Correspondence to: H.F. Willard     

Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae

Combining phylogenetic reconstructions of species relationships with comparative genomic approaches is a powerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct the history of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis of plastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii, N. forgetiana and N. bonariensis (called the n = 9 group) and another containing N. plumbaginifolia and N. longiflora (called the n = 10 group). Despite little plastid DNA sequence divergence, we observed, via fluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosome numbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA (rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. We compared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in the distribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNA associated with divergence of the species in section Alatae. We suggest that sequence homogenization has replaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs more slowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (at least two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapid rate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not only associated with polyploidy.     

Concerted evolution of light satellite DNA in genus Mus implies amplification and homogenization of large blocks of repeats

Light satellite DNA components present in species belonging to the genus Mus and to related murids were studied using the Southern blot technique. The results show species variations in both the amount and periodic structure of the repeating units, which suggests that families of related higher-order repeats developed in a common ancestor and were then amplified and/or deleted to different extents during the subsequent evolutionary period. Although the patterns generated by a series of type B enzymes (restriction enzymes that possess sites in a limited number of segments making up the total satellite DNA) in the species closely related to the M. musculus complex were very similar, sequence analysis of cloned unit repeats in two of these species (M. musculus domesticus and M. spretoides) showed near fixation of species-diagnostic variant nucleotides. This suggests that the important amplification and homogenization events that occurred after the divergence of M. spretus must have involved large blocks of sequences.     

Patterns of intra- and interarray sequence variation in alpha satellite from the human X chromosome: evidence for short-range homogenization of tandemly repeated DNA sequences

A number of processes, such as sequence conversion, unequal crossingover, and molecular drive, have been postulated to explain the homogenization of tandemly repeated DNA families. To investigate the nature and extent of such processes in the alpha satellite family of centromeric DNA, we determined the nucleotide sequence of approximately 700 bp from each of 40 representative alpha satellite repeats from six sources of human X chromosomes, obtaining a total of approximately 28 kb of sequence data. Sequence divergence among the repeats examined was low, with an average pairwise difference of approximately 1%. Pairwise comparisons of all repeats indicate that the degree of similarity for those repeats in physical proximity (within approximately 15 kb) of each other is significantly greater than that for randomly located repeats, from either the same or different X chromosomes, suggesting that the mechanisms predicted to homogenize these arrays are effectively short-range in action. Analysis of individual patterns of sequence variation allows the assignment of haplotypes for five high-copy-number diagnostic positions and reveals distinct positions of equilibrium and disequilibrium within the repeat. These analyses address hypotheses about the origin of the observed patterns of variation throughout alpha satellite evolution.     

Repetitive DNA and chromosome evolution in plants

Most higher plant genomes contain a high proportion of repeated sequences. Thus repetitive DNA is a major contributor to plant chromosome structure. The variation in total DNA content between species is due mostly to variation in repeated DNA content. Some repeats of the same family are arranged in tandem arrays, at the sites of heterochromatin. Examples from the Secale genus are described. Arrays of the same sequence are often present at many chromosomal sites. Heterochromatin often contains arrays of several unrelated sequences. The evolution of such arrays in populations is discussed. Other repeats are dispersed at many locations in the chromosomes. Many are likely to be or have evolved from transposable elements. The structures of some plant transposable elements, in particular the sequences of the terminal inverted repeats, are described. Some elements in soybean, antirrhinum and maize have the same inverted terminal repeat sequences. Other elements of maize and wheat share terminal homology with elements from yeast, Drosophila, man and mouse. The evolution of transposable elements in plant populations is discussed. The amplification, deletion and transposition of different repeated DNA sequences and the spread of the mutations in populations produces a turnover of repetitive DNA during evolution. This turnover process and the molecular mechanisms involved are discussed and shown to be responsible for divergence of chromosome structure between species. Turnover of repeated genes also occurs. The molecular processes affecting repeats imply that the older a repetitive DNA family the more likely it is to exist in different forms and in many locations within a species. Examples to support this hypothesis are provided from the Secale genus.     

