Comparative analysis of intra-individual and inter-species DNA sequence variation in salmonid ribosomal DNA cistrons

This study examines sequence divergence in three spacer regions of the ribosomal DNA (rDNA) cistron, to test the hypothesis of unequal mutation rates. Portions of two transcribed spacers (ITS-1 and 5' ETS) and the non-transcribed spacer (NTS) or intergenic spacer (IGS) formed the basis of comparative analyses. Sequence divergence was measured both within an individual lake trout (Salvelinus namaycush) and among several related salmonid species (lake trout; brook trout, Salvelinus fontinalis; Arctic char, Salvelinus alpinus; Atlantic salmon, Salmo salar; and brown trout, Salmo trutta). Despite major differences in the length of the rDNA cistron within individual lake trout, minimal sequence difference was detected among cistrons. Interspecies comparisons found that molecular variation in the rDNA spacers did not conform to the predicted pattern of evolution (ITS spacers相似文献   

Phylogenetic utility of the external transcribed spacer (ETS) of 18S-26S rDNA: congruence of ETS and ITS trees of Calycadenia (Compositae)

The 3' region of the external transcribed spacer (ETS) of 18S-26S nuclear ribosomal DNA was sequenced in 19 representatives of Calycadenia/Osmadenia and two outgroup species (Compositae) to assess its utility for phylogeny reconstruction compared to rDNA internal transcribed spacer (ITS) data. Universal primers based on plant, fungal, and animal sequences were designed to amplify the intergenic spacer (IGS) and an angiosperm primer was constructed to sequence the 3' end of the ETS in members of tribe Heliantheae. Based on these sequences, an internal ETS primer useful across Heliantheae sensu lato was designed to amplify and sequence directly the 3' ETS region in the study taxa, which were the subjects of an earlier phylogenetic investigation based on ITS sequences. Size variation in the amplified ETS region varied across taxa of Heliantheae sensu lato from approximately 350 to 700 bp, in part attributable to an approximately 200-bp tandem duplication in a common ancestor of Calycadenia/Osmadenia. Phylogenetic analysis of the 200-bp subrepeats and examination of apomorphic changes in the duplicated region demonstrate that the subrepeats in Calycadenia/Osmadenia have evolved divergently. Phylogenetic analyses of the entire amplified ETS region yielded a highly resolved strict consensus tree that is nearly identical in topology to the ITS tree, with strong bootstrap and decay support on most branches. Parsimony analyses of combined ETS and ITS data yielded a strict consensus tree that is better resolved and generally better supported than trees based on either data set analyzed separately. We calculated an approximately 1.3- to 2.4-fold higher rate of sequence evolution by nucleotide substitution in the ETS region studied than in ITS-1 + ITS-2. A similar disparity in the proportion of variable (1.3 ETS:1 ITS) and potentially informative (1.5 ETS:1 ITS) sites was observed for the ingroup. Levels of homoplasy are similar in the ETS and ITS data. We conclude that the ETS holds great promise for augmenting ITS data for phylogenetic studies of young lineages.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Structure,molecular evolution,and phylogenetic utility of the 5(') region of the external transcribed spacer of 18S-26S rDNA in Lessingia (Compositae,Astereae)

The 18S-26S nuclear rDNA external transcribed spacer (ETS) has recently gained attention as a region that is valuable in phylogenetic analyses of angiosperms primarily because it can supplement nucleotide variation from the widely used and generally shorter internal transcribed spacers (ITS-1 and ITS-2) and thereby improve phylogenetic resolution and clade support in rDNA trees. Subrepeated ETS sequences (often occurring in the 5(') region) can, however, create a challenge for systematists interested in using ETS sequence data for phylogeny reconstruction. We sequenced the 5(')ETS for members of Lessingia (Compositae, Astereae) and close relatives (26 taxa total) to characterize the subrepeat variation across a group of closely related plant lineages and to gain improved understanding of the structure, molecular evolution, and phylogenetic utility of the region. The 5(')ETS region of Lessingia and relatives varied in length from approximately 245 to 1009 bp due to the presence of a variable number of subrepeats (one to eight). We assessed homology of the subrepeats using phylogenetic analysis and concluded that only two of the subrepeats and a portion of a third ( approximately 282 bp in total) were orthologous across Lessingia and could be aligned with confidence and included in further analyses. When the partial 5(')ETS data were combined with 3(')ETS and ITS data in phylogenetic analyses, no additional resolution of relationships among taxa was obtained beyond that found from analysis of 3(')ETS + ITS sequences. Inferred patterns of concerted evolution indicate that homogenization is occurring at a faster rate in the 3(')ETS and ITS regions than in the 5(')ETS region. Additionally, homogenization appears to be acting within but not among subrepeats of the same rDNA array. We conclude that challenges in assessing subrepeat orthology across taxa greatly limit the utility of the 5(')ETS region for phylogenetic analyses among species of Lessingia.     

