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.

A novel arrangement of sequence elements surrounding the rDNA promoter and its spacer duplications in tsetse species.

Variation in organization and sequence of the rDNA of six species of tsetse fly (Glossina) has been investigated. Several novel tsetse-specific features have been uncovered. Like many other species the spacer is composed of subrepeats, which in some species contain duplications of the true promoter at the spacer-ETS boundary. In tsetse, however, the first 90 base-pairs of the external transcribed spacer (ETS) (that is, +1 to +90 after transcription initiation) is the 3' end of the last subrepeat. The absence of a "unique" region between the last subrepeat and the ETS suggests that the tsetse rDNA unit may consist of multiple true promoters, that is there is no single ETS boundary. Furthermore, interspecific comparisons show that the 90 base-pair region is part of a conserved 202 base-pair region, consisting of 72 base-pairs upstream from the initiation site and a further 40 base-pairs downstream, which is shared by all promoters other than the last. In genera other than tsetse, subrepeat lengths between species are generally similar; in tsetse they differ due to (1) variation in copy-number of the subsubrepeat motif A9T6CAG, and (2) the presence of large regions flanked by direct simple repeats such as GA5 or TGGTCTC. Slippage-like mechanisms are probably responsible for (1), and recombination and subsequent excision involving the direct repeats for (2). Different structural and sequence variants are seen to be homogenized in the family and fixed in each species, reflecting continual unequal crossing-over. However, notwithstanding this process of differentiation, the available comparisons also reveal that there are two small conserved regions between Glossina and Drosophila: one is part of the promoter and the other is an ETS processing site. Such intergeneric and interspecific differences are discussed in relation to the problem of the maintenance of several essential functions within the rDNA repeating unit despite the continual differentiation of the unit into novel arrangements.

Conservation of major nuclease S1-sensitive sites in the non-conserved spacer region of ribosomal DNA in Drosophila species.

We have analysed nuclease S1-sensitive sites in cloned ribosomal DNA repeats from Drosophila melanogaster, D. hydei and D. virilis. All species contain major S1-sensitive sites in the spacer near the region of transcription termination, albeit with somewhat different positions and sensitivities. The same sites are also sensitive to the single-strand specificity of Bal31 nuclease at neutral pH. Additional major sites exist at each end of the intervening sequence within the 28 S gene of non-transcribed intervening-sequence-positive ribosomal DNA units of D. hydei. Only minor sites, however, were detected in the Pol I promoter regions. This is in contrast to Pol II transcribed genes, where S1 hypersensitivity becomes apparent at the 5' ends during gene expression. We have sequenced and mapped the S1 sites in the D. hydei spacer. They consist mainly of alternating A and T nucleotides that could form small cruciform structures. Cross-hybridization at low stringencies between the relevant S1-sensitive spacer regions of the three species indicates that the sites lie within very divergent sequences. We discuss the potential functional significance of S1 sites in rDNA spacers and intervening sequences, and the manner in which they might be maintained during rDNA sequence divergence.

Evolutionary divergence of promoters and spacers in the rDNA family of four Drosophila species. Implications for molecular coevolution in multigene families.

The organization and sequence of the rDNA multigene family of four Drosophila species (melanogaster, orena, virilis and hydei) have been compared in order to understand the quality and quantity of the differences which are involved with interspecific divergence of promoters and the polymerase I complexes (molecular coevolution). Each species has an intergenic spacer (IGS) made up of subrepeats which contain duplications of the promoter. Major structural and point-mutational differences exist, most of which have been spread by unequal crossingover through the family and species. Structural differences involve the types, lengths and copy-number of the IGS subrepeats, and the lengths and position of "unique" regions between blocks of repeats. The 240 base-pair repeat array shared by D. melanogaster and D. orena has been replaced by a 220 base-pair repeat, and the 95 and 330 base-pair arrays are absent altogether in D. virilis and D. hydei. The length of the "unique" region between the 240/220 base-pair arrays and the start of transcription varies, with the unusual situation of the last of the 220 repeats ending at the external transcribed spacer (ETS) boundary in D. virilis. Other structural differences involve regions of high cryptic simplicity arising from slippage in D. virilis and D. hydei IGSs. Sequence analysis of IGS and the ETSs indicates that the rDNA is not uniformly divergent throughout its length. Apart from the genes, there are regions of relatively high conservation covering the promoter regions and at some but not all potential RNA processing sites. The conserved promoter regions are more extensive within each pair of species D. melanogaster versus D. orena and D. virilis versus D. hydei, in keeping with their phylogenetic distances. Slippage-like mechanisms are involved with large numbers of deletions/insertions that make up the ETS differences between the species. Patterns of shared mutations between IGS subrepeats indicate stages of transition during rDNA differentiation by continual homogenization. The simultaneous operation of different turnover mechanisms, at different periodicities and rates, generates a complex picture of reorganization, some of which would influence the process of molecular coevolution in the family.

