Unique structure in the intergenic and 5' external transcribed spacer of the ribosomal RNA gene from the pea aphid Acyrthosiphon pisum.

We analyzed the DNA sequence of the 5' external transcribed spacer (ETS) and part of the intergenic transcribed spacer (IGS) of the aphid ribosomal RNA gene (rDNA). The 5' ETS of aphid rDNA consists of 843 nucleotides with a G/C content of 69 mol/100 mol, far higher than that of any other known 5' ETS for insect rDNA. The IGS of aphid rDNA contained a characteristic array of repeated sequences of 247 nucleotides. The repeated sequences were identical. It was shown that the number of the repeating sequence is heterogeneous.     

Preribosomal RNA processing in Xenopus oocytes does not include cleavage within the external transcribed spacer as an early step.

Recently it has been reported that U3 snRNA is necessary for: (a) internal cleavage at +651/+657 within the external transcribed spacer (ETS) of mouse precursor ribosomal RNA (pre-rRNA); and (b) cleavage at the 5' end of 5.8S rRNA in Xenopus oocytes. To study if U3 snRNA plays a role at more than one processing site in the same system, we have investigated whether internal cleavage sites exist within the ETS of Xenopus oocyte pre-rRNA. The ETS of Xenopus pre-rRNA contains the consensus sequence for the mammalian early processing site (+651/+657 in mouse pre-rRNA), but freshly prepared RNA from Xenopus oocytes has no cuts in this region. The only putative cleavage sites we found in the ETS of Xenopus oocyte pre-rRNA are a cluster further downstream of the mouse early processing site consensus sequence. This cluster is not homologous to the mouse +651/+657 sites because unlike the latter it is (a) not abolished by disruption of U3 snRNA, (b) not cleaved during early steps of pre-rRNA processing, and (c) lacks sequence similarity to the +651/+657 consensus. Therefore, pre-rRNA of Xenopus oocytes does not cleave within the ETS as an early step in rRNA processing. We conclude that cleavage within the ETS is not an obligatory early step needed for the rest of rRNA maturation.     

Human 18 S ribosomal RNA sequence inferred from DNA sequence. Variations in 18 S sequences and secondary modification patterns between vertebrates.

We have determined the DNA sequences encoding 18 S ribosomal RNA in man and in the frog, Xenopus borealis. We have also corrected the Xenopus laevis 18 S sequence: an A residue follows G-684 in the sequence. These and other available data provide a number of representative examples of variation in primary structure and secondary modification of 18 S ribosomal RNA between different groups of vertebrates. First, Xenopus laevis and Xenopus borealis 18 S ribosomal genes differ from each other by only two base substitutions, and we have found no evidence of intraspecies heterogeneity within the 18 S ribosomal DNA of Xenopus (in contrast to the Xenopus transcribed spacers). Second, the human 18 S sequence differs from that of Xenopus by approx. 6.5%. About 4% of the differences are single base changes; the remainder comprise insertions in the human sequence and other changes affecting several nucleotides. Most of these more extensive changes are clustered in a relatively short region between nucleotides 190 and 280 in the human sequence. Third, the human 18 S sequence differs from non-primate mammalian sequences by only about 1%. Fourth, nearly all of the 47 methyl groups in mammalian 18 S ribosomal RNA can be located in the sequence. The methyl group distribution corresponds closely to that in Xenopus, but there are several extra methyl groups in mammalian 18 S ribosomal RNA. Finally, minor revisions are made to the estimated numbers of pseudouridines in human and Xenopus 18 S ribosomal RNA.     

Nucleotide sequence study of mouse 5.8S ribosomal RNA.

The primary structure of 5.8S mouse ribosomal RNA has been studied and compared to the structures previously established for other animal species. The results obtained show that mouse 5.8S ribosomal RNA yields pancreatic oligonucleotides with the same nucleotide sequence as the homologous oligonucleotides from rat cells. Furthermore T1 oligonucleotides of 5.8S ribosomal RNA from rat, mouse and human cells behave identically on fingerprinting fractionation and have the same composition as judged by pancreatic digestion. These results strongly suggest that the primary structures of 5.8S ribosomal RNA from rat, mouse and human cells are identical. This identity of structure is also found when the presence of several modified bases (psi and methylated bases) is considered. The findings emphasize the remarkable evolutionary stability of ribosomal gene structure. Comparison of the terminal regional of 5.8S RNA with those of 18S RNA reveals differences which imply a more complex mechanism underlying the maturation of 45S precursor RNA than the finding of identical structure would have suggested.     

