Miroslav Plohl

Satellite DNA evolution.

Satellite DNAs represent the most abundant fraction of repetitive sequences in genomes of almost all eukaryotic species. Long arrays of satellite DNA monomers form densely packed heterochromatic genome compartments and also span over the functionally important centromere locus. Many specific features can be ascribed to the evolution of tandemly repeated genomic components. This chapter focuses on the structural and evolutionary dynamics of satellite DNAs and the potential molecular mechanisms responsible for rapid changes of the genomic areas they constitute. Monomer sequences of a satellite DNA evolve concertedly through a process of molecular drive in which mutations are homogenized in a genome and fixed in a population. This process results in divergence of satellite sequences in reproductively isolated groups of organisms. However, some satellite DNA sequences are conserved over long evolutionary periods. Since many satellite DNAs exist in a genome, the evolution of species-specific satellite DNA composition can be directed by copy number changes within a library of satellite sequences common for a group of species. There are 2 important features of these satellite DNAs: long time sequence conservation and, at the same time, proneness to rapid changes through copy number alterations. Sequence conservation may be enhanced by constraints such as those imposed on functional motifs and/or architectural features of a satellite DNA molecule. Such features can limit the selection of sequences able to persist in a genome, and can direct the evolutionary course of satellite DNAs spanning the functional centromeres.

Centromere identity from the DNA point of view

The centromere is a chromosomal locus responsible for the faithful segregation of genetic material during cell division. It has become evident that centromeres can be established literally on any DNA sequence, and the possible synergy between DNA sequences and the most prominent centromere identifiers, protein components, and epigenetic marks remains uncertain. However, some evolutionary preferences seem to exist, and long-term established centromeres are frequently formed on long arrays of satellite DNAs and/or transposable elements. Recent progress in understanding functional centromere sequences is based largely on the high-resolution DNA mapping of sequences that interact with the centromere-specific histone H3 variant, the most reliable marker of active centromeres. In addition, sequence assembly and mapping of large repetitive centromeric regions, as well as comparative genome analyses offer insight into their complex organization and evolution. The rapidly advancing field of transcription in centromere regions highlights the functional importance of centromeric transcripts. Here, we comprehensively review the current state of knowledge on the composition and functionality of DNA sequences underlying active centromeres and discuss their contribution to the functioning of different centromere types in higher eukaryotes.

Sequence of PRAT Satellite DNA "Frozen" in Some Coleopteran Species

Abstract. The intriguing diversity of highly abundant satellite repeats found even among closely related species can result from processes leading to dramatic changes in copy number of a particular sequence in the genome and not from rapid accumulation of mutations. To test this hypothesis, we investigated the distribution of the PRAT satellite DNA family, a highly abundant major satellite in the coleopteran species Palorus ratzeburgii, in eight species belonging to the related genera (Tribolium, Tenebrio, Latheticus), the subfamily (Pimeliinae), and the family (Chrysomelidae). Dot blot analysis and PCR assay followed by Southern hybridization revealed that the PRAT satellite, in the form of low-copy number repeats, was present in all tested species. The PRAT satellite detected in the species Pimelia elevata has been sequenced, and compared with previously cloned PRAT monomers from Palorus ratzeburgii and Palorus subdepressus. Although the two Palorus species diverged at least 7 Myr ago, and the subfamily Pimeliinae separated from the genus Palorus 50–60 Myr ago, all PRAT clones exhibit high mutual homology, with average variability relative to the common consensus sequence of 1.3%. The presence of ancestral mutations found in PRAT clones from all three species as well as the absence of species diagnostic mutations illustrate extremely slow sequence evolution. This unexpectedly high conservation of PRAT satellite DNA sequence might be induced by a small bias of turnover mechanisms favoring the ancestral sequence in the process of molecular drive.

Complex structural features of satellite DNA sequences in the genus Pimelia (Coleoptera: Tenebrionidae): random differential amplification from a common 'satellite DNA library'.