Secondary structure constraints on the evolution of Drosophila 28 S ribosomal RNA expansion segments.

Eukaryotic ribosomal RNA genes contain rapidly evolving regions of unknown function termed expansion segments. We present the comparative analysis of the primary and secondary structure of two expansion segments from the large subunit rRNA gene of ten species of Drosophila and the tsetse fly species Glossina morsitans morsitans. At the primary sequence level, most of the differences observed in the sequences obtained are single base substitutions. This is in marked contrast with observations in vertebrate species in which the insertion or deletion of repetitive motifs, probably generated by a DNA-slippage mechanism, is a major factor in the evolution of these regions. The secondary structure of the two regions, supported by multiple compensatory base changes, is highly conserved between the species examined and supports the existence of a general folding pattern for all eukaryotes. Intriguingly, the evolutionary rate of expansion segments is very slow relative to other genic and non-genic regions of the Drosophila genome. These results suggest that the evolution of expansion segments in the rDNA multigene family is a balance between the homogenization of new mutations by unequal crossing over and a combination of selection against some such mutations per se and selection for subsequent compensatory mutations, in order to maintain a particular RNA secondary structure.

The cohesive population genetics of molecular drive.

The long-term population genetics of multigene families is influenced by several biased and unbiased mechanisms of nonreciprocal exchanges (gene conversion, unequal exchanges, transposition) between member genes, often distributed on several chromosomes. These mechanisms cause fluctuations in the copy number of variant genes in an individual and lead to a gradual replacement of an original family of n genes (A) in N number of individuals by a variant gene (a). The process for spreading a variant gene through a family and through a population is called molecular drive. Consideration of the known slow rates of nonreciprocal exchanges predicts that the population variance in the copy number of gene a per individual is small at any given generation during molecular drive. Genotypes at a given generation are expected only to range over a small section of all possible genotypes from one extreme (n number of A) to the other (n number of a). A theory is developed for estimating the size of the population variance by using the concept of identity coefficients. In particular, the variance in the course of spreading of a single mutant gene of a multigene family was investigated in detail, and the theory of identity coefficients at the state of steady decay of genetic variability proved to be useful. Monte Carlo simulations and numerical analysis based on realistic rates of exchange in families of known size reveal the correctness of the theoretical prediction and also assess the effect of bias in turnover. The population dynamics of molecular drive in gradually increasing the mean copy number of a variant gene without the generation of a large variance (population cohesion) is of significance regarding potential interactions between natural selection and molecular drive.

Multigene family of ribosomal DNA in Drosophila melanogaster reveals contrasting patterns of homogenization for IGS and ITS spacer regions. A possible mechanism to resolve this paradox.

The multigene family of rDNA in Drosophila reveals high levels of within-species homogeneity and between-species diversity. This pattern of mutation distribution is known as concerted evolution and is considered to be due to a variety of genomic mechanisms of turnover (e.g., unequal crossing over and gene conversion) that underpin the process of molecular drive. The dynamics of spread of mutant repeats through a gene family, and ultimately through a sexual population, depends on the differences in rates of turnover within and between chromosomes. Our extensive molecular analysis of the intergenic spacer (IGS) and internal transcribed spacer (ITS) spacer regions within repetitive rDNA units, drawn from the same individuals in 10 natural populations of Drosophila melanogaster collected along a latitudinal cline on the east coast of Australia, indicates a relatively fast rate of X-Y and X-X interchromosomal exchanges of IGS length variants in agreement with a multilineage model of homogenization. In contrast, an X chromosome-restricted 24-bp deletion in the ITS spacers is indicative of the absence of X-Y chromosome exchanges for this region that is part of the same repetitive rDNA units. Hence, a single lineage model of homogenization, coupled to drift and/or selection, seems to be responsible for ITS concerted evolution. A single-stranded exchange mechanism is proposed to resolve this paradox, based on the role of the IGS region in meiotic pairing between X and Y chromosomes in D. melanogaster.

