Unusually high numbers of ribosomal RNA genes in copepods (Arthropoda: Crustacea) and their relationship to genome size.

We report on copy numbers of 18S ribosomal RNA genes in three species of copepods (Crustacea: Copepoda), two of which possess an unusual arrangement in which 5S genes are included within the 18S-5.8S-28S repeat unit. Slot blots of genomic and standard DNA were hybridized with an 18S rRNA gene probe constructed from one of the marine species and hybridization was quantified using chemiluminescence. Diploid 18S rRNA gene copy numbers are estimated as ca. 15 300 and 33 500 in the marine species Calanus finmarchicus (13.0 pg DNA in 2C adult nuclei) and C. glacialis (24.2 pg DNA), respectively, and ca. 840 and 730 in two freshwater populations of Mesocyclops edax (both ca. 3 pg DNA) from Virginia and Nova Scotia, respectively. The roughly proportional relationship between 2C somatic nuclear DNA contents and rRNA gene copy number in the sibling species C. finmarchicus and C. glacialis may reflect polytenic replication of entire genomes during abrupt speciation events. Copy numbers may also reflect differential losses during embryonic chromatin diminution.     

Variation among alfalfa somaclones in copy number of repeated DNA sequences.

Repeated DNA sequences of alfalfa (Medicago sativa L.) somaclonal variants were analyzed to determine if changes in copy number had occurred during tissue culture. DNA clones containing highly repeated nuclear sequences from the diploid line HG2 (2x = 16) were slot blotted and probed with labeled DNAs from HG2 and several somaclones of HG2. Two DNA clones that differed visually in hybridization intensity among the plant DNAs and one clone that had constant hybridization intensity were selected and used as probes on Southern blots and slot blots containing equal quantities of DNAs from HG2 and 15 régénérants. Statistically significant differences were detected in the copy number of two anonymous DNA sequences initially selected as variable and in the copy number of sequences homologous to pea ribosomal DNA. Based on Southern blot analysis, these sequences appeared to be arranged as tandem repeats. The cloned sequence initially selected as stable did not vary significantly in copy number and it appeared to be arranged as a dispersed repeat. Both increases and decreases in copy number of repeated sequences were observed in plants from successive regeneration cycles. Results from this study indicate that specific repeated nuclear DNA sequences have changed copy number in plants regenerated from tissue culture.     

Comparative study of molecular characteristics of X and Y nucleolar organizers of various lines of Drosophila melanogaster

We have determined for the X chromosomes of 10 laboratory strains and the Y chromosomes of 4 of them both the total number of ribosomal units and the relative percentages of uninterrupted (ins-), type 1 (ins1: with distinction between small ins1S and large ins1L) and type 2 (ins2) interrupted ribosomal units. These studies were made with the DNA extracted from third instar larval diploid tissues (brains and imaginal discs) of X/X female lines or XNO-/Y male lineages (devoid of X ribosomal genes) whose members possess copies of the same initial X or Y chromosome. Between the X chromosomes as well as the Y chromosomes an approximately equal to 2-fold variation was observed in the total number of ribosomal genes: from approximately equal to 200 to 420 for the X chromosomes and from approximately equal to 150 to 330 for the Y ones. The Y chromosomes are devoid of insertion 1 interrupted units, but one can observe some variation in the percentage and hence the absolute numbers of uninterrupted and insertion 2 interrupted units. Among the X chromosomes a very large variation exists between the percentage and absolute number of all the ribosomal unit types; it is to be noted especially that the number of uninterrupted units, which are the only kind of ribosomal genes actively transcribed, can vary from about 20 to 140 without any differences in the development of the different strains.     