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.     

Phylogenetic Utility of the External Transcribed Spacer (ETS) of 18S–26S rDNA: Congruence of ETS and ITS Trees ofCalycadenia(Compositae)

The 3′ region of the external transcribed spacer (ETS) of 18S–26S nuclear ribosomal DNA was sequenced in 19 representatives ofCalycadenia/Osmadeniaand two outgroup species (Compositae) to assess its utility for phylogeny reconstruction compared to rDNA internal transcribed spacer (ITS) data. Universal primers based on plant, fungal, and animal sequences were designed to amplify the intergenic spacer (IGS) and an angiosperm primer was constructed to sequence the 3′ end of the ETS in members of tribe Heliantheae. Based on these sequences, an internal ETS primer useful across Heliantheaesensu latowas designed to amplify and sequence directly the 3′ ETS region in the study taxa, which were the subjects of an earlier phylogenetic investigation based on ITS sequences. Size variation in the amplified ETS region varied across taxa of Heliantheaesensu latofrom approximately 350 to 700 bp, in part attributable to an approximately 200-bp tandem duplication in a common ancestor ofCalycadenia/Osmadenia.Phylogenetic analysis of the 200-bp subrepeats and examination of apomorphic changes in the duplicated region demonstrate that the subrepeats inCalycadenia/Osmadeniahave evolved divergently. Phylogenetic analyses of the entire amplified ETS region yielded a highly resolved strict consensus tree that is nearly identical in topology to the ITS tree, with strong bootstrap and decay support on most branches. Parsimony analyses of combined ETS and ITS data yielded a strict consensus tree that is better resolved and generally better supported than trees based on either data set analyzed separately. We calculated an approximately 1.3- to 2.4-fold higher rate of sequence evolution by nucleotide substitution in the ETS region studied than in ITS-1 + ITS-2. A similar disparity in the proportion of variable (1.3 ETS:1 ITS) and potentially informative (1.5 ETS:1 ITS) sites was observed for the ingroup. Levels of homoplasy are similar in the ETS and ITS data. We conclude that the ETS holds great promise for augmenting ITS data for phylogenetic studies of young lineages.     

Efficient 5' ETS walking from conserved 18S rDNA sequences of the dinoflagellates <Emphasis Type="Italic">Alexandrium</Emphasis> and <Emphasis Type="Italic">Akashiwo sanguinea</Emphasis> (Dinophyceae)

An external transcribed spacer (ETS) walking PCR technique was developed for the isolation of unknown sequences adjacent to the 18S rDNA. This strategy relied on four "walking primers", which were designed to bind unknown sequences upstream from the 18S rDNA, and a specially programmed series of thermocycles. This method was successful in the isolation of the 5' ETS regions from harmful dinoflagellates, including Alexandrium affine, A. catenella, A. minutum, A. tamarense, and Akashiwo sanguinea. Mono-directional sequencing reactions revealed the PCR products to be 392–962 nucleotides in length, and the 5' ETS in these products were longer than 362 bp. These are the first such sequences available for A. sanguinea and the Alexandrium. In comparisons of the ETS sequences, genetic distance was considerably high within the Alexandrium. Furthermore, the sequences were significantly variable among the different strains of identical species: genetic distance was recorded at 0.0420 for A. tamarense strains and as less than 0.7841 within strains of A. sanguinea. The 5'-start nucleotide of 18S rDNA was variable between the two genera: the five species of Alexandrium contained a T base, and A. sanguinea contained an A base. These results demonstrate the effectiveness of the ETS walking PCR method. This method will be valuable in directional ETS walking from known regions to unknown regions, particularly concerning the boundary sequences of rRNA genes.