One-Step and Stepwise Magnification of a BOBBED LETHAL Chromosome in DROSOPHILA MELANOGASTER

Bobbed lethal (bbl) chromosomes carry too few ribosomal genes for homozygous flies to be viable. Reversion of bbl chromosomes to bb or nearly bb+ occurs under magnifying conditions at a low frequency in a single generation. These reversions occur too rapidly to be accounted for by single unequal sister chromatid exchanges and seem unlikely to be due to multiple sister strand exchanges within a given cell lineage. Analysis of several one-step revertants indicates that they are X-Y recombinant chromosomes which probably arise from X-Y recombination at bb. The addition of ribosomal genes from the Y chromosome to the bbl chromosome explains the more rapid reversion of the bbl chromosome than is permitted by single events of unequal sister chromatid exchange. Analysis of stepwise bbl magnified chromosomes, which were selected over a period of 4-9 magnifying generations, shows ribosomal gene patterns that are closely similar to each other. Similarity in rDNA pattern among stepwise magnified products of the same parental chromosome is consistent with reversion by a mechanism of unequal sister strand exchange.     

Differential magnification of rDNA gene types in bobbed mutants of Drosophila melanogaster

Summary In Drosophila melanogaster a partial loss of ribosomal genes leads to the bobbed phenotype. Magnification is a heritable increase in rDNA that may occur in males carrying a deleted X chromosome with a strong bobbed phenotype. The restriction patterns of X chromosome total rDNA, insertions and spacers from magnified bobbed strains were compared with those of the original bobbed mutations. It was found that magnification modifies restriction patterns and differentially affects gene types, increasing specific genes lacking insertions (INS^-). Increases in copy number of genes with type I insertions are generally lower than the total number of INS^- genes, while type II insertion genes are not perceptibly increased. The recovery of homogeneous progeny from a single premagnified male indicates that the magnification event might take place and become stable very early in the germ line, arguing against magnification being due to extrachromosomal amplification. Additionally, some gene types increase 3.5-fold while others are eliminated, indicating that they could not result from a single unequal cross-over. These results are in good agreement with the existence of partial clustering of rDNA genes according to type, and suggest that magnification could result from local amplification of genes.     

Autosomal Modifiers of the Bobbed Phenotype Are a Major Component of the Rdna Magnification Paradox in DROSOPHILA MELANOGASTER

rDNA magnification in Drosophila melanogaster is defined experimentally as the ability of bb/Ybb- males to produce exceptional progeny that are wild type with respect to rDNA associated phenotypes. Here, we show that some of these bobbed-plus progeny result not from genetic reversion at the bb locus but rather from variants at two or more autosomal loci that ameliorate the bobbed phenotype of rDNA deficient males in Drosophila. In doing so we resolve several aspects of a long-standing paradox concerning the phenomenon of rDNA magnification. This problem arose from the use of two genetic assays, which were presumed to be identical, but paradoxically, produced conflicting data on both the kinetics of reversion and the stability of magnified bb+ chromosomes. We resolve this problem by demonstrating that in one assay bobbed-plus progeny arise primarily by genetic reversion at the bobbed locus, whereas in the other assay bobbed-plus progeny arise both by reversion and by an epistatic effect of autosomal modifiers on the bobbed phenotype. We further show that such modifiers can facilitate the appearance of phenotypically bobbed-plus progeny even under conditions where genetic reversion is blocked by magnification defective mutants. Finally, we present a speculative model relating the action of these modifiers to the large increases in rDNA content observed in males undergoing magnification.     

Sister chromatid exchange and the evolution of rDNA spacer length

The structures of rDNA spacers from several species have been characterized and virtually all have internally repeated sequences. Different numbers of these internal repeats are responsible for most spacer length variation. Because unequal recombination between these internal repeats will cause new length variation, while unequal exchange between rDNA copies will homogenize the variants, we modeled the interaction of these two processes. Two models were used to simulate both types of unequal exchange at the sister chromatid level. Both models indicate that a narrow range of relative recombination frequencies is required to produce levels of variability comparable to those published. One model puts a lower limit on the number of internal repeats, and the other puts both a lower and upper limit on the number of repeats. The model with both maximum and minimum constraints produces a distribution closer to actual spacer distributions. These results imply that small changes in recombination rates can generate the differences in numbers of length variants observed in different species.     