Evolution of DNA in Heterochromatin: the Drosophila Melanogaster Sibling Species Subgroup as a Resource

The Drosophila melanogasterspecies subgroup is a closely-knit collection of eight sibling species whose relationships are well defined. These species are too close for most evolutionary studies of euchromatic genes but are ideal to investigate the major changes that occur to DNA in heterochromatin over short periods during evolution. For example, it is not known whether the locations of genes in heterochromatin are conserved over this time. The 18S and 28S ribosomal RNA genes can be considered as genuine heterochromatic genes. In D. melanogasterthe rRNA genes are located at two sites, one each on the X and Y chromosome. In the other seven sibling species, rRNA genes are also located on the sex chromosomes but the positions often vary significantly, particularly on the Y. Furthermore, rDNA has been lost from the Y chromosome of both D. simulansand D. sechellia, presumably after separation of the line leading to present-day D. mauritiana.We conclude that changes to chromosomal position and copy number of rDNA arrays occur over much shorter evolutionary timespans than previously thought. In these respects the rDNA behaves more like the tandemly repeated satellite DNAs than euchromatic genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intra-Genomic Variation in the Ribosomal Repeats of Nematodes

Ribosomal loci represent a major tool for investigating environmental diversity and community structure via high-throughput marker gene studies of eukaryotes (e.g. 18S rRNA). Since the estimation of species’ abundance is a major goal of environmental studies (by counting numbers of sequences), understanding the patterns of rRNA copy number across species will be critical for informing such high-throughput approaches. Such knowledge is critical, given that ribosomal RNA genes exist within multi-copy repeated arrays in a genome. Here we measured the repeat copy number for six nematode species by mapping the sequences from whole genome shotgun libraries against reference sequences for their rRNA repeat. This revealed a 6-fold variation in repeat copy number amongst taxa investigated, with levels of intragenomic variation ranging from 56 to 323 copies of the rRNA array. By applying the same approach to four C. elegans mutation accumulation lines propagated by repeated bottlenecking for an average of ~400 generations, we find on average a 2-fold increase in repeat copy number (rate of increase in rRNA estimated at 0.0285-0.3414 copies per generation), suggesting that rRNA repeat copy number is subject to selection. Within each Caenorhabditis species, the majority of intragenomic variation found across the rRNA repeat was observed within gene regions (18S, 28S, 5.8S), suggesting that such intragenomic variation is not a product of selection for rRNA coding function. We find that the dramatic variation in repeat copy number among these six nematode genomes would limit the use of rRNA in estimates of organismal abundance. In addition, the unique pattern of variation within a single genome was uncorrelated with patterns of divergence between species, reflecting a strong signature of natural selection for rRNA function. A better understanding of the factors that control or affect copy number in these arrays, as well as their rates and patterns of evolution, will be critical for informing estimates of global biodiversity.     

Genomic dosages of active rRNA genes in coke-oven workers

Genomic dosage (copy number) of active ribosomal genes was evaluated using visual semi-quantitative method determining the sizes of Ag-NORs in acrocentric chromosomes after selective silver nitrate staining. A relationship between the length of service and the active ribosomal gene copy number was established: the highest numbers of active rRNA genes were observed in coke-oven workers with a length of service exceeding 20 years. An inverse relationship between the individual doses of active ribosomal genes and toxicogenetic susceptibility of the workers to the occupational factors was also revealed.     

Evidence Against Amplification of Four Genes in Giant Cells Induced by Meloidogyne incognita

Giant-cell DNA was isolated from pea (Pisum sativum) inoculated with Meloidogyne incognita and used in slot blots to test for selective sequence amplification. Four sequences representing low (ribulose 1,5-bisphosphate carboxylase and actin), mid-level (histone 3), and highly repetitive (large ribosomal repeat) sequence DNA were used as probes. Known amounts of root-tip DNA and giant-cell DNA were blotted onto hybridization membranes and probed. The signal strength on autoradiographs containing equal amounts of root-tip DNA and giant-cell DNA were compared with a scanning densitometer. No difference in signal strength between equal amounts of root-tip DNA and giant-cell DNA was found. Thus, for the probes tested, there is no difference in copy number and, hence, no selective DNA sequence amplification has occurred.     