Monitoring the rate and dynamics of concerted evolution in the ribosomal DNA repeats of Saccharomyces cerevisiae using experimental evolution

Concerted evolution describes the unusual evolutionary pattern exhibited by certain repetitive sequences, whereby all the repeats are maintained in the genome with very similar sequences but differ between related species. The pattern of concerted evolution is thought to result from continual turnover of repeats by recombination, a process known as homogenization. Approaches to studying concerted evolution have largely been observational because of difficulties investigating repeat evolution in an experimental setting with large arrays of identical repeats. Here, we establish an experimental evolution approach to look at the rate and dynamics of concerted evolution in the ribosomal DNA (rDNA) repeats. A small targeted mutation was made in the spacer of a single rDNA unit in Saccharomyces cerevisiae so we could monitor the fate of this unit without the need for a selectable marker. The rate of loss of this single unit was determined, and the frequency of duplication was also estimated. The results show that duplication and deletion events occur at similar rates and are very common: An rDNA unit may be gained or lost as frequently as once every cell division. Investigation of the spatial dynamics of rDNA turnover showed that when the tagged repeat unit was duplicated, the copy predominantly, but not exclusively, ended up near to the tagged repeat. This suggests that variants in the rDNA spread in a semiclustered fashion. Surprisingly, large deletions that remove a significant fraction of total rDNA repeats were frequently found. We propose these large deletions are a driving force of concerted evolution, acting to increase homogenization efficiency over-and-above that afforded by turnover of individual rDNA units. Thus, the results presented here enhance our understanding of concerted evolution by offering insights into both the spatial and temporal dynamics of the homogenization process and suggest an important new aspect in our understanding of concerted evolution.     

Kinetic basis of spontaneous mutation. Misinsertion frequencies, proofreading specificities and cost of proofreading by DNA polymerases of Escherichia coli

Repeating members of multiple-copy sequence families display high levels of sequence homogeneity. In order to examine the rates at which this is achieved, and to compare the rates with those assessed for the ribosomal DNA and histone gene families (Coen et al., 1982, accompanying paper), we have examined the patterns of variation in the Drosophila melanogaster species subgroup for the “complex” noncoding families of high copy-number. Our analysis reveals that the evolution of some of the families has involved the gradual replacement of ancestral repeats by variant repeats, independently within each species. Hybridizations between genomes at different levels of stringency indicate the presence of two basic ancestral families (the “500” and “360” families) within the subgroup. The majority of repeats representative of these families can be characterized by restriction sites and patterns of organization that are uniquely diagnostic for each species, excepting the two most closely related species. Drosophila mauritiana and Drosophila simulans. Another family (the “180” family) is confined to the one species. Drosophila orena, with features suggestive of a more rapid origin. The wide karyotypic distribution of some members of the 500 and 180 families, revealed by hybridization in situ, shows that chromosomes are evolving in concert with respect to gradual and rapidly evolving families. The distribution of sequence and pattern variation within the subgroup shows that the time required for gradual fixation (concerted evolution) of variants within large families, distributed throughout the karyotype, is longer than that required for the smaller and chromosomally restricted families of rDNA and histone genes (Coen et al., 1982). We discuss the forces that might either accelerate or retard the fixation of variants in karyotypically dispersed families.     

Unisexuality and Molecular Drive: Bag320 Sequence Diversity in <Emphasis Type="Italic">Bacillus</Emphasis> Taxa (Insecta Phasmatodea)

Satellite DNA variability follows a pattern of concerted evolution through homogenization of new variants by genomic turnover mechanisms and variant fixation by chromosome redistribution into new combinations with the sexual process. Bacillus taxa share the same Bag320 satellite family and their reproduction ranges from strict bisexuality (B. grandii) to automictic (B. atticus) and apomictic (B. whitei = rossius/ grandii; B. lynceorum = rossius/grandii/atticus) unisexuality. Thelytokous reproduction clearly allows uncoupling of homogenization from fixation. Both trends and absolute values of satellite variability were analyzed in all Bacillus taxa but B. rossius, on 906 sequenced monomers at all level of comparisons: intraspecimen, intrapopulation, interpopulation, intersubspecies, and interspecies. For unisexuals, allozymic and mitochondrial clones were also taken into account. Different reproductive modes (sexual/parthenogenetic) appear to explain observed variability trends, supporting Dover's hypothesis of sexuality acting as a driving force in the fixation of sequence variants, but the present analyses also highlight current spreading of new variants in B. grandii maretimi specimens and point to a biased sequence inheritance at the time of hybrid onset in the apomictic hybrids B. whitei and B. lynceorum. Evidence of biased gene conversion events suggests that, given enough time, sequence homogenization can take place in a unisexual such as B. lynceorum. On the contrary, the absolute values of sequence diversity in each taxon are linked to the species' range, time of divergence, and repeat copy number and, possibly, to transposon features. Satellite dynamics appears therefore to be the outcome of both general molecular processes and specific organismal traits.     

