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.     

Heterogeneity and organization of the ribosomal RNA genes ofCucurbita maxima

Thirty-six clones were recovered fromCucurbita maxima genomic DNA which had been enriched for rDNA and cleaved at the unique repeat unitHind III site. Twenty-nine of these, which contain complete rDNA units, were compared to a standard whose intergenic spacer (IGS) nucleotide sequence has been determined. Twenty-one are identical in length and restriction site pattern. Eight which differ from the standard in length do so because of addition or deletion of varying numbers of IGS subrepetitive units of two different classes, with four of the length variants being different in both of these classes. Seven clones were isolated which contain incomplete repeat units, six of which are composites of rDNA and non-rDNA material. They have been cleaved at the unique rDNAHind III site at one end and at a non-rDNAHind III site at the other. We consider it most likely that these are derived from the termini of repeat unit tandem arrays, although other explanations are possible. Twelve individual plants of two different cultivars were examined for heterogeneity of IGS length distribution. They all appear to be identical in this regard.     

Intragenomic length variation of the ribosomal DNA intergenic spacer in a malaria vector,Anopheles sinensis

We determined the complete sequences of six size variants of intergenic spacer (IGS) region from one individual of the malaria vector mosquito species, Anopheles sinensis. All six size variants observed in this study show almost the same basic primary structure in which three repeat regions (A, B, and C) are interspersed by highly conserved nonrepeating sections. In contrast to the well-ordered subrepeating patterns found in A and C, the repeat region B displays extremely variable and complicated profiles in the number and arrangement of subrepeat units among different size classes. It is apparent that the prominent level of length difference in the repeat regions B and C is responsible for the intragenomic length variations of the IGS molecule observed in the present study. High level of sequence homology and regularly arranged repeating pattern of 11 to 14 bp motif sequences harbored within the B repeat region allow us to consider that these motif sequences may be associated with their potential role as a recombination site. Compared to those previously published in other mosquito species, the IGS of A. sinensis showed a very unique structural format in subrepeat patterns of the IGS region. This result suggests that the structure and sequence profiles of the IGS region would provide useful information for the exploitation of a convenient molecular marker to identify morphologically complicated species complex and to characterize the genetic variation of population. This suggestion is far from being conclusive at present, but a further genetic study will bring more compelling evidences for this pending issue.     

Variation of nuclear ribosomal RNA genes in Eragrostis tef (Zucc.) Trotter.

Variation in the ribosomal RNA genes (rDNA) was examined to assess the genetic variability among 314 plants representing 28 accessions of Eragrostis tef, an important food crop. A restriction site map was constructed for the species by localization of the BamHI, BglII, DraI, EcoRI, EcoRV, NdeI, SacI, SpeI, XbaI, and XhoI sites. A comparison of this map with those of other grasses showed conservation of sites, especially in the coding region. However, a unique EcoRI site combined with a BamHI site in the 18S region may be of diagnostic value for the species. A BamHI fragment that spans the intergenic spacer was used as an indicator of length variation of rDNA repeat units. rDNA repeat units in E. tef ranged in size from 8.4 to 11.07 kbp. Considerable size variation of rDNA repeats was present among accessions, between individual plants within some accessions, and within single plants. A total of 19 spacer length (sl) phenotypes was observed in 16 accessions in which 11-42 plants were analyzed. A single restriction site polymorphism was detected in PI442115 that was also distinguished by having a single sl variant. Variation in the rRNA genes is a useful indicator of genetic diversity in E. tef germplasm.     

Comparative analysis of the intergenic spacer regions and population structure of the species complex of the pathogenic yeast Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI +5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.     

Ribosomal DNA variation within and among individuals ofLisianthius (Gentianaceae) populations

Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of theLisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site differences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18S and 5.8S rRNA genes, a region inLisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.     

Ribosomal DNA variation in foxtail millet, Setaria italica (L.) P. Beauv., and a survey of variation from Europe and Asia

 Restriction fragment length polymorphism (RFLP) and the structure of ribosomal RNA genes (rDNA) were investigated in 117 landraces of foxtail millet, Setaria italica (L.) P. Beauv. Five RFLP phenotypes were found when the genomic DNA was digested with BamHI; these were named types I–V. Of these types I, II and III were the most frequent. Type I was mainly distributed in the temperature zone, type II in the Taiwan-Philippines Islands and type III in South Asia. Restriction mapping of the cloned rDNA and comparison with RFLP phenotypes showed that the different types originated from a polymorphism in the length within the intergenic spacer (IGS) and BamHI site changes within the IGS. Received: 28 August 1996 / Accepted: 28 February 1997     

Functional and ecological significance of rDNA intergenic spacer variation in a clonal organism under divergent selection for production rate

