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.     

The Complex Satellite DNA of Tropaeolum majus L.: Partial Characterization of Isolated and of Cloned Restriction Fragments

Satellite DNA (satDNA) was purified by repeated neutral CsCl,ethidium bromide/CsCl and actinomycin D/CsCl density centrifugationfrom total nuclear DNA of Tropaeolum majus. Digestion patternsof satDNA and main band DNA as generated with 22 different restrictionenzymes were partially characterized and hybridized by the Southernblot technique with Hpa II-generated satDNA fragments and aribosomal DNA fragment. Some restriction fragments were sequencedby the chemical method, either directly, or after cloning, andpossible secondary structures were determined. The results showthat the satellite is of high complexity and evidently composedof different, interspersed repeats, and is also interspersedwith ribosomal sequences. Some sequences of the satellite aredispersed throughout the Tropaeolum genome, and even found inthe chloroplast genome. Most of the restriction enzymes cutthe satDNA into many fragments of lengths between 14 bp and3000 bp. Hpa II cut the satellite DNA into at least 63 differentsized fragments (more than is known from any other plant satDNA).Sal I cut the satDNA into a single fragment of 1040 bp in size,which probably represented the superrepeat unit. Sequence analysisof satDNA restriction fragments and of cloned fragments isolatedat different times, indicated a rapid diversification in vivoand in vitro. Computer modeling of most likely secondary structuressuggest the occurrence of palindromic loops (cruciform structures).These could be the basis of rapid diversification in being sitesof preferred intragenomic recombination and rearrangement. Cloning, palindromic sequences, restriction analysis, satellite DNA complexity, Tropaeolum majus L, nasturtium     

Is Radon Emission in Caves Causing Deletions in Satellite DNA Sequences of Cave-Dwelling Crickets?

The most stable isotope of radon, ²²²Rn, represents the major source of natural radioactivity in confined environments such as mines, caves and houses. In this study, we explored the possible radon-related effects on the genome of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae) sampled in caves with different concentrations of radon. We analyzed specimens from ten populations belonging to two genetically closely related species, D. geniculata and D. laetitiae, and explored the possible association between the radioactivity dose and the level of genetic polymorphism in a specific family of satellite DNA (pDo500 satDNA). Radon concentration in the analyzed caves ranged from 221 to 26000 Bq/m³. Specimens coming from caves with the highest radon concentration showed also the highest variability estimates in both species, and the increased sequence heterogeneity at pDo500 satDNA level can be explained as an effect of the mutation pressure induced by radon in cave. We discovered a specific category of nuclear DNA, the highly repetitive satellite DNA, where the effects of the exposure at high levels of radon-related ionizing radiation are detectable, suggesting that the satDNA sequences might be a valuable tool to disclose harmful effects also in other organisms exposed to high levels of radon concentration.     

The non-regular orbit: three satellite DNAs in Drosophila martensis (buzzatii complex, repleta group) followed three different evolutionary pathways

The genome of species from the buzzatii cluster (buzzatii complex, repleta group) is hosted by a number of satellite DNAs (satDNAs) showing contrasting structural characteristics, genomic organization and evolution, such as pBuM-alpha (~190 bp repeats), pBuM-alpha/beta (~370 bp repeats) and the DBC-150 (~150 bp repeats). In the present study, we aimed to investigate the evolution of these three satDNAs by looking for homologous sequences in the genome of the closest outgroup species: Drosophila martensis (buzzatii complex). After PCR, we isolated and sequenced 9 alpha, 8 alpha/beta and 11 DBC-150 sequences from this species. The results were compared to all pBuM and DBC-150 sequences available in literature. After D. martensis split from the buzzatii cluster some 6 Mya, the three satDNAs evolved differently in the genome of D. martensis by: (1) maintenance of a collection of major types of ancestral repeats in the genome (alpha); (2) fixation for a single major type of ancestral repeats (alpha/beta) or (3) fixation for new divergent species-specific repeat types (DBC-150). Curiously, D. seriema and D. martensis, although belonging to different and allopatric clusters, became independently fixed for the same major type of alpha/beta ancestral repeats, illustrating a rare case of parallelism in satDNA evolution. The contrasting pictures illustrate the diversity of evolutionary pathways a satDNA can follow, defining a “non-regular orbit” with outcomes difficult to predict.     

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.     