Differential rates of local and global homogenization in centromere satellites from Arabidopsis relatives

Higher eukaryotic centromeres contain thousands of satellite repeats organized into tandem arrays. As species diverge, new satellite variants are homogenized within and between chromosomes, yet the processes by which particular sequences are dispersed are poorly understood. Here, we isolated and analyzed centromere satellites in plants separated from Arabidopsis thaliana by 5-20 million years, uncovering more rapid satellite divergence compared to primate alpha-satellite repeats. We also found that satellites derived from the same genomic locus were more similar to each other than satellites derived from disparate genomic regions, indicating that new sequence alterations were homogenized more efficiently at a local, rather than global, level. Nonetheless, the presence of higher-order satellite arrays, similar to those identified in human centromeres, indicated limits to local homogenization and suggested that sequence polymorphisms may play important functional roles. In two species, we defined more extensive polymorphisms, identifying physically separated and highly distinct satellite types. Taken together, these data show that there is a balance between plant satellite homogenization and the persistence of satellite variants. This balance could ultimately generate sufficient sequence divergence to cause mating incompatibilities between plant species, while maintaining adequate conservation within a species for centromere activity.     

Organization and evolutionary progress of a dispersed repetitive family of sequences in widely separated rodent genomes

The genomes of Mus musculus and other rodent species share a long conserved family of sequences that are dispersed and abundant (approx. 20,000 copies), and that have several novel features of organization and evolution. EcoR1 restriction of M. musculus DNA reveals a prominent 1350 bp² set of sequences. Two nonhomologous sequences of 850 and 500 bp, representing almost the total population of the 1350 bp repeats, were used to examine the detailed organization of the dispersed family and its surrounding sequences using a combination of restriction analysis and “Southern” hybridization. The 1350 bp sequence is contained within a longer repeating unit of approximately 3 kb that is dispersed amongst a wide variety of non-homologous and seemingly non-repetitive sequences. At some sites within the 3 kb repeat, considerable sequence heterogeneity has been found between members of the family, such that the family can be divided into largely non-overlapping subsets (or “segments”) according to the positioning of HinIII sites. Underlying the segmental organization there is a low background overlap of each segment with every other. Some but not all members of the family and its variants have been located on the X-chromosome in a Chinese hamster, M. musculus, X chromosome cell line: suggesting a wide genomic dispersion of the family. Homologous repeated sequences to the M. musculus 1350 bp repeat have been identified in species of Mus and Apodemus, with strikingly similar features of organization and dispersion. In M. spretus a 1350 bp sequence is contained within a dispersed repeat of at least 2·9 kb. However, the majority of M. spretus repeats contain an additional restriction site not present in the equivalent M. musculus array, suggesting a mechanism of widespread substitution or “conversion” of one variant by another in each genome. Apodemus sylvaticus possesses two dispersed and homologous families of 1350 bp and 1850 bp repetition, respectively, which contain sequences that have diverged from M. musculus to differing extents. A. mystacinus possesses only one family of dispersed and homologous repeats of 1850 bp. The majority of members within each Apodemus homologous family also contain characteristic variant restriction-site arrangements. The mechanisms underlying the spread of such variants within each array; the generation of segmental patterns; and the evolutionary conservation of this mouse interspersed family (MIF-1) are discussed in relation to the present knowledge of the organization and activity of other dispersed sequence families.     

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.     