It has recently been hypothesized that variation in the intergenic spacer (IGS) of rDNA has considerable developmental, evolutionary and ecological significance through effects on growth rate and body C : N : P stoichiometry resulting from the role of the IGS in production of rRNA. To test these ideas, we assessed changes in size and structure of the repetitive region of the IGS, juvenile growth rate (JGR), RNA and phosphorus (P) contents in clonal lineages of Daphnia pulex derived from a single female and subjected to divergent selection on weight-specific fecundity (WSF). As a result of selection, WSF diverged rapidly, with significant reductions within two generations. Other significant changes accompanying shifts in WSF were that juveniles produced by low-WSF females grew more rapidly and had higher RNA and P contents. An increased predominance of long IGS variants was observed in lineages with elevated JGRs and low WSF. The observed variations in IGS length were related to the number of subrepeat units carrying a promoter sequence in the repetitive region. These results strongly support the hypothesized relationships, indicate a genetic mechanism for the evolution of such associations and demonstrate that Daphnia (and perhaps other parthenogens) possess considerable potential for rapid adaptive change in major life-history traits.     

Organization of 5S ribosomal DNA of Melitaea trivia

Two length variants of 5S rDNA repeated units were detected in the genome of East European butterfly Melitaea trivia. Both repeat variants contain the 5S rRNA coding region of the same length of 120 bp, but possess the intergenic spacer region (IGS) of different size, 78 and 125 bp, respectively. The level of sequence similarity between the two 5S rDNA variants amounts to 43.9-45.5% in the IGS, whereas the coding region appears to be more conservative. In the IGS, microsatellite sequence motives were found; amplification of these motives could be involved in the evolution of the 5S rDNA.     

Genetic diversity and recombination within populations of Fusarium pseudograminearum from western Canada.

Genetic diversity within populations of Fusarium pseudograminearum isolated from wheat grains from the Canadian provinces of Alberta and Saskatchewan was investigated. Three restriction enzymes (EcoRI, HaeIII, and PstI) were used to carry out restriction analysis of the nuclear ribosomal DNA (nrDNA) intergenic spacer region (IGS region) and eight primers were used to generate inter-simple sequence-repeat (ISSR) molecular markers. Our study indicated substantially high genetic diversity within these two populations, but low genetic differentiation and frequent gene flow among populations. The IGS data showed no genetic distinction between the two Alberta populations and only minor genetic differentiation between the Saskatchewan and Alberta populations. Analysis of molecular variance indicated that most genetic variability resulted from differences among isolates within populations. Multilocus linkage disequilibrium analysis suggested a panmictic population genetic structure and the occurrence of significant recombination in F. pseudograminearum. Regular gene flow and random mating between isolates from different populations could result in novel genotypes with both improved pathological and biological traits.     

Genetic changes within an aphid clone: homogenization of rDNA intergenic spacers after insecticide selection

A single asexual maternal lineage (i.e. clone) of the greenbug aphid, Schizaphis graminum (Rondani) was repeatedly selected with the insecticide disulfoton ( O , O -diethyl S- [2-(ethylthio)ethyl] phosphorodioate). A parallel colony of the non-selected clone was also maintained. After approximately 200 generations (4 years) of continuous selection, both the selected and non-selected clones were assayed for changes in intergenic spacer (IGS) length variants of the rRNA cistron. No changes in sets of IGS variants were detected in the non-selected clone. However, the selected clone was found to have lost three variants present in the non-selected clone. This probably occurred by unequal cross-over between sister chromatids, whereby the cistron became homogenized by an increase of frequency of two smaller variants. This documents a large-scale genetic change occurring within the rRNA cistron in a parthenogenetically reproducing aphid.  © 2003 The Linnean Society of London. Biological Journal of the Linnean Society , 2003, 79 , 101–105.     

The organization of ribosomal genes in vertebrates

The organization of the repeat unit of the ribosomal genes has been determined in 15 different species of vertebrates. The EcoRI and BamHI restriction maps of the rDNA from single individuals of different species of fishes, amphibians, and reptiles have been analysed. Two rDNA clones from Xenopus laevis (representing one complete repeat unit) were used as probes in Southern blots to detect restriction fragments containing ribosomal genes. The results obtained indicate that the transcribed regions are highly conserved in length and sequence inside the same zoological class. These regions are less conserved when species from different classes are compared but a general trend has been observed. In contrast, the length and sequence of the spacer regions are very variable, even within the same zoological class. Different types of heterogeneity have been observed; examples range from a single type of ribosomal repeat unit within a species to the absence of any detectable regular tandem array of units.     