Characterization of an <Emphasis Type="Italic">Eco</Emphasis>RI family of satellite DNA from two species

We have cloned and sequenced a 321bp band of repetitive DNA from Eptesicus fuscus and E. serotinus observed after gel electrophoresis of EcoRI digested genomic DNA in both species. Southern blot analysis of genomic DNA (from both species) digested with the same enzyme showed the existence of a ladder pattern indicating that the repetitive DNA is arrayed in tandem. The repetitive sequences have a monomer unit of 321bp which is composed of two subunits of 160bp, suggested by the existence of a 160bp band in the ladder of E. fuscus and by the presence of some direct repeats found in the analysis of the consensus sequence. Analysis of the methylation status demonstrated that cytosines in CCGG sequences in this satellite DNA are methylated in E. fuscus but not in the E. serotinus. Alignment of the sequenced clones showed that several nucleotide positions are diagnostic species-specific and consequently the phylogenetic analysis grouped the monomer units from both species in two clearly separated groups.     

In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501

Summary The tobacco (Nicotiana tabacum) nuclear genome contains long tracts of DNA (i.e. in excess of 18 kb) with high sequence homology to the tobacco plastid genome. Five lambda clones containing these nuclear DNA sequences encompass more than one-third of the tobacco plastid genome. The absolute size of these five integrants is unknown but potentially includes uninterrupted sequences that are as large as the plastid genome itself. An additional sequence was cloned consisting of both nuclear and plastid-derived DNA sequences. The nuclear component of the clone is part of a family of repeats, which are present in about 400 locations in the nuclear genome. The homologous sequences present in chromosomal DNA were very similar to those of the corresponding sequences in the plastid genome. However significant sequence divergence, including base substitutions, insertions and deletions of up to 41 bp, was observed between these nuclear sequences and the plastid genome. Associated with the larger deletions were sequence motifs suggesting that processes such as DNA replication slippage and excision of hairpin loops may have been involved in deletion formation.     

Palindromic repeated sequences (PRSs) in the mitochondrial genome of rice: evidence for their insertion after divergence of the genus Oryza from the other Gramineae

We have identified a family of small repeated sequences (from 60 to 66 bp in length) in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare). There are at least ten copies of these sequences and they are distributed throughout the mitochondrial genome. Each is potentially capable of forming a stem-and-loop structure and we have designated them PRSs (palindromic repeated sequences). Their features are reminiscent of the small dispersed repeats in the mitochondrial DNA (mtDNA) of some lower eukaryotes, such as Saccharomyces cerevisiae, Neurospora crassa and Chlamydomonas reinhardtii. Some of the PRSs of rice mtDNA are located in the intron of the gene for ribosomal protein S3 (rps3) and in the flanking sequence of the gene for chloroplast-like tRNA^Asn (trnN). An analysis of PCR-amplified fragments of these regions from the DNA of some Gramineae suggests that the PRSs were inserted into these regions of the Oryza mtDNA after the divergence of Oryza from the other Gramineae.     

Centromeric and non-centromeric satellite DNA organisation differs in holocentric <Emphasis Type="Italic">Rhynchospora</Emphasis> species

Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Unusual genome organization of the marine invertebrate Rapana thomasiana Grosse (Gastropoda)

The genome organization of the marine snail Rapana thomasiana Grosse (Gastropoda), genome size 2.7 pg, was studied by reassociation kinetics, S₁-nuclease assay, and restriction enzyme analysis. The slow-reassociating (single-copy) fraction represented only 21% of the genome. The average length of 80% of the single-copy sequences was less than 700 bp and the remaining 20% no longer than 1,400 bp. Longer stretches of unique DNA were not observed. The genome contained an unusually high percent-age of inverted repeats: at standard fragment length the zero-time binding fraction amounted to 25% of the genome. Foldback structures ranging from 200 bp to more than 10 kb were observed after S₁-nuclease treatment. They were randomly distributed throughout at least 85% of the genome, and the spacings between them were estimated to be about 1,600 bp on the average. The middle-repetitive DNA (45% of the genome) contained two kinetic components, repeated 430 and 65,000 times per genome, respectively. It was found that the majority of the repetitive sequences are about 300 bp long. Longer repeats (about 2,000 bp) were also observed, comprising a small portion of the genome. The inverted repeats, the middle-repetitive, and the singly-copy sequences were fully interspersed in the genome, thus indicating that R. thomasiana DNA is not organized in either the Xenopus or the Drosophila pattern type. — R. thomasiana is the only mollusc so far in which a satellite DNA has been found. It is organized in tandem repeats of 1,460 bp with a very complex organization but a low degree of divergence.     