Reciprocal Recombination and the Evolution of the Ribosomal Gene Family of Drosophila Melanogaster

The role of reciprocal recombination in the coevolution of the ribosomal RNA gene family on the X and Y chromosomes of Drosophila melanogaster was assessed by determining the frequency and nature of such exchange. In order to detect exchange events within the ribosomal RNA gene family, both flanking markers and restriction fragment length polymorphisms within the tandemly repeated gene family were used. The vast majority of crossovers between flanking markers were within the ribosomal RNA gene region, indicating that this region is a hotspot for heterochromatic recombination. The frequency of crossovers within the ribosomal RNA gene region was approximately 10(-4) in both X/X and X/Y individuals. In conjunction with published X chromosome-specific and Y chromosome-specific sequences and restriction patterns, the data indicate that reciprocal recombination alone cannot be responsible for the observed variation in natural populations.     

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymerase chain reaction (repPCR), we are able to amplify a representative sampling of multiple repetitive units simultaneously, allowing rapid analysis of chromosomal subsets. Direct sequence analysis of repPCR amplified alpha satellite from chromosomes 17 and X reveals positions of sequence heterogeneity as two bands at a single nucleotide position on a sequencing ladder. The use of TdT in the sequencing reactions greatly reduces the background associated with polymerase pauses and stops, allowing visualization of heterogeneous bases found in as little as 10% of the repeat units. Confirmation of these heterogeneous positions was obtained by comparison to the sequence of multiple individual cloned copies obtained both by PCR and non-PCR based methods. PCR amplification of alpha satellite can also reveal multiple repeat units which differ in size. Analysis of repPCR products from chromosome 17 and X allows rapid determination of the molecular basis of these repeat unit length variants, which appear to be a result of unequal crossing-over. The application of repPCR to the study of tandemly repeated DNA should allow in-depth analysis of intra- and interchromosomal variation and unequal crossing-over, thus providing insight into the biology and genetics of these large families of DNA.     

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.     

Extensive sequence homologies between Y and other human chromosomes

Twenty-six human Y-chromosome-derived DNA sequences, free of repetitive material, were used to probe male and female genomic blots. We present data from a detailed analysis and chromosomal location of the bands detected by such probes, which demonstrate extensive DNA sequence homology between the mammalian sex chromosomes and autosomes. Under stringent conditions, nine Y-derived probes reacted exclusively with the Y chromosome, 12 probes detected homologous sequences present on both the Y and the X, four probes detected homologies between Y and autosome(s) without any X counterpart and, finally, one probe hybridized to homologous sequences on Y, X and autosome(s). These data are consistent with the hypothesis of a common evolutionary origin for the mammalian sex chromosomes and reveal structural similarities between Y-located and autosomal non-repetitive sequences.     

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.     

Genomic dosages of active rRNA genes in coke-oven workers

Genomic dosage (copy number) of active ribosomal genes was evaluated using visual semi-quantitative method determining the sizes of Ag-NORs in acrocentric chromosomes after selective silver nitrate staining. A relationship between the length of service and the active ribosomal gene copy number was established: the highest numbers of active rRNA genes were observed in coke-oven workers with a length of service exceeding 20 years. An inverse relationship between the individual doses of active ribosomal genes and toxicogenetic susceptibility of the workers to the occupational factors was also revealed.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1702–1708.Original Russian Text Copyright © 2004 by Minina, Druzhinin.     

Chromosome Rearrangement by Ectopic Recombination in Drosophila Melanogaster: Genome Structure and Evolution

Ectopic recombination between interspersed repeat sequences generates chromosomal rearrangements that have a major impact on genome structure. A survey of ectopic recombination in the region flanking the white locus of Drosophila melanogaster identified 25 transposon-mediated rearrangements from four parallel experiments. Eighteen of the 25 were generated from females carrying X chromosomes heterozygous for interspersed repeat sequences. The cytogenetic and molecular analyses of the rearrangements and the parental chromosomes show: (1) interchromosomal and intrachromosomal recombinants are generated in about equal numbers; (2) ectopic recombination appears to be a meiotic process that is stimulated by the interchromosomal effect to about the same degree as regular crossing over; (3) copies of the retrotransposon roo were involved in all of the interchromosomal exchanges; some copies were involved much more frequently than others in the target region; (4) homozygosis for interspersed repeat sequences and other sequence variations significantly reduced ectopic recombination.     

