Conservation of intergenic spacer length in ribosomal DNA of the tailed frog, Ascaphus truei

We have examined the organization of cloned rDNA [encoding ribosomal RNA (rRNA)] repeat units from the tailed frog, Ascaphus truei, and have compared rDNA spacer lengths in the genomes of eleven individuals from two widely-separated populations. This comparison has shown that the A. truei spacer is always very short (about 1.5 kb) and that it is remarkably constant in length. In none of the individuals tested were more than two spacer-length classes found and the maximum difference in spacer length found in comparisons both within single animals and across both populations was about 120 bp. We point out those structural features that may contribute to the unusual stability of this spacer and the consequent absence of the extensive length heterogeneities found amongst rDNA repeat units in most genomes.     

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.     

Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica; cloning and analysis of two size classes of repeats

Southern blotting of DNA from the ascomycetous yeast Yarrowia lipolytica revealed two major size classes of DNA units coding for rRNAs, which differ in length by about 1000 bp. We have cloned an rDNA unit of each size class. R-looping experiments revealed that the rRNA genes of both units are uninterrupted; subsequent heteroduplex analysis showed that the size difference both units is located within the nontranscribed spacer. Sequence analysis revealed that a major part of these spacers consists of a complex pattern of repetitions in periodicities of up to about 150 bp and that the difference between both rDNA units are located mainly in this repetitive region. Apart from different lengths of the repetitive regions, both rDNA units also reveal extended microheterogeneity within their homologous parts. Furthermore, no gene for 5S rRNA was observed in the spacer region. Therefore, the organization of the spacer of Yarrowia rDNA is clearly different from that of Saccharomyces cerevisiae.     

Cerastoderma glaucum 5S ribosomal DNA: characterization of the repeat unit, divergence with respect to Cerastoderma edule, and PCR-RFLPs for the identification of both cockles.

The 5S rDNA repeat unit of the cockle Cerastoderma glaucum from the Mediterranean and Baltic coasts was PCR amplified and sequenced. The length of the units was 539-568 bp, of which 120 bp were assigned to the 5S rRNA gene and 419-448 bp to the spacer region, and the G/C content was 46%-49%, 54%, and 44%-47%, respectively. Two types of units (A and B), differing in the spacer, were distinguished based on the percentage of differences and clustering in phylogenetic trees. A PCR assay with specific primers for each unit type indicated that the occurrence of both units is not restricted to the sequenced individuals. The 5S rDNA units of C. glaucum were compared with new and previously reported sequences of Cerastoderma edule. The degree of variation observed in C. edule was lower than that in C. glaucum and evidence for the existence of units A and B in C. edule was not found. The two cockles have the same coding region but displayed numerous fixed differences in the spacer region and group separately in the phylogenetic trees. Digestion of the 5S rDNA PCR product with the restriction enzymes HaeIII and EcoRV revealed two RFLPs useful for cockle identification.     

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.     

Heterogeneity in cucumber ribosomal DNA

Summary Restriction and hybridization analysis of cucumber native ribosomal (r) DNA purified from actinomycin-D/CsCl gradients suggested that the repeat units were heterogeneous in both length and sequence. Several full length rDNA repeat units were cloned and five are described which account for all the EcoR I and Xba I fragments present in native DNA. One of a number of BamH I sites found in the clones is not found in a proportion of native rDNA because of base modification. Restriction maps are described for the representative clones and aligned with R-loop maps obtained from electron microscope analysis of each type of repeat unit hybridized under R-loop conditions to pure 18S and 25S rRNAs. The major heterogeneity is explained by differences in length of the external spacer region and by a proportion of the repeat units showing a restriction fragment length polymorphism on EcoR I digestion. The regions coding for 18S and 25S rRNA are uninterrupted and highly conserved.     

Molecular characterization of ribosomal gene variation within and among NORs segregating in specialized populations of chicken.

The molecular organization of the 18S, 5.8S, and 28S ribosomal RNA gene repeat units, located at the single nucleolus organizer region (NOR) locus in the chicken, was investigated in genetically distinct populations of research and commercial chickens. Substantial gene repeat variation within and among NORs was documented. Intact ribosomal gene repeat size ranged from 11 kb to over 50 kb. Unique combinations of ribosomal genes, of different size, were specific to particular populations. It was determined that the basis for the ribosomal gene repeat size variation was intergenic spacer (IGS) length heterogeneity. Interestingly, in different populations, the location of the variation that contributes to length heterogeneity was specific to particular IGS subregions. In addition to IGS variation, an inbred line of Red Jungle Fowl exhibited coding region variation. Ribosomal gene copy number variation was also studied, and line averages ranged from 279 to 368. Average rDNA array size (a function of copy number and gene repeat length) was calculated for each of the populations and found to vary over a range of two megabases, from 5 to 7 Mb.     