RIBOSOMAL DNA AND RAPD VARIATION IN THE RARE PLANT FAMILY LACTORIDACEAE

Lactoris fernandeziana, endemic to the island of Masatierra in the Juan Fernandez Archipelago, is the only living member of the primitive angiosperm family, Lactoridaceae. The species was surveyed for ribosomal DNA (rDNA) and RAPD (Random Amplified Polymorphic DNA) variation. Previous analyses of allozymes had revealed no variation within the species. Variation was found for length in the intergenic spacer and for restriction sites in the 18S–25S genes of rDNA, and for the presence of amplified bands using 16 primers. Different rDNA repeat lengths and restriction site variants were detected within individuals as well as within and among populations. The level of variation in RAPDs is low relative to other Juan Fernandez endemic species surveyed, and nearly all variants were restricted to single populations. The rDNA length variants were distributed throughout the island, whereas the rDNA restriction site variants and RAPD markers indicated minor genetic differences among the populations.     

Study of polymorphism in human rRNA gene clusters. Population and familial analysis of basic types of RFLP

The frequency and average amount of copy number per genome were defined for standard and a number of new variants of BamHI 5'-NTS RFLP from populations of Moscow, Riga and individuals with Down syndrome. It was demonstrated that the populations studied differ neither in population frequency nor in the average amount of copy number of the variants. New variants were detected in the EcoRI 3'-NTS RFLP system and their amplification, as well as discordance among MZ twins. Possible target for methylation in the HindII site of 3' end of 28S rRNA gene was revealed. Analysis of data obtained demonstrated inefficiency of using the RFLP systems in systematic mapping of NOR-chromosomes. Our data also suggested a possible role of amplification of one copy repeated unit rRNA genes in their evolution.     

Monitoring the mode and tempo of concerted evolution in the Drosophila melanogaster rDNA locus

Non-LTR retrotransposons R1 and R2 have persisted in rRNA gene loci (rDNA) since the origin of arthropods despite their continued elimination by the recombinational mechanisms of concerted evolution. This study evaluated the short-term evolutionary dynamics of the rDNA locus by measuring the divergence among replicate Drosophila melanogaster lines after 400 generations. The total number of rDNA units on the X chromosome of each line varied from 140 to 310, while the fraction of units inserted with R1 and R2 retrotransposons ranged from 37 to 65%. This level of variation is comparable to that found in natural population surveys. Variation in locus size and retrotransposon load was correlated with large changes in the number of uninserted and R1-inserted units, yet the numbers of R2-inserted units were relatively unchanged. Intergenic spacer (IGS) region length variants were also used to evaluate changes in the rDNA loci. All IGS length variants present in the lines showed significant increases and decreases of copy number. These studies, combined with previous data following specific R1 and R2 insertions in these lines, help to define the type and distribution, both within the locus and within the individual units, of recombinational events that give rise to the concerted evolution of the rDNA locus.     

Nuclear Ribosomal DNA as a Probe for Genetic Variability in the Japanese Pear Pathotype of Alternaria alternata

A restriction fragment length polymorphism analysis of nuclear ribosomal RNA genes (rDNA) was used to measure the amount and distribution of genetic variability in populations of the Japanese pear pathotype of Alternaria alternata on both micro- and macrogeographical scales. A total of 322 isolates were obtained from 13 areas in Aichi, Gifu, and Tottori Prefectures in central and western Japan. The restriction fragment length polymorphism analysis revealed that the pathogen populations contained at least eight rDNA variants. The eight variant types differed in the lengths and in the presence of the restriction sites in spacer DNA outside the coding regions for rRNAs. A total of 271 isolates were classified into the eight types. The remaining 51 isolates were determined to have mixed rDNA types. Single pear fields typically contained two to five types of rDNA variants. The frequencies of rDNA variants in 11 populations in Tottori Prefecture were compared; in this prefecture orchards containing the susceptible pear are common. Except for one collection site, there were no significant differences in the composition of the rDNA variants among the populations. This suggests that dispersal of inocula has occurred frequently in Tottori Prefecture. In contrast, significantly different distributions were observed in the three prefectures, indicating that gene flow between prefectures might be limited by geographical isolation. DNA fingerprints resulting from hybridization with a moderately repetitive DNA sequence of the fungus revealed greater genetic variability and geographical differences in genetic population structure even within the same rDNA type.     

Characterization of a rat tRNA gene cluster containing the genes for tRNAAsp, tRNAGly and tRNAGlu, and pseudogenes.

The putative genes for tRNAGAUAsp(C), tRNAGGAGly(G) and tRNAGAGGlu are in a cluster on the rat chromosome and are present exclusively in a 3.3 kb region cleaved with a restriction endonuclease EcoRI. The cluster reiterates about 10 times on the haploid DNA. Four lambda clones each containing an independent repeating unit were isolated from a rat gene library. The studies on the cloned DNA revealed that the length of the repeating unit including the 3.3 kb EcoRI fragment was at least 13.5 kb. Nucleotide sequence analysis of the 3.3 kb DNA in the isolated clones showed sequence variations among the repeating units and incomplete genes for tRNAGly and tRNAGlu within the clusters.