Coevolution in bicoid-dependent promoters and the inception of regulatory incompatibilities among species of higher Diptera.

To what extent and in what way do gene promoters and their transacting regulatory proteins coevolve? In this and in earlier publications we show that the Bicoid-dependent promoters of the segmentation genes hunchback and tailless in species of higher Diptera (Drosophila, Musca, Calliphora, and Lucilia) are different with respect to the copy number, spacing, sequence, and orientation of Bicoid binding sites. At the same time there are significant amino acid differences in the Bicoid homeodomain. To test these interspecific differences, we used a series of functional assays, starting with the analysis of Bicoid binding affinities of individual sites, through to transgene rescue experiments, to compare within-species with between-species mixtures of Bicoid homeodomains and hunchback or tailless promoters. We observed that components taken from different species interact with less efficiency compared with those taken from within the same species. Our interpretation is that such interspecific incompatibilities are a consequence of interactive genetic elements coevolving one with another, hence maintaining functional compatibility within each species. At the same time such a process allows differences to accumulate between species regarding the precise molecular basis whereby the common function is effected.

Divergent structure and function of the bicoid gene in Muscoidea fly species.

We have investigated the evolution of the bicoid (bcd) gene in fly species of the Muscoidea Superfamily. We obtained the complete bcd sequence from the housefly Musca domestica and found polymorphism in the coding region among Musca strains. In addition to Musca, we cloned most of the bcd coding sequences from two blowfly species Calliphora vicina and Lucilia sericata. The 5' and 3' regulatory regions flanking the Musca bcd gene are widely diverged in sequence from Drosophila; however, some important sequence motifs identified in Drosophila bcd are present. The predicted RNA secondary structures of the 3' UTRs are similar, despite sequence divergence. Comparison of Bicoid (Bcd) proteins shows a serine-rich domain of unknown function is present in the Muscoidea species, but is absent in other species. The in vivo function of bcd in Musca was tested by RNAi to mimic loss of function phenotype. We obtained a head defect phenotype similar to weak bcd alleles of Drosophila. Although our comparisons initially suggest functional conservation between species, closer inspection reveals significant differences. Divergence of structural motifs, such as regulatory elements in flanking regions and conservation of protein domains in some species but not in others, points to functional divergence between species. We suggest that the larger embryonic size in Muscoidea species restricts the morphogenetic activity of a weak Bcd activator, which has evolved a more specialized role in head determination and lost some functions in thoracic development.

Sequence and expression of the hunchback gene in Lucilia sericata: a comparison with other Dipterans.

We have found that the hunchback (hb)gene from Lucilia sericata is conserved in its functional domains in comparison with related flies, although there is divergence in the protein outside these regions. The expression patterns of Lucilia hb in early embryos are broadly similar to other higher Dipterans. However, in the posterior region we report blastoderm and post-gastrulation expression patterns, which are diverged from Musca and Drosophila. These patterns are reminiscent of hb expression in more primitive insects and could be indicative of changes in the regulation of hb in Lucilia by the terminal system.

Rapid restructuring of bicoid-dependent hunchback promoters within and between Dipteran species: implications for molecular coevolution.

Interacting genetic elements need to coevolve if their joint function is to be maintained; for example, the correct binding of transcriptional regulators to defined binding sites in gene promoters needs to be maintained during evolution to ensure proper function. As part of a wider investigation into the molecular coevolution of the Dipteran homeodomain-bearing regulator bicoid (bcd) and Bcd-dependent promoters, we present data on the functional, structural, and sequence differences between the promoters of the segmentation gene hunchback (hb), in several species of Cyclorrhaphan (higher) Diptera. The result of phenocopying hb mutations using RNA interference (RNAi) in Musca domestica shows broadly similar functions to the hb gene in Drosophila melanogaster. However, the Bcd-binding sites in the hb promoters of Drosophila, Musca, and the two blowfly species Lucilia sericata and Calliphora vicina differ in copy number, sequence, orientation, and spacing. Furthermore, all promoters are subject to rapid turnover by slippage-like processes leading to high densities of short repetitive motifs. A study of polymorphism among six strains of M. domestica reveals that turnover by slippage also occurs in the promoter, untranslated leader, and exonic coding sequences of hb, but to different extents. We discuss these results in terms of the known interspecific differences in bcdand the potential coevolution of selected compensatory mutations in trans and cis in response to continuous promoter restructuring.

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.

Evolutionary flux of P element regulation in a Drosophila melanogaster hybrid dysgenesis cline.