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.     

Investigations of 5S rDNA of Vitis vinifera L.: sequence analysis and physical mapping.

Here we report the first results of a study of 5S rDNA of Vitis vinifera. 5S rDNA sequences from seven genotypes were amplified by PCR, cloned, and sequenced. Three types of repeats were found. Two variants, denominated long repeat and short repeat, appeared to be the main components of the 5S rDNA of this species, since they were found in all genotypes analyzed. They differed markedly from each other in both the length and the nucleotide composition of the spacers. The third variant, classified as DEL short repeat, differs from the short repeat owing to a large deletion in the spacer region. It appears to be the most recent repeat type, since it was identified in only one genotype. The organization of the 5S rDNA repeat unit variants was investigated by amplifying the genomic DNA with primers designed on the sequence of the long and short spacers. The PCR-amplified fragments showed that the long repeat is associated with the other two repeats, indicating that in V. vinifera different repeat units coexist within the same tandem array. FISH analysis demonstrated that 5S rRNA genes are localized at a single locus. The variability of 5S rDNA repeats is discussed in relation to the putative allopolyploid origin of V. vinifera.     

Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species

Nicotiana tabacum (tobacco) is an allotetraploid derived from ancestors of the modern diploids, N. sylvestris and N. tomentosiformis. We identified and characterized two distinct families of 5S ribosomal DNA (rDNA) in N. tabacum; one family had an average 431 bp unit length and the other a 646 bp unit length. In the diploid species, N. sylvestris and N. tomentosiformis, the 5S rDNA unit lengths are 431 bp and 644 bp respectively. The non-coding spacer sequence of the short unit in tobacco had high sequence homology to the spacer of N. sylvestris5S rDNA, while the longer spacer of tobacco had high homology with the 5S spacer of N. tomentosiformis. This suggests that the two 5S families in tobacco have their origin in the diploid ancestors. The longer spacer sequence had a GC rich sub-region (called the T-genome sub-region) that was absent in the short spacer. Pulsed field gel analysis and fluorescent in situ hybridization to tobacco metaphase chromosomes showed that the two families of 5S rDNA units are spatially separate at two chromosomal loci, on chromosomes S8 (short family) and T8 (long family). The repeat copy number at each chromosomal locus showed heterogeneity between different tobacco cultivars, with a tendency for a decrease in the copy number of one family to be compensated by an increase in the copy number of the second family. Sequence analysis reveals there is as much diversity in 5S family units within the diploid species as there is within the T and S-genome 5S family units respectively, suggesting 5S diversification within each family had occurred before tobacco speciation. There is no evidence of interlocus homogenization of the two 5S families in tobacco. This is therefore substantially different to 18-26S rDNA where interlocus gene conversion has substantially influenced most sequences of S and T genome origin; possible reasons are discussed.     

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.     

rRNA genes from the lower chordate Herdmania momus: structural similarity with higher eukaryotes.

Ascidians, primitive chordates that have retained features of the likely progenitors to all vertebrates, are a useful model to study the evolutionary relationship of chordates to other animals. We have selected the well characterized ribosomal RNA (rRNA) genes to investigate this relationship, and we describe here the cloning and characterization of an entire ribosomal DNA (rDNA) tandem repeat unit from a lower chordate, the ascidian Herdmania momus. rDNA copy number and considerable sequence differences were observed between two H. momus populations. Comparison of rDNA primary sequence and rRNA secondary structures from H. momus with those from other well characterized organisms, demonstrated that the ascidians are more closely related to other chordates than invertebrates. The rDNA tandem repeat makes up a larger percentage (7%) of the genome of this animal than in other higher eukaryotes. The total length of the spacer and transcribed region in H. momus rDNA is small compared to most higher eukaryotes, being less than 8 kb, and the intergenic spacer region consists of smaller internal repeats. Comparative analysis of rDNA sequences has allowed the construction of secondary structures for the 18S, 5.8S and 26S rRNAs.     

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.     