Recombination between the X and Y chromosomes and the Sxr region of the mouse.

The Sxr (sex-reversed) region that carries a copy of the mouse Y chromosomal testis-determining gene can be attached to the distal end of either the Y or the X chromosome. During male meiosis, Sxr recombined freely between the X and Y chromosomes, with an estimated recombination frequency not significantly different from 50% in either direction. During female meiosis, Sxr recombined freely between the X chromosome to which it was attached and an X-autosome translocation. A male mouse carrying the original Sxra region on its Y chromosome, and the shorter Sxrb variant on the X, also showed 50% recombination between the sex chromosomes. Evidence of unequal crossing-over between the two Sxr regions was obtained: using five markers deleted from Sxrb, 3 variant Sxr regions were detected in 159 progeny (1.9%). Four other variants (one from the original cross and three from later generations) were presumed to have been derived from illegitimate pairing and crossing-over between Sxrb and the homologous region on the short arm of the Y chromosome. The generation of new variants throws light on the arrangement of gene loci and other markers within the short arm of the mouse Y chromosome.     

Highly efficient sex chromosome interchanges produced by I-CreI expression in Drosophila

The homing endonuclease I-CreI recognizes a site in the gene encoding the 23S rRNA of Chlamydomonas reinhardtii. A very similar sequence is present in the 28S rRNA genes that are located on the X and Y chromosomes of Drosophila melanogaster. In this work we show that I-CreI expression in Drosophila is capable of causing induced DNA damage and eliciting cell cycle arrest. Expression also caused recombination between the X and Y chromosomes in the heterochromatic regions where the rDNA is located, presumably as a result of a high frequency of double-strand breaks in these regions. Approximately 20% of the offspring of males expressing I-CreI showed exceptional inheritance of X- and Y-linked markers, consistent with chromosome exchange at rDNA loci. Cytogenetic analysis confirmed the structures of many of these products. Exchange between the X and Y chromosomes can be induced in males and females to produce derivative-altered Y chromosomes, attached-XY, and attached-X chromosomes. This method has advantages over the traditional use of X rays for generating X-Y interchanges because it is very frequent and it generates predictable products.     

Interchromosomal duplications on the Bactrocera oleae Y chromosome imply a distinct evolutionary origin of the sex chromosomes compared to Drosophila

Background

Diptera have an extraordinary variety of sex determination mechanisms, and Drosophila melanogaster is the paradigm for this group. However, the Drosophila sex determination pathway is only partially conserved and the family Tephritidae affords an interesting example. The tephritid Y chromosome is postulated to be necessary to determine male development. Characterization of Y sequences, apart from elucidating the nature of the male determining factor, is also important to understand the evolutionary history of sex chromosomes within the Tephritidae. We studied the Y sequences from the olive fly, Bactrocera oleae. Its Y chromosome is minute and highly heterochromatic, and displays high heteromorphism with the X chromosome.

Methodology/Principal Findings

A combined Representational Difference Analysis (RDA) and fluorescence in-situ hybridization (FISH) approach was used to investigate the Y chromosome to derive information on its sequence content. The Y chromosome is strewn with repetitive DNA sequences, the majority of which are also interdispersed in the pericentromeric regions of the autosomes. The Y chromosome appears to have accumulated small and large repetitive interchromosomal duplications. The large interchromosomal duplications harbour an importin-4-like gene fragment. Apart from these importin-4-like sequences, the other Y repetitive sequences are not shared with the X chromosome, suggesting molecular differentiation of these two chromosomes. Moreover, as the identified Y sequences were not detected on the Y chromosomes of closely related tephritids, we can infer divergence in the repetitive nature of their sequence contents.

Conclusions/Significance

The identification of Y-linked sequences may tell us much about the repetitive nature, the origin and the evolution of Y chromosomes. We hypothesize how these repetitive sequences accumulated and were maintained on the Y chromosome during its evolutionary history. Our data reinforce the idea that the sex chromosomes of the Tephritidae may have distinct evolutionary origins with respect to those of the Drosophilidae and other Dipteran families.     