Nonuniform concerted evolution and chloroplast capture: heterogeneity of observed introgression patterns in three molecular data partition phylogenies of Asian Mitella (saxifragaceae)

Interspecific hybridization is one of the major factors leading to phylogenetic incongruence among loci, but the knowledge is still limited about the potential of each locus to introgress between species. By directly sequencing three DNA regions: chloroplast DNAs (matK gene and trnL-F noncoding region), the nuclear ribosomal external transcribed spacer (ETS) region, and internal transcribed spacer (ITS) regions, we construct three phylogenetic trees of Asian species of Mitella (Saxifragaceae), a genus of perennials in which natural hybrids are commonly observed. Within this genus, there is a significant topological conflict between chloroplast and nuclear phylogenies and also between the ETS and the ITS, which can be attributed to frequent hybridization within the lineage. Chloroplast DNAs show the most extensive introgression pattern, ITS regions show a moderate pattern, and the ETS region shows no evidence of introgression. Nonuniform concerted evolution best explains the difference in the introgression patterns between the ETS region and ITS regions, as the sequence heterogeneity of the ITS region within an individual genome is estimated to be twice that of an ETS in this lineage. Significant gene conversion patterns between two hybridizing taxa were observed in contiguous arrays of cloned ETS-ITS sequences, further confirming that only ITS regions have introgressed bidirectionally. The relatively slow concerted evolution in the ITS regions probably allows the coexistence of multiple alleles within a genome, whereas the strong concerted evolution in the ETS region rapidly eliminates heterogeneous alleles derived from other species, resulting in species delimitations highly concordant with those based on morphology. This finding indicates that the use of multiple molecular tools has the potential to reveal detailed organismal evolution processes involving interspecific hybridization, as an individual locus varies greatly in its potential to introgress between species.     

Human 28S ribosomal RNA sequence heterogeneity.

DNA sequencing of several cloned human 28S ribosomal RNA gene fragments has revealed sequence heterogeneity (1) but it was not clear whether these are inactive pseudogenes or are active genes that are transcribed and represented in ribosomes. S1 nuclease analysis allowed us to examine the population of ribosomal RNA molecules of a cell, and we found that 28S rRNA is a heterogeneous assortment of molecules in both mono- and polysomal preparations. Sequence variation, although largely concentrated in variable regions of the molecule, apparently also occurs in the conserved regions.     

Structure and molecular evolution of the ribosomal DNA external transcribed spacer in the cockroach genus Blattella

The ribosomal DNA (rDNA) cluster of insects contains several hundred repeating structural-functional units and, therefore, is a typical example of a multigene family. Eukaryotic ribosomal RNA (rRNA) genes (18S, 5.8S, and 28S like) are arranged in tandemly repeated clusters in the nucleolus organizers, separated by several spacers, namely the nontranscribed spacer, the external transcribed spacer (ETS), and the internal transcribed spacers. The nucleotide sequences of the ETS of the three closely related Blattella cockroach species, Blattella germanica (Linnaeus, 1767), Blattella asahinai (Mizukubo, 1981), and Blattella lituricollis (Walker, 1868), were determined and compared. The three species had relatively similar ETS lengths, and sequence differences among them could be explained by two types of rearrangements, namely deletions of subrepeats and nucleotide substitutions. Minor ETS variants in B. germanica differed from the major variant in the same way that the major ETS variants of the three Blattella species differed from each other. Concerted evolution and the birth-and-death models, which are often invoked to explain the diversity and evolution of the multigene families of rDNA clusters, are discussed in the light of our data. A new model is proposed to explain the evolutionary reorganization of the ETS region: evolution of rDNA by "magnification-and-fixation" is characterized by magnification of minor subrepeats, which become adaptive in a new rapidly changed environment, and subsequent fixation of this variant type as a major component of the multigene family of a new species.     

A U3 small nuclear ribonucleoprotein-requiring processing event in the 5'' external transcribed spacer of Xenopus precursor rRNA.

A processing site has been identified within the 5' external transcribed spacer (ETS) of Xenopus laevis and X. borealis pre-RNAs, and this in vivo processing can be reproduced in vitro. It involves a stable and specific association of the pre-rRNA with factors in the cell extract, including at least four RNA-contacting polypeptides, yielding a distinct complex that sediments at 20S. Processing also requires the U3 small nuclear RNA. This processing, at residue +105 of the 713-nucleotide X. laevis 5' ETS, is highly reminiscent of the initial processing cleavage of mouse pre-rRNA within its 3.5-kb 5' ETS, previously thought to be mammal specific. The frog and mouse processing signals share a short essential sequence motif, and mouse factors can faithfully process the frog pre-rRNA. This conservation suggests that this 5' ETS processing site serves an evolutionarily selective function.     

Evolutionary profiling of the U49 snoRNA gene

Small nucleolar RNAs (snoRNAs) are involved in precursor ribosomal RNA (pre-rRNA) processing and rRNA base modifications (2'-O-ribose methylation and pseudouridylation). Their genomic organization show great flexibility: some are individually or polycistronically transcribed, while others are encoded within introns of other genes. Here, we present an evolutionary analysis of the U49 gene in seven species. In all species analyzed, U49 contains the typical hallmarks of C and D box motifs, and a conserved 12-15 nt sequence complementary to rRNA that define them as homologs. In mouse, human, and Drosophila U49 is found encoded within introns of different genes