The major satellites of the nine species of the subgenera Pimelia s. str. and Amblyptera characterised in this paper are composed of longer monomers (500 and 700 bp) than those described previously in 26 Pimelia s. str. taxa (357 bp, a sequence called PIM357). Sequence analysis reveals partial similarity among these satellites and with the PIM357 monomers. The discrepancy between the phylogeny obtained based on three mitochondrial and two nuclear markers and that deduced from satellite DNA (stDNA) sequences suggests that the different Pimelia satellites were already present in a common ancestor forming what has been called a ‘satellite DNA library’. Thus, the satellite profiles in the living species result from a random amplification of sequences from that ‘library’ during diversification of the species. However, species-specific turnover in the sequences has occurred at different rates. They have included abrupt replacements, a gradual divergence and, in other cases, no apparent change in sequence composition over a considerable evolutionary time. The results also suggest a common evolutionary origin of all these Pimelia satellite sequences, involving several rearrangements. We propose that the repeat unit of about 500 bp has originated from the insertion of a DNA fragment of 141 bp into the PIM357 unit. The 705-bp repeats have originated from a 32-bp direct duplication and the insertion of a 141-bp fragment in inverted orientation relative to a basic structure of 533 bp.

Preservation and High Sequence Conservation of Satellite DNAs Suggest Functional Constraints

Due to a high evolutionary turnover many satellite DNAs are restricted to a group of closely related species. Here we demonstrate that the satellite DNA family PSUB, abundant in the beetle Palorus subdepressus, is distributed in a low number of copies among diverse taxa of Coleoptera (Insecta), some of them separated for an evolutionary period of up to 60 Myr. Comparison of PSUB cloned from the species Tribolium brevicornis with the PSUB family previously characterized in Palorussubdepressus revealed high sequence conservation and absence of fixed species-specific mutations. The most polymorphic sites are those with ancestral mutations shared among clones of both species. Since the ancestral mutations contribute significantly to overall diversity, it could be proposed that a similar mutational profile already existed in an ancestral species. The pattern of variability along the satellite monomer is characterized by the presence of conserved and variable regions. The nonrandom pattern of variability as well as the absence of sequence divergence is also discerned for PRAT satellite DNA, cloned previously from two Palorus species and a distantly related Pimelia elevata. Since PRAT and PSUB are present in parallel in diverse taxa of Coleoptera, we propose that their long evolutionary preservation suggests a possible functional significance. This indication is additionally supported not only by the high evolutionary conservation of the sequences, but also by the presence of significantly conserved and variable regions along the monomers.

Similarity of Structural Features and Evolution of Satellite DNAs from Palorus subdepressus (Coleoptera) and Related Species

Abstract. A novel highly abundant satellite DNA comprising 20% of the genome has been characterized in Palorus subdepressus (Insecta, Coleoptera). The 72-bp-long monomer sequence is composed of two copies of T2A5T octanucleotide alternating with 22-nucleotide-long elements of an inverted repeat. Phylogenetic analysis revealed clustering of monomer sequence variants into two clades. Two types of variants are prevalently organized in an alternating pattern, thus showing a tendency to generate a new complex repeating unit 144 bp in length. Fluorescent in situ hybridization revealed even distribution of the satellite in the region of pericentric heterochromatin of all 20 chromosomes. P. subdepressus satellite sequence is clearly species specific, lacking similarity even with the satellite from congeneric species P. ratzeburgii. However, on the basis of similarity in predicted tertiary structure induced by intrinsic DNA curvature and in repeat length, P. subdepressus satellite can be classified into the same group with satellites from related tenebrionid species P. ratzeburgii, Tenebrio molitor, and T. obscurus. It can be reasonably inferred that repetitive sequences of different origin evolve under constraints to adopt and conserve particular features. Obtained results suggest that the higher-order structure and repeat length, but not the nucleotide sequence itself, are maintained through evolution of these species.

A GC-rich satellite DNA and karyology of the bivalve mollusk Donax trunculus: a dominance of GC-rich heterochromatin

We characterized the DTF2 satellite DNA family of the clam Donaxtrunculus and compared its chromosomal localization with cytogenetic data revealed by fluorochrome banding, C-banding, and 28S rDNA FISH. In contrast to the other satellites detected previously in this species, DTF2 is an abundant (2%) GC-rich satellite that exhibits CpG methylation. Sequence characteristics of DTF2 indicate that its evolution is not affected by constraints that might indicate some functional interactions. Fluorescence in situ hybridization revealed subtelomeric location of this satellite on a subset of 14 out of 19 D. trunculus chromosome pairs. The chromomycin A3 (CMA) staining of GC-rich regions on D. trunculus chromosomes revealed a complex banding pattern that overlaps completely with C-bands. In total, only three bands show subtelomeric location, while 13 bands are located interstitially, one of them being coincident with the 28S rDNA hybridization signal. No bands, either CMA positive (GC-rich) or DAPI positive (AT-rich) were detected at centromeric chromosomal positions. Only two of the CMA-positive bands co-localize with the DTF2 satellite, showing a) the presence of small islands of GC-rich repetitive sequences that remained undetected by CMA/C-banding and b) the abundance of DTF2-divergent GC-rich sequences at interstitial chromosomal locations.