Double crossing over: elementary events and the sequence of their occurrence

Analysis of the crossing over increment in the structurally normal chromosome of Drosophila caused by a rearrangement in nonhomologous chromosome (interchromosomal effect on crossing over, IEC) was carried out based on the author's personal and literature data. The IEC in the left arm of chromosome 2 caused by inversions in chromosomes X and 3, as well as the IEC in X chromosome caused by inversions in chromosomes 2 and 3, were examined. The IEC-induced increment of crossing over results from the increase of the number of double exchanges under the constant or reduced number of single exchanges. Tetrad analysis showed that the given alternation of the crossing over processes could occur only in the case of conversion of the tetrads with single exchanges into the tetrads with double exchanges. In other words, the events leading to the formation of double exchanges occur consecutively. The borders of the IEC-induced double exchanges can be seen all over the chromosome body. However, the IEC-induced increase of chromosome recombination length occurs only in the proximal region (in rare cases, in proximal and distal regions) of the chromosome arm. This means that a double exchange is formed when the first event with predominant location in the middle of the arm is supplemented with the second event predominantly localized at the arm T end, most frequently in the proximal region. The pattern of the IEC-induced double exchange formation can be satisfactorily described in terms of the contact model of the crossing over. According to the model, an elementary crossing-over event is the local contact between the homologues. Neither single exchange nor a double-stranded DNA break can serve as an elementary event in the process of any multiple exchange formation.     

Evolutionary dynamics of a satellite DNA in the tiger beetle species pair Cicindela campestris and C. maroccana.

Satellite repeat elements are an abundant component of eukaryotic genomes, but not enough is known about their evolutionary dynamics and their involvement in karyotype and species differentiation. We report the nucleotide sequence, chromosomal localization, and evolutionary dynamics of a repetitive DNA element of the tiger beetle species pair Cicindela maroccana and Cicindela campestris. The element was detected after restriction digest of C. maroccana total genomic DNA with EcoRI as a single band and its multimers on agarose gels. Cloning and sequencing of several isolates revealed a consensus sequence of 383 bp with no internal repeat structure and no detectable similarity to any entry in GenBank. Hybridization of the satellite unit to C. maroccana mitotic and meiotic chromosomes revealed the presence of this repetitive DNA in the centromeres of all chromosomes except the Y chromosome, which exhibited only a very weak signal in its short arm. PCR-based tests for this satellite in related species revealed its presence in the sister species C. campestris, but not in other closely related species. Phylogenetic analysis of PCR products revealed well-supported clades that generally separate copies from each species. Because both species exhibit the multiple X chromosome karyotypic system common to Cicindela, but differ in their X chromosome numbers (four in C. maroccana vs. three in C. campestris), structural differences could also be investigated with regard to the position of satellites in a newly arisen X chromosome. We find the satellite in a centromeric position in all X chromosomes of C. maroccana, suggesting that the origin of the additional X chromosome involves multiple karyotypic rearrangements.     

The use of repetitive DNA in cytogenetic studies of plant sex chromosomes

The structure of sex chromosomes in plants was analyzed by fluorescent in situ hybridization (FISH) with repetitive DNAs. FISH probes were successfully obtained from DNA libraries that were amplified from microdissected sex chromosomes. Some probes hybridized to the subtelomeric regions, where many kinds of repetitive DNAs are located with intrachromosomal similarity of their repeat units rather than interchromosomal similarity. For example, FISH with the subtelomeric repetitive sequence can easily show the location of the pseudoautosomal region (PAR) on the X chromosome of Silene latifolia. The other probes were localized on the interstitial region of the sex chromosomes. The interstitial region contains chloroplast DNAs or neighboring sequences of the internal telomeres, suggesting insertion or translocation occurred during differentiation of the sex chromosome. These data are very informative for understanding the structure of the plant sex chromosomes and their evolutionary process.