The 10 kb Drosophila virilis 28S rDNA intervening sequence is flanked by a direct repeat of 14 base pairs of coding sequence

Most repeat units of rDNA in Drosophila virilis are interrupted in the 28S rRNA coding region by an intervening sequence about 10 kb in length; uninterrupted repeats have a length of about 11 kb. We have sequenced the coding/intervening sequence junctions and flanking regions in two independent clones of interrupted rDNA, and the corresponding 28S rRNA coding region in a clone of uninterrupted rDNA. The intervening sequence is terminated at both ends by a direct repeat of a fourteen nucleotide sequence that is present once in the corresponding region of an intact gene. This is a phenomenon associated with transposable elements in other eukaryotes and in prokaryotes, and the Drosophila rDNA intervening sequence is discussed in this context. We have compared more than 200 nucleotides of the D. virilis 28S rRNA gene with sequences of homologous regions of rDNA in Tetrahymena pigmentosa (Wild and Sommer, 1980) and Xenopus laevis (Gourse and Gerbi, 1980): There is 93% sequence homology among the diverse species, so that the rDNA region in question (about two-thirds of the way into the 28S rRNA coding sequence) has been very highly conserved in eukaryote evolution. The intervening sequence in T. pigmentosa is at a site 79 nucleotides upstream from the insertion site of the Drosophila intervening sequence.     

Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis

In a search for specific molecular markers for population analysis of Puccinia striiformis f. sp. tritici, the ribosomal DNA (rDNA) intergenic spacer (IGS) 1 region (rDNA-IGS1, between the 28S and the 5S rDNA genes) was amplified, cloned, and sequenced. It was found to exhibit multiple bands and length polymorphism. Surprisingly, single isolates were found to possess between three to five different IGS1 haplotypes. Bands were cloned and sequenced, and two highly variable regions (α and β) were found between conserved regions, with repeat units interspersed in both types of regions. There were 14 different repeat units, and these were sometimes grouped further into four combinations of repeat units, with a few individual nucleotides (A or C) inserted between the repeats. Among three geographically dispersed isolates, the variable region α was divided into eight types, and the variable region β was divided into two types based on repeat units. Most of the 14 repeat units were shared by the variable and the conserved regions. Among the three isolates, there were a total of 12 IGS1 haplotypes, but some of these were shared between isolates such that there were only eight unique haplotypes. The occurrence of multiple haplotypes within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of this pathogen.     

Heterogeneity of pumpkin ribosomal DNA

The ribosomal DNA (rDNA) of Cucurbita pepo L. has been found to consist of tandemly arrayed repeat units, most of which are 10 kilobases in length. Thirty-six repeat units, cloned into the HindIII site of pACYC 177, fall into seven classes which differ from each other in length and/or nucleotide sequence. Most of the heterogeneity occurs in noncoding portions of the repeat unit although there is some nucleotide sequence variation in the coding portion as well. Heterogeneity of base modification was observed in genomic rDNA of which two examples are: (a) all of the repeat units have three BamHI sites, one of which is unavailable for restriction in about half of the units and (b) all of the CCGG sites except one are methylated at the internal cytidine in many of the units; a second site is unmethylated in some of the units and in a very few units a third site remains unmethylated.     

Low genetic variation in Swedish populations of the rare speciesVicia pisiformis (Fabaceae) revealed with rflp (rDNA) and RAPD

Nine Swedish populations, 1–5 individuals/population, and one cultivated individual of the rare speciesVicia pisiformis were investigated for genetic variation. In hybridizations with two rDNA probes using 8 restriction enzymes, only two individuals belonging to one population were polymorphic. A map of the rDNA gene cluster was constructed for four of the restriction enzymes used. Two of the polymorphic sites were mapped and were found to be located outside regions coding for rRNA, presumably caused by single point mutations or small deletions. The repeat length of the rDNA region was c. 10,000 bp, which corresponds well with the size found for other species belonging toFabaceae. No length polymorphism was found in the intergenic spacer, contrary to the situation found for most other plant species investigated for rDNA variation. The haplotype diversity for the species (Hsp Shannon) was very low (0.055). Within-population values (Hpop) was 0 for all populations except the variable one, which had 0.301. PCR amplification with 6 random primers also revealed very low levels of genetic diversity. A polymorphism was observed in a limited number of individuals for four populations. Hsp was 0.065 and was 0.050. The average D value (Wetton) for the PCR haplotypes was 0.99.     

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.     

Cloning and characterization of ribosomal RNA genes from wheat and barley.

Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.