Clines of P-induced hybrid dysgenesis provide a means for monitoring the evolution of transposition repression over space and time. We have studied the molecular and phenotypic profiles of flies taken from a 2900 km cline along the eastern coast of Australia, which had previously been characterized over 10 years ago as having P populations in the north, Q populations at central sites and M' populations in the south. We have found that Q and M' populations of flies have increased their range within the cline at the expense of P lines. Q populations were found to be in the north of the cline and M' populations in the south. Some of the northern Q lines transmit repression through both sexes and type I deletion elements have been isolated from them. We suggest that these elements are responsible for Q type repression. The results support our model that populations made up of Q individuals with strong biparentally transmitted repression form an evolutionarily stable strategy for the repression of hybrid dysgenesis in Drosophila melanogaster.

The ancient and divergent origins of the human pathogenic trypanosomes, Trypanosoma brucei and T. cruzi.

This study presents new findings concerning the evolution of the human pathogens, Trypanosoma brucei and T. cruzi, which suggest that these parasites have divergent origins and fundamentally different patterns of evolution. Phylogenetic analysis of 18S rRNA sequences places T. brucei in a clade comprising exclusively mammalian trypanosomes of African origin, suggesting an evolutionary history confined to Africa. T. cruzi (from humans and sylvatic mammals) clusters with trypanosomes specific to Old and New World bats, T. rangeli and a trypanosome species isolated from an Australian kangaroo. The origins of parasites within this clade, other than some of those from bats, lie in South America and Australia suggesting an ancient southern super-continent origin for T. cruzi, possibly in marsupials; the only trypanosomes from this clade to have spread to the Old World are those infecting bats, doubtless by virtue of the mobility of their hosts. Viewed in the context of palaeogeographical evidence, the results date the divergence of T. brucei and T. cruzi to the mid-Cretaceous, around 100 million years before present, following the separation of Africa, South America and Euramerica. The inclusion in this study of a broad range of trypanosome species from various different hosts has allowed long phylogenetic branches to be resolved, overcoming the limitations of many previous studies. Moreover, T. brucei and the other mammalian tsetse-transmitted trypanosomes appear, from these data, to be evolving several times faster than T. cruzi and its relatives.

High sequence turnover in the regulatory regions of the developmental gene hunchback in insects.

Extensive sequence analysis of the developmental gene hunchback and its 5' and 3' regulatory regions in Drosophila melanogaster, Drosophila virilis, Musca domestica, and Tribolium castaneum, using a variety of computer algorithms, reveals regions of high sequence simplicity probably generated by slippage-like mechanisms of turnover. No regions are entirely refractory to the action of slippage, although the density and composition of simple sequence motifs varies from region to region. Interestingly, the 5' and 3' flanking regions share short repetitive motifs despite their separation by the gene itself, and the motifs are different in composition from those in the exons and introns. Furthermore, there are high levels of conservation of motifs in equivalent orthologous regions. Detailed sequence analysis of the P2 promoter and DNA footprinting assays reveal that the number, orientation, sequence, spacing, and protein-binding affinities of the BICOID-binding sites varies between species and that the 'P2' promoter, the nanos response element in the 3' untranslated region, and several conserved boxes of sequence in the gene (e.g., the two zinc-finger regions) are surrounded by cryptically-simple-sequence DNA. We argue that high sequence turnover and genetic redundancy permit both the general maintenance of promoter functions through the establishment of coevolutionary (compensatory) changes in cis- and trans-acting genetic elements and, at the same time, the possibility of subtle changes in the regulation of hunchback in the different species.

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.

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.

Natural repressors of P-induced hybrid dysgenesis in Drosophila melanogaster: a model for repressor evolution.

Type I repressors control P element transposition and comprise full length elements and elements with small 3' deletions in the final exon. Using a sensitive assay for measuring the strength of repression of P element transposition in somatic and germline tissues, we have isolated and characterized a naturally occurring type I repressor element from a Q population of Drosophila melanogaster. We demonstrate that the almost complete repression of transposition in this population is a mixture of KP elements with intermediate levels of repression, and the strong contribution of a single 2.6 kb P element deletion derivative, which we call SR (Strong Repressor). A deletion in the final intron of SR allows for the constitutive production of a putative 75 kDa repressor protein in germline tissues in addition to the production of the 66 kDa repressor in the soma, which would result in a biparental mode of inheritance of repression. Based on the four observed classes of natural Q populations, we propose a model in which populations containing SR-like elements, capable of producing strong type I repressor constitutively, have a selective advantage over populations which rely either on maternally transmitted P cytotype or on KP-induced weak levels of repression. Such populations may subsequently spread and constitute an evolutionary stable strategy for the repression of hybrid dysgenesis in Drosophila melanogaster.