Patterns of variation in the intergenic spacers of ribosomal DNA in Drosophila melanogaster support a model for genetic exchanges during X-Y pairing.

Detailed analysis of variation in intergenic spacer (IGS) and internal transcribed spacer (ITS) regions of rDNA drawn from natural populations of Drosophila melanogaster has revealed contrasting patterns of homogenization although both spacers are located in the same rDNA unit. On the basis of the role of IGS regions in X-Y chromosome pairing, we proposed a mechanism of single-strand exchanges at the IGS regions, which can explain the different evolutionary trajectories followed by the IGS and the ITS regions. Here, we provide data from the chromosomal distribution of selected IGS length variants, as well as the detailed internal structure of a large number of IGS regions obtained from specific X and Y chromosomes. The variability found in the different internal subrepeat regions of IGS regions isolated from X and Y chromosomes supports the proposed mechanism of genetic exchanges and suggests that only the "240" subrepeats are involved. The presence of a putative site for topoisomerase I at the 5' end of the 18S rRNA gene would allow for the exchange between X and Y chromosomes of some 240 subrepeats, the promoter, and the ETS region, leaving the rest of the rDNA unit to evolve along separate chromosomal lineages. The phenomenon of localized units (modules) of homogenization has implications for multigene family evolution in general.

Amplification of KP elements associated with the repression of hybrid dysgenesis in Drosophila melanogaster.

Mobile P elements in Drosophila melanogaster cause hybrid dysgenesis if their mobility is not repressed. One type of repression, termed P cytotype, is a complex interaction between chromosomes carrying P elements and cytoplasm and is transmitted through the cytoplasm only of females. Another type of repression is found in worldwide M' strains that contain approximately 30 copies per individual of one particular P element deletion-derivative termed the KP element. This repression is transmitted equally through both sexes. In the present study we show that biparentally transmitted repression increases in magnitude together with a rapid increase in KP copy-number in genotypes starting with one or a few KP elements and no other deletion-derivatives. Such correlated increases in repression and KP number per genome occur only in the presence of complete P elements, supporting the interpretation that they are probably a consequence of the selective advantage enjoyed by flies carrying the highest numbers of KP elements. Analysis of Q strains also reveals the presence of qualitative differences in the way the repression of dysgenesis is transmitted. In general, Q strains not containing KP elements have the P cytotype mode of repression, whereas Q strains with KP elements transmit repression through both sexes. This difference among Q strains further supports the existence of at least two types of repression of P-induced hybrid dysgenesis in natural populations of D. melanogaster.

Comparison of bicoid-dependent regulation of hunchback between Musca domestica and Drosophila melanogaster.

Co-evolution between developmental regulatory elements is an important mechanism of evolution. This work compares the hunchback-bicoid interaction in the housefly Musca domestica with Drosophila melanogaster. The Musca HUNCHBACK protein is 66% conserved and partially rescues a hunchback mutant, yet the BICOID-dependent promoter (P2) of Musca hunchback is unexpectedly diverged from D. melanogaster. Introduced into D. melanogaster, this promoter drives a normal P2 pattern during the syncytial blastoderm stage but is expressed ectopically at the anterior pole of the embryo at later stages. We also report differences in the early expression of hunchback in Musca. We suggest that conservation of the morphogenetic function of bicoid in different sized embryos of higher diptera requires co-evolution of bicoid and its target binding sites.

Evolving the improbable.

