Two-dimensional RFLP analyses reveal megabase-sized clusters of rRNA gene variants in Arabidopsis thaliana, suggesting local spreading of variants as the mode for gene homogenization during concerted evolution

Eukaryotic genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNA (rRNA genes or rDNA) are virtually identical within a species, yet they evolve rapidly between species, a phenomenon known as concerted evolution. The mechanisms by which sequence homogenization and fixation of new rRNA gene variants occurs within a genome are not clear. In diploid Arabidopsis thaliana , approximately 1500 rRNA genes are tandemly arrayed at two nucleolus organizer regions, one on chromosome 2 ( NOR2 ), the other on chromosome 4 ( NOR4 ). This paper shows that NOR2 and NOR4 are similar in size, each spanning approximately 3.5–4.0 Mbp. Using two-dimensional mapping techniques involving a combination of pulsed-field and conventional gel electrophoresis, the distributions of four distinct rRNA gene variants at NOR2 and NOR4 have been determined. rRNA genes at NOR4 are homogeneous with respect to a Hin dIII site occurring once per gene. In contrast, fewer than 10% of the rRNA genes at NOR2 are Hin dIII-bearing variants. A single intergenic spacer length is found among rRNA genes at NOR2 but three classes of spacer length variants are present at NOR4 . The NOR4 variants are not intermingled with one another; instead, they are highly clustered over distances as large as 1.5 Mbp. These data suggest that in the concerted evolution of rRNA genes, homogenization is a consequence of local spreading of new rRNA gene variants.     

Use of RFLPs larger than 100 kbp to map the position and internal organization of the nucleolus organizer region on chromosome 2 in Arabidopsis thaliana

In Arabidopsis thaliana ribosomal RNA genes (rRNA genes or rDNA) are grouped in two nucleolus organizer regions (NORs) that together comprise approximately 6% of the genome. The map positions of the NORs relative to other genetic markers are unknown. It was found that the restriction endonuclease HindIII cuts once in some but not all rRNA genes to yield strain-specific RFLPs of 100–700 kb that could be visualized by pulsed-field gel electrophoresis and Southern blotting. The HindIII RFLPs of the A. thaliana strains Columbia and Landsberg segregated among recombinant inbred lines derived from a cross between these two strains. Linkage analysis placed the NOR bearing the polymorphic HindIII sites to the top of the upper arm of chromosome 2. The name NOR2 is suggested for this locus. HindIII-bearing rRNA genes are interspersed with clusters of HindIII-less genes throughout NOR2. The observed clustering is most consistent with unequal crossing-over, or nearest-neighbor gene conversion, as the mechanism(s) that spread rRNA gene variants throughout an NOR. No meiotic cross-over events yielding a ‘hybrid’ NOR with multiple RFLPs from both parents were observed among the 47 recombinant inbred lines examined. However, the appearance of novel HindIII profiles in approximately 40% of the recombinant inbred lines demonstrates that fluctuations in the distribution of rRNA gene variants occur frequently and can be readily detected on pulsed-field gels.     

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.     

Distribution of 5S ribosomal RNA genes in somatic and germ cells of the house cricket,Acheta domesticus

In the house cricket,Acheta domesticus, the 110 genes per haploid genome encoding 18S and 28S rRNA are contained within rDNA repeats which are amplified during oogenesis. The 5S rRNA coding sequences of this cricket are found in two sizes of 5S DNA repeating units (measuring 2.1 and 3.0 kb). The 3.0 kb repeats account for more than 90% of the totalAcheta 5S DNA. We have determined the number of cricket 5S rRNA genes by RNA-DNA hybridization analysis: 310 5S DNA repeats/haploid genome clearly approximates the number of 18S and 28S rRNA genes. Because of the relatively low copy number of 5S rRNA genes the possibility of 5S DNA amplification in oocytes ofA. domesticus was also examined. Although amplification of rDNA is readily detectable, amplification of 5S DNA is not observed in oocytes ofA. domesticus. Unlike the genes coding for 18S and 28S rRNA which are localized at a single chromosomal site in the genome ofA. domesticus, the 5S rRNA genes occupy numerous sites distributed along the length of most chromosomes.     

Mapping of the nucleolus organizer region on chromosome 4 inArabidopsis thaliana

InArabidopsis thaliana the ribosomal RNA genes (rRNA genes or rDNA) are clustered in tandemly repeated blocks in two nucleolus organizer regions (NORs). Cytogenetic analysis has shown that the NORs are localized on chromosome 2 (NOR 2) and 4 (NOR 4). Recently the map position of NOR 2 was determined using a RFLP which was larger than 100 kb. In the course of a fingerprint analysis of differentArabidopsis ecotypes we have detected four rDNA polymorphisms between the ecotypes Landsberg (La) and Niederzenz (Nd). Mapping of these polymorphisms using established segregating F₂ populations reveals that all polymorphisms detected are dominant. Three of them map to the locus on the second chromosome that has been shown to harbour the NOR 2. The fourth polymorphism can be unambigously assigned to the upper arm of the fourth chromosome. This is the first polymorphism found which originates in the second rDNA cluster ofArabidopsis thaliana. It enables localization of NOR 4 and thus completes the mapping of rDNA genes in the NORs ofArabidopsis.     

Mapping of the nucleolus organizer region on chromosome 4 inArabidopsis thaliana

InArabidopsis thaliana the ribosomal RNA genes (rRNA genes or rDNA) are clustered in tandemly repeated blocks in two nucleolus organizer regions (NORs). Cytogenetic analysis has shown that the NORs are localized on chromosome 2 (NOR 2) and 4 (NOR 4). Recently the map position of NOR 2 was determined using a RFLP which was larger than 100 kb. In the course of a fingerprint analysis of differentArabidopsis ecotypes we have detected four rDNA polymorphisms between the ecotypes Landsberg (La) and Niederzenz (Nd). Mapping of these polymorphisms using established segregating F₂ populations reveals that all polymorphisms detected are dominant. Three of them map to the locus on the second chromosome that has been shown to harbour the NOR 2. The fourth polymorphism can be unambigously assigned to the upper arm of the fourth chromosome. This is the first polymorphism found which originates in the second rDNA cluster ofArabidopsis thaliana. It enables localization of NOR 4 and thus completes the mapping of rDNA genes in the NORs ofArabidopsis.     

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.     

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.     

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.     

Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.