ITS2 ribosomal DNA sequence variation of the bumblebee,Bombus ardens (hymenoptera: Apidae)

The bumblebee species,Bombus, is an invaluable natural resource for greenhouse pollination. Low levels of genetic variation ofBombus ardens have been reported in a previous mitochondrial (mt) gene study. In this study, we sequenced the complete internal transcribed spacer 2 (ITS2) of the nuclear rDNA obtained from 100B. ardens individuals collected from several Korean localities, in an effort to assess its usefulness in characterizing the genetic diversity and relationships among populations of B. ardens. The ITS2 sequences ofB. ardens were shown to be longest among known insects, ranging in size from 1,971–1,984 bp. The sequences harbor four duplicated repeats-≈27 bp repeats, ≈20 bp repeats, ≈33 bp repeats, and ≈34 bp repeats-which have never before been reported in other insect ITS2 rDNA. The maximum sequence divergence of 1.01% among 96 sequence types confirmed the applicability of this molecule to the study of intraspecific variation, revealing higher sequence variation as compared to the previously studied mt COI gene. Overall, a very high per generation migration ratio (Nm = 5.83 ≈ infinite) and a very low level of genetic fixation (FST =0 –0.08) were noted to exist among populations ofB. ardens. The high estimation of gene flow among most populations-in particular, between the remote island Ulleungdo and several inland populations-suggest that historical events may be more responsible for the contemporary population structure of B. ardens. The finding of the lowest genetic diversity (π) in the population on Ulleungdo Island (π = 0.007434) may be reflective of a relatively small population size and the geographical isolation of the population as compared with other inland populations.     

High-resolution mapping of repetitive DNA by in situ hybridization: molecular and chromosomal features of prominent dispersed and discretely localized DNA families from the wild beet species Beta procumbens

Members of three prominent DNA families of Beta procumbens have been isolated as Sau3A repeats. Two families consisting of repeats of about 158 bp and 312 bp are organized as satellite DNAs (Sau3A satellites I and II), whereas the third family with a repeat length of 202 bp is interspersed throughout the genome. Multi-colour fluorescence in situ hybridization was used for physical mapping of the DNA families, and has shown that these tandemly organized families occur in large heterochromatic and DAPI positive blocks. The Sau3A satellite I hybridized exclusively around or near the centromeres of 10, 11 or 12 chromosomes. The Sau3A satellite family I showed high intraspecific variability and high-resolution physical mapping was performed on pachytene chromosomes using differentially labelled repeats. The physical order of satellite subfamily arrays along a chromosome was visualized and provided evidence that large arrays of plant satellite repeats are not contiguous and consist of distinct subfamily domains. Re-hybridization of a heterologous rRNA probe to mitotic metaphase chromosomes revealed that the 18S-5.8S-25S rRNA genes are located at subterminal position on one chromosome pair missing repeat clusters of the Sau3A satellite family I. It is known that arrays of Sau3A satellite I repeats are tightly linked to a nematode (Heterodera schachtii) resistance gene and our results show that the gene might be located close to the centromere. Large arrays of the Sau3A satellite II were found in centromeric regions of 16 chromosomes and, in addition, a considerable interspersion of repeats over all chromosomes was observed. The family of interspersed 202 bp repeats is uniformly distributed over all chromosomes and largely excluded from the rRNA gene cluster but shows local amplification in some regions. Southern hybridization has shown that all three families are specific for genomes of the section Procumbentes of the genus Beta.     

Molecular and cytogenetic characterization of an AT-rich satellite DNA family in <Emphasis Type="Italic">Urvillea chacoensis</Emphasis> Hunz. (Paullinieae,Sapindaceae)

Urvillea chacoensis is a climber with 2n = 22 and some terminal AT-rich heterochromatin blocks that differentiate it from other species of the genus. The AT-rich highly repeated satellite DNA was isolated from U. chacoensis by the digestion of total nuclear DNA with HindIII and XbaI and cloned in Escherichia coli. Satellite DNA structure and chromosomal distribution were investigated. DNA sequencing revealed that the repeat length of satDNA ranges between 721 and 728 bp, the percentage of AT-base pairs was about 72–73% and the studied clones showed an identity of 92.5–95.9%. Although this monomer has a tetranucleosomal size, direct imperfect repetitions of ~180 bp subdividing it in four nucleosomal subregions were observed. The results obtained with FISH indicate that this monomer usually appears distributed in the terminal regions of most chromosomes and is associated to heterochromatin blocks observed after DAPI staining. These observations are discussed in relation to the satellite DNA evolution and compared with other features observed in several plant groups.     