The debate about the evolutionary roles of natural selection and molecular drive cannot be resolved by assigning probabilities with hind-sight to unique events in the evolution of complex adaptations. Hoyle dismisses natural selection by calculating, erroneously, the improbability of assembling by chance a string of amino acids in the order in which they occur in a given protein. Dawkins dismisses molecular drive on the same false grounds of the improbability of making the right decision, by chance, at each step in a long series of steps in the evolution of a given trait. Calculation of the probability, at any given step, of a successful match between fortuitous genetic variation and fortuitous environmental heterogeneity requires detailed knowledge of all parameters at that step. Such information is an essential requirement for quantifying the roles of natural selection and molecular drive in the evolution of one actual series of steps out of many potential series.

DNA turnover and the molecular clock.

Many detailed studies on the mechanisms by which different components of eukaryotic nuclear genomes have diverged reveal that the majority of sequences are seemingly not passively accumulating base substitutions in a clocklike manner solely determined by laws of diffusion at the population level. It appears that variation in the rates, units, biases, and gradients of several DNA turnover mechanisms are contributing to the course of DNA divergence. Turnover mechanisms have the potential to retard, maintain, or accelerate the rate of DNA differentiation between populations. Furthermore, examples are known of coding and noncoding DNA subject to the simultaneous operation of several turnover mechanisms leading to complex patterns of fine-scale restructuring and divergence, generally uninterpretable using selection and/or neutral drift arguments in isolation. Constancy in the rate of divergence, where observed over defined periods of time, could be a reflection of constancy in the rates and units of turnover. However, a consideration of the generally large disparity between rates of turnover and mutation reveals that DNA clocks, which would be independently driven by turnover in separate genomic components, would tend to be episodic. The utility of any given DNA sequence for measuring time and species relationships, like individual proteins, is proportional to the extent to which all contributing forces to the evolution of the sequence, internal and external, are understood.

Observing development through evolutionary eyes: a practical approach.

An argument is made that only through a detailed comparison of mutational mechanisms underlying the evolution of the genetic systems governing development, can the 'logic' of individual development be fully comprehended. To do this, it is essential to choose two or more genes (or their products) that interact in the establishment of a given function, and to compare the molecular basis of that interaction in closely related species. The rationale to this approach arises from observations of molecular co-evolution between interacting partners involved with given functions which have led to species specificity in the manner in which such functions are effected. Molecular coevolution reveals that divergence in sequence can be tolerated whilst biological functions are maintained, not because it is neutral and dispensable but because successful, compensatory changes can evolve in eukaryotic genomes that are in continuous states of flux.

Molecular coevolution among cryptically simple expansion segments of eukaryotic 26S/28S rRNAs.

The set of "expansion segments" of any eukaryotic 26S/28S ribosomal RNA (rRNA) gene is responsible for the bulk of the difference in length between the prokaryotic 23S rRNA gene and the eukaryotic 26S/28S rRNA gene. The expansion segments are also responsible for interspecific fluctuations in length during eukaryotic evolution. They show a consistent bias in base composition in any species; for example, they are AT rich in Drosophila melanogaster and GC rich in vertebrate species. Dot-matrix comparisons of sets of expansion segments reveal high similarities between members of a set within any 28S rRNA gene of a species, in contrast to the little or spurious similarity that exists between sets of expansion segments from distantly related species. Similarities among members of a set of expansion segments within any 28S rRNA gene cannot be accounted for by their base-compositional bias alone. In contrast, no significant similarity exists within a set of "core" segments (regions between expansion segments) of any 28S rRNA gene, although core segments are conserved between species. The set of expansion segments of a 26S/28S gene is coevolving as a unit in each species, at the same time as the family of 28S rRNA genes, as a whole, is undergoing continual homogenization, making all sets of expansion segments from all ribosomal DNA (rDNA) arrays in a species similar in sequence. Analysis of DNA simplicity of 26S/28S rRNA genes shows a direct correlation between significantly high relative simplicity factors (RSFs) and sequence similarity among a set of expansion segments. A similar correlation exists between RSF values, overall rDNA lengths, and the lengths of individual expansion segments. Such correlations suggest that most length fluctuations reflect the gain and loss of simple sequence motifs by slippage-like mechanisms. We discuss the molecular coevolution of expansion segments, which takes place against a background of slippage-like and unequal crossing-over mechanisms of turnover that are responsible for the accumulation of interspecific differences in rDNA sequences.