Long-term conservation vs high sequence divergence: the case of an extraordinarily old satellite DNA in bivalve mollusks

The ubiquity of satellite DNA (satDNA) sequences has raised much controversy over the abundance of divergent monomer variants and the long-time nucleotide sequence stability observed for many satDNA families. In this work, we describe the satDNA BIV160, characterized in nine species of the three main bivalve clades (Protobranchia, Pteriomorphia and Heteroconchia). BIV160 monomers are similar in repeat size and nucleotide sequence to satDNAs described earlier in oysters and in the clam Donax trunculus. The broad distribution of BIV160 satDNA indicates that similar variants existed in the ancestral bivalve species that lived about 540 million years ago; this makes BIV160 the most ancient satDNA described so far. In the species examined, monomer variants are distributed in quite a complex pattern. This pattern includes (i) species characterized by a specific group of variants, (ii) species that share distinct group(s) of variants and (iii) species with both specific and shared types. The evolutionary scenario suggested by these data reconciles sequence uniformity in homogenization-maintained satDNA arrays with the genomic richness of divergent monomer variants formed by diversification of the same ancestral satDNA sequence. Diversified repeats can continue to evolve in a non-concerted manner and behave as independent amplification-contraction units in the framework of a ‘library of satDNA variants’ representing a permanent source of monomers that can be amplified into novel homogeneous satDNA arrays. On the whole, diversification of satDNA monomers and copy number fluctuations provide a highly dynamic genomic environment able to form and displace satDNA sequence variants rapidly in evolution.

A novel interspersed type of organization of satellite DNAs in Tribolium madens heterochromatin.

Analysis of arrangement of satellite DNA sequences in Tribolium madens (Insecta, Coleoptera) by Southern analysis of pulsed-field blots and two colour FISH on extended chromosomes and DNA fibres revealed a novel type of heterochromatin organization. Two satellite DNAs, distributed over the whole pericentromeric heterochromatin of all chromosomes form clusters, ranging in size from 150 kb up to several Mb. Within the clusters, both satellites are in the form of highly interspersed, short homogeneous arrays which vary in size with a lowest length limit of only few kb. The longest arrays composed of a single satellite are relatively short, up to 70 kb for satellite I, and up to 45 kb for satellite II. Only a small fraction of about 15% of satellite II is organized in long tandem repeats, while the rest is in the form of only a few repeats intermingled with satellite I. The results indicate that large clusters composed of interspersed arrays of both satellites represent a major component of T. madens heterochromatin, which is mostly devoid of long regions of other sequences. The same organizational pattern probably also includes a region of the functional centromere. We propose that such an organizational pattern of DNA sequences in heterochromatin might be common in genomes characterized by a high rate of interchromosomal exchange. This pattern of organization is different from that in other animal as well as plant species analysed up to now, in which every satellite in heterochromatin is organized in a small number of large separate domains.

Conservation of satellite DNA in species of the genus Pimelia (Tenebrionidae, Coleoptera).

Satellite DNA has been characterized in six allopatric species from the genus Pimelia: P. interjecta, P. integra, P. variolosa and P. baetica, inhabiting Iberian Peninsula, and P. elevata and P. criba, endemic to Balearic Islands Ibiza and Mallorca, respectively. All species show the presence of a single satellite DNA of a basic monomer length of 357 bp and A+T content of 69%, comprising a considerable amount of the genome (39%-45%, corresponding to about 4.5 x 10(5) copies per haploid genome). The sequence analysis of 22 cloned repeats reveals very high intra- and interspecific sequence similarity. Phylogenetic analysis separates the satellite sequences into two clusters, each comprising clones from three species exclusively. Within the clusters, satellite clones are not grouped species-specifically, except those of P. integra where species-diagnostic nt substitutions are detected with a pattern that could be produced by gene conversion. Such high sequence conservation could be related to preservation of satellite DNA curvature, resulting in a higher order helical structure, proposed to act as a specific protein binding domain.