R2 dynamics in Triops cancriformis (Bosc, 1801) (Crustacea,Branchiopoda, Notostraca): turnover rate and 28S concerted evolution

The R2 retrotransposon is here characterized in bisexual populations of the European crustacean Triops cancriformis. The isolated element matches well with the general aspects of the R2 family and it is highly differentiated from that of the congeneric North American Triops longicaudatus. The analysis of 5′ truncations indicates that R2 dynamics in T. cancriformis populations show a high turnover rate as observed in Drosophila simulans. For the first time in the literature, though, individuals harboring truncation variants, but lacking the complete element, are found. Present results suggest that transposition-mediated deletion mechanisms, possibly involving genomic turnover processes acting on rDNAs, can dramatically decrease the copy number or even delete R2 from the ribosomal locus. The presence of R2 does not seem to impact on the nucleotide variation of inserted 28S rDNA with respect to the uninserted genes. On the other hand, a low level of polymorphism characterizes rDNA units because new 28S variants continuously spread across the ribosomal array. Again, the interplay between transposition-mediated deletion and molecular drive may explain this pattern.     

Phylogenetic study of Class Armophorea (Alveolata,Ciliophora) based on 18S-rDNA data

The 18S rDNA phylogeny of Class Armophorea, a group of anaerobic ciliates, is proposed based on an analysis of 44 sequences (out of 195) retrieved from the NCBI/GenBank database. Emphasis was placed on the use of two nucleotide alignment criteria that involved variation in the gap-opening and gap-extension parameters and the use of rRNA secondary structure to orientate multiple-alignment. A sensitivity analysis of 76 data sets was run to assess the effect of variations in indel parameters on tree topologies. Bayesian inference, maximum likelihood and maximum parsimony phylogenetic analyses were used to explore how different analytic frameworks influenced the resulting hypotheses. A sensitivity analysis revealed that the relationships among higher taxa of the Intramacronucleata were dependent upon how indels were determined during multiple-alignment of nucleotides. The phylogenetic analyses rejected the monophyly of the Armophorea most of the time and consistently indicated that the Metopidae and Nyctotheridae were related to the Litostomatea. There was no consensus on the placement of the Caenomorphidae, which could be a sister group of the Metopidae + Nyctorheridae, or could have diverged at the base of the Spirotrichea branch or the Intramacronucleata tree.     

Restriction site and length polymorphism of the rDNA unit in the cultivated basidiomycetePleurotus cornucopiae

In the ribosomal DNA unit ofPleurotus cornucopiae, the rDNA coding regions are in the order 5, 5S-18S-5.8S-25S, 3, with the 5 location of the 5S gene differing from its 3 location found in other basidiomycetes. The most discriminating probe used to study the rDNA polymorphism consisted of a fragment that included the 5S, 18S and part of the 5.8S and 25S genes flanking three intergenic sequences. A high degree of rDNA polymorphism was observed in the sevenP. cornucopiae dikaryons studied. For the first time within a basidiomycete species, the restrictions maps distinguished two types of rDNA units (I and II). In each rDNA type, length variations in the external intergenic sequence IGS 1 located between the 25S and 5S genes allowed characterization of two different rDNA units in type I and four rDNA units in type II. This suggested that theP. cornucopiae rDNA units were derived from two kinds of ancestors (type I and II) by insertion or deletion events (100–700 bp) in the IGS 1. In four dikaryotic strains, two rDNA units of the same type (I or II) differing only by the IGS 1 length, were found in a similar number of copies, and presented a meiotic segregation in homokaryotic progeny. In one progeny, some homokaryotic strains possessed two different rDNA units: one with a high copy number and another with a lower one, showing that two different rDNA units could coexist in a single nucleus.     

Phylogenetic Relationships of Globodera millefolii,G. artemisiae,and Cactodera salina Based on ITS Region of Ribosomal DNA

Globodera millefolii and G. artemisiae are interesting because their type localities (Estonia and Russia, respectively) are geographically distant from those of the potato cyst nematodes and other Globodera species that seem to have originated in the Western world, and because the type host for each is a member of Compositae rather than Solanaceae. Sequence data for ITS1, ITS2, and 5.8S ribosomal DNA (ITS rDNA) for G. millefolii and G. artemisiae were nearly identical to sequence data for Cactodera salina from the rhizosphere of the estuary plant Salicornia bigelovii in Sonora, Mexico. The ITS rDNA sequences of these three species were all about 94% similar to those of two other Cactodera species for which ITS rDNA data were obtained. Phylogenetic analysis indicated that, based on the ITS rDNA data, G. millefolii and G. artemisiae are more closely related phylogenetically to the Cactodera species than to other nominal Globodera species. The molecular data further suggest that the genus Cactodera may comprise two or more morphologically similar but separate groups.     

Ring Chromosomes and rDNA Magnification in Drosophila

Tartof showed that ribosomal gene magnification in Drosophila was inhibited in a ring X chromosome. The present studies extend this observation by showing that ring X chromosomes are lost meiotically in male Drosophila undergoing ribosomal gene magnification as evidenced by the recovery of a lower number of ring-bearing progeny under magnifying conditions compared with nonmagnifying conditions. Associated with ring chromosome loss is a highly significant increase in the number of double-sized dicentric ring chromosomes in meiotic cells from magnifying males. These observations explain the failure of ring X chromosomes to magnify and imply that magnification in rod chromosomes occurs via a mechanism of unequal sister chromatid exchange. Our results support the hypothesis that the primary event of magnification is a sister chromatid exchange in the rDNA, that the frequency of sister strand exchanges is increased in magnifying flies, that a significant number of exchanges in magnifying flies occurs meiotically and that some of the exchanges are nonreciprocal. We have also found that autosomal mutations can affect both the frequency of abnormal ring structures and the ability of ring X chromosomes to magnify.     

Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli,Avoiding Dangers of Runaway Overreplication

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the “natural” CRC limit of ∼8 (cells divide more slowly); the “functional” CRC limit of ∼22 (cells divide extremely slowly); and the “tolerance” CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.     

Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

Regulation of rDNA stability by sumoylation

Repair of DNA lesions by homologous recombination relies on the copying of genetic information from an intact homologous sequence. However, many eukaryotic genomes contain repetitive sequences such as the ribosomal gene locus (rDNA), which poses a risk for illegitimate recombination. Therefore, the eukaryotic cell has evolved mechanisms to favor equal sister chromatid exchange (SCE) and suppress unequal SCE, single-strand annealing and break-induced replication. In the budding yeast Saccharomyces cerevisiae, the tight regulation of homologous recombination at the rDNA locus is dependent on the Smc5–Smc6 complex and sumoylation of Rad52, which directs DNA double-strand breaks in the rDNA to relocalize from within the nucleolus to the nucleoplasm before association with the recombination machinery. The relocalization before repair is important for maintaining rDNA stability. The focus of this review is the regulation of recombinational DNA repair at the rDNA locus by sumoylation and the Smc5–Smc6 complex in S. cerevisiae.     

Magnification in Drosophila: evidence for an inducible rDNA-specific recombination system

An experiment is described that provides evidence for an exchange mechanism to explain the increase in ribosomal gene number that occurs during bobbed magnification. We show that bobbed and bobbed-lethal alleles do not magnify in closed X chromosomes, but that a spontaneous ring opening restores normal magnification. The results provide strong evidence that the elementary magnifying event is unequal sister chromatid exchange, and can be interpreted in the framework of an inducible rDNA-specific recombination system as the basis of ribosomal gene magnification.     

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.     

First report of Xiphinema brevicolle Lordello et Costa, 1961 (Nematoda, Longidoridae) in Japan

Mixed populations of Xiphinema americanum-group species were detected from a root zone soil sample of Japanese holly, Ilex crenata, during a survey for plant-parasitic nematodes of commercial ornamental plant nurseries in Chiba Prefecture, Japan. From the result of the morphological study, the species were identified as Xiphinema brevicolle and Xiphinema sp. This is the first record of Xiphinema brevicolle in Japan. Morphometrics of Xiphinema brevicolle generally agree with those of the type specimens and the topotype specimens. Xiphinema sp. morphometrically resembles Xiphinema paramonovi except for tail length. The mitochondrial COI region, the nuclear 18S rDNA and the nuclear large subunit rDNA D2/D3 region of the species were sequenced and compared in the molecular study. For the COI region, PCR primers were newly designed to obtain longer sequences, ca. 900 bp, than previously used. Sequence identities of COI, 18S and D2/D3 regions between these two populations were 84.0-84.1%, 99.9% and 98.1-98.2%, respectively. Phylogenetic analyses of maximum likelihood trees were carried out to compare genetic relationships among the group and some suggestions were made on the Xiphinema brevicolle-subgroup.     

The Molecular through Ecological Genetics of Abnormal Abdomen. II. Ribosomal DNA Polymorphism Is Associated with the Abnormal Abdomen Syndrome in DROSOPHILA MERCATORUM

Restriction endonuclease cleavage analyses of cloned and genomic DNA samples indicate that the structure of the DNA encoding the large cytoplasmic RNAs (rDNAs) is altered in Drosophila mercatorum lines which exhibit an abnormal abdomen (aa) phenotype. In a majority of the rDNA repeat units from aa flies, the 28S coding sequence is interrupted by a large [5-6 kilobase pairs (kbp)] insert. A subclone containing this inserted DNA (ins 3) hybridizes primarily to rDNA-containing sequences in in situ and genomic blot hybridization experiments. Additionally, genomic nitrocellulose blot hybridization analyses show that ins- containing rDNA repeat units are clustered in a spontaneously arising aa mutant. This rDNA alteration in D. mercatorum flies with the aa phenotype more closely resembles the bobbed (bb) defect of D. hydei than the bb defect of D. melanogaster, which involves alterations in rDNA copy number. By analogy with the other Drosophila systems, we propose that the altered D. mercatorum rDNA repeat units are defective in rRNA production at a critical stage. The lowered levels of rRNA ultimately would limit the concentration of ribosomes needed to produce large quantities of a protein (in these cases, juvenile hormone esterase) needed for normal development.