Population and species variation of minisatellite DNA in Plantago

Three Plantago species were surveyed for within- and between-population variation at DNA sequences detected with the M13 minisatellite probe. The levels and patterns of variation detected by this probe correspond to those expected from the mating systems of the species. The highly-selfing species P. major has a relatively low variability of minisatellite sequences within populations and considerable differentiation between populations. The outcrossing species P. lanceolata exhibits higher minisatellite variability within populations and moderate differentiation between populations. P. coronopus, with a mixed mating system, has levels of variation intermediate between P. major and P. lanceolata. The levels of variation within and between populations corresponds, in general, to the levels of allozyme variation determined in an earlier study. Mating system and population structure appear to have a major influence on M13-detected fragment variation.     

Phylogeographic relationships among worldwide populations of the cosmopolitan marine species, the striped gray mullet (Mugil cephalus), investigated by partial cytochrome b gene sequences

Several factors such as geographical barriers, demographic history, biological and ecological traits may contribute to delineate the evolutionary history of a species. The gray mullet, Mugil cephalus, represents an interesting case of a marine species with a coastal ecology and a cosmopolitan distribution. In this study, partial cytochrome b sequences were resolved for 177 M. cephalus specimens sampled from 14 different geographic sites, in order to investigate the genetic divergence and the phylogeographic relationship among populations at a global scale. Demographic parameters were also assessed. Analysis of partial cyt b sequences showed high levels of differentiation among populations from distant geographic areas as most populations harbored private alleles and showed reciprocal monophyly. Both phylogenetic trees and haplotype network indicate the East Australian haplotypes as the closest to the ancestor sequences, which leads to the hypothesis of an Indo-West Pacific center of origin for M. cephalus. The analyses of the molecular variance showed that the genetic variation in M. cephalus is mainly harbored among populations rather than within populations. The high variability indices (h, π) calculated on M. cephalus individuals pooled together and the very low variability indices detected at some geographic sampling sites suggest that gray mullet populations have undergone a long-term reproductive isolation characterized by events of population abundance reductions which may have favored genetic differentiation among populations.     

A genome-specific repeat sequence from kiwifruit (Actinidia deliciosa var. deliciosa)

Summary Six members of a family of moderately repetitive DNA sequences from kiwifruit (Actinidia deliciosa var. deliciosa) have been cloned and characterized. The repeat family is composed of elements that have a unit length of 463 bp, are highly methylated, occur in tandem arrays of at least 50 kb in length, and constitute about 0.5% of the kiwifruit genome. Individual elements diverge in nucleotide sequence by up to 5%, which suggests that the repeat sequence is evolving rapidly. Homologous sequences were found in A. deliciosa var. chlorocarpa. The repeat sequence was not found under low stringency hybridization conditions in the diploid A. chinensis, the species most closely related to the hexaploid kiwifruit, or in eight other Actinidia species. However, homologous repeats were detected in a tetraploid species, A. chrysantha. The results provide the first molecular evidence to suggest that kiwifruit may be an allopolyploid species.     

The organisation,nucleotide sequence,and chromosomal distribution of a satellite DNA from Allium cepa

We have investigated the organisation, nucleotide sequence, and chromosomal distribution of a tandemly repeated, satellite DNA from Allium cepa (Liliaceae). The satellite, which constitutes about 4% of the A. cepa genome, may be resolved from main-band DNA in antibiotic-CsCl density gradients, and has a repeat length of about 375 base pairs (bp). A cloned member of the repeat family hybridises exclusively to chromosome telomeres and has a non-random distribution in interphase nuclei. We present the nucleotide sequences of three repeats, which differ at a large number of positions. In addition to arrays made up of 375-bp repeats, homologous sequences are found in units with a greater repeat length. This divergence between repeats reflects the heterogeneity of the satellite determined using other criteria. Possible constraints on the interchromosomal exchange of repeated sequences are discussed.