Evolution of genetic redundancy for advanced players

An ever expanding database on the sequence organization and repetition of genic and non-genic components of nuclear and organelle genomes reveals that the vast majority of sequences are subject to one or other mechanism of DNA turnover (gene conversion, unequal crossing over, slippage, retrotransposition, transposition and others). Detailed studies, using novel methods of experimental detection and analytical procedures, show that such mechanisms can operate one on top of another and that wide variations in their unit lengths, biases, polarities and rates create bizarre and complex patterns of genetic redundancy. The ability of these mechanisms to operate both within and between chromosomes implies that realistic models of the evolutionary dynamics of redundancy, and of the potential interaction with natural selection in a sexual species, need to consider the diffusion of variant repeats across multiple chromosome lineages, in a population context. Recently, important advances in both experimental and analytical approaches have been made along these lines. There is increasing awareness that genetic redundancy and turnover induces a molecular co-evolution between functionally interacting genetic systems in order to maintain essential functions.     

Intraspecific Concerted Evolution of the rDNA ITS1 in Anopheles farauti Sensu Stricto (Diptera: Culicidae) Reveals Recent Patterns of Population Structure

We examined the intraindividual variation present in the first ribosomal internal transcribed spacer (ITS1) of Anopheles farauti to determine the level of divergence among populations for this important malarial vector. We isolated 187 clones from 70 individuals and found regional variation among four internal tandem repeats. The data were partitioned prior to analysis given the presence of a paralogous ITS2 sequence, called the 5'-subrepeat, inserted in the ITS1 of most clones. A high level of homogenization and population differentiation was observed for this repeat, which indicates a higher rate of turnover relative to the adjacent 'core' region. Bayesian analysis was performed using several substitutional models on both a combined and a partitioned data set. On the whole, the ITS1 phylogeny and geographic origin of the samples appear to be congruent. Some interesting exceptions indicate the spread of variant repeats between populations and the retention of ancestral polymorphism. Our data clearly demonstrate concerted evolution at the intraspecific level despite intraindividual variation and a complex internal repeat structure from a species that occupies a continuous coastal distribution. A high rate of genomic turnover in combination with a high level of sequence divergence appears to be a major factor leading to its concerted evolution within these populations.     

Unexpectedly slow homogenisation within a repetitive DNA family shared between two subspecies of tsetse fly

Summary Repetitive DNA families in sexual species are subject to a variety of turnover mechanisms capable of homogenising newly arising mutations. Very high levels of homogeneity in DNA families in some species ofDrosophila indicate that the rate of turnover is fast relative to that of mutation. To gauge the generality of such phenomena, we cloned and sequenced individual members of homologous repetitive DNA families from two subspecies of tsetse fly,Glossina morsitans centralis andG. morsitans morsitans. Unexpectedly high levels of variation were found within each subspecies, averaging 24% and 31%, respectively. Contiguous repeats and repeats cloned at random were comparably divergent. Nevertheless, it was possible to identify three instances of apparent homogenisation, each being, remarkably, of an insertion/deletion nature. We conclude that the rate of turnover in the tsetse families is comparable to that of most mutations, and discuss the possible parameters affecting flux in these families.     

Concerted evolution of satellite DNA in Sarcocapnos: a matter of time

SarkOne is a genus-specific satellite-DNA family, isolated from the genomes of the species of the genus Sarcocapnos. This satellite DNA is composed of repeats with a consensus length of 855 bp and a mean G+C content of 52.5%. We have sequenced a total of 189 SarkOne monomeric repeats belonging to a total of seven species of the genus Sarcocapnos. The comparative analysis of these sequences both at the intraspecific and the interspecific levels have revealed divergence patterns between species are proportional to between-species divergence according to the phylogeny of the genus. Our study demonstrates that the molecular drive leading to the concerted-evolution pattern of this satellite DNA is a time-dependent process by which new mutations are spreading through genomes and populations at a gradual pace. However, time is a limiting factor in the observation of concerted evolution in some pairwise comparisons. Thus, pairwise comparisons of species sharing a recent common ancestor did not reveal nucleotide sites in transitional stages higher than stage III according to the Strachan's model. By contrast, there was a gradation in the percentage of upper transition stages (IV, V, VI) the more phylogenetically distant the species were. In addition, closely related species shared a high number of polymorphic sites, but these types of sites were not common when comparing more distant species. All these data are discussed in the light of current life-cycle models of satellite-DNA evolution.