PCR amplification of intergenic spacers in the ribosomal DNA of Drosophila melanogaster reveals high levels of turnover in length and copy-number of spacers in geographically separated populations.

A recently described PCR-based method for the analysis of intergenic spacer (IGS) length variation in the ribosomal (r) DNA of Drosophila melanogaster was used to analyse the distribution of IGS length variants in the rDNA of a number of recently collected D. melanogaster populations. One group of populations, from Europe and North Africa, was shown to have low intrapopulation IGS length variation following maintenance of massed populations in the laboratory for an extended period. However, a greater degree of IGS profile variability was detected at a number of levels in the majority of laboratory-maintained isofemale lines from two of these populations plus a second group of populations which were collected more recently from the eastern coast of Australia; all of which were immediately divided into isofemale lines following collection. Interestingly, PCR analysis of pooled DNA extracts from 30 individuals of either sex showed almost identical PCR profiles from each of the Australian populations. These preliminary results are discussed with regard to the possible combinations of forces (natural selection, neutral drift and genomic molecular drive) on the patterns of IGS length variation.

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.

Rapid evolution of a heteroplasmic repetitive sequence in the mitochondrial DNA control region of carnivores.

We describe a repetitive DNA region at the 3' end of the mitochondrial DNA (mtDNA) control region and compare it in 21 carnivore species representing eight carnivore families. The sequence and organization of the repetitive motifs can differ extensively between arrays; however, all motifs appear to be derived from the core motif "ACGT." Sequence data and Southern blot analysis demonstrate extensive heteroplasmy. The general form of the array is similar between heteroplasmic variants within an individual and between individuals within a species (varying primarily in the length of the array, though two clones from the northern elephant seal are exceptional). Within certain families, notably ursids, the array structure is also similar between species. Similarity between species was not apparent in other carnivore families, such as the mustelids, suggesting rapid changes in the organization and sequence of some arrays. The pattern of change seen within and between species suggests that a dominant mechanism involved in the evolution of these arrays is DNA slippage. A comparative analysis shows that the motifs that are being reiterated or deleted vary within and between arrays, suggesting a varying rate of DNA turnover. We discuss the evolutionary implications of the observed patterns of variation and extreme levels of heteroplasmy.

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.

Elephant seal genetic variation and the use of simulation models to investigate historical population bottlenecks.

Because the northern elephant seal (Mirounga angustirostrus) was heavily exploited during the 19th century, it experienced an extreme population bottleneck. Since then, under legislative protection in the United States and Mexico, northern elephant seals have recovered dramatically in number, although their genomic diversity was greatly reduced, apparently as a consequence of the bottleneck. In this study we investigated DNA sequence diversity in two mtDNA regions (the control region and 16S RNA) and found low genetic variation in the northern elephant seal: there were only two control region haplotypes (sequence difference = 1%), which was consistent with an extreme founder event in the recent history of the northern species. We also reaffirmed the lack of allozyme diversity in this species. In contrast, the southern elephant seal (M. leonina), which though similarly exploited never fell below 1,000 animals, had 23 control region mtDNA haplotypes (average sequence difference = 2.3%). To investigate the extent of the founder event in the northern elephant seal we devised a simulation model based on extensive demographic data. This allowed a statistical analysis of the likely outcome of bottlenecks of different size and duration. Given these historical data, our results indicate (within 95% confidence) a bottleneck of less than 30 seals and 20-year duration, or, if hunting was the primary pressure on the population, a single-year bottleneck of less than 20 seals.

Generation of VNTRs and heteroplasmy by sequence turnover in the mitochondrial control region of two elephant seal species.

We describe an unusual repetitive DNA region located in the 3' end of the light (L)-strand in the mitochondrial control region of two elephant seal species. The array of tandem repeats shows both VNTR (variable-number tandem repeat) and sequence variation and is absent from 12 compared mammalian species, except for the occurrence in the same location of a distinct repetitive region in rabbit mtDNA and a similar repeat in the harbor seal. The sequence composition and arrangement of the repeats differ considerably between the northern elephant seal (Mirounga angustirostris) and the southern species (M. leonina) despite an estimated divergence time of 1 MY (based on an mtDNA-RNA gene and the nonrepetitive control region). Analysis of repeat sequence relationships within and between species indicate that divergence in sequence and structure of repeats has involved both slippage-like and unequal crossingover processes of turnover, generating very high levels of divergence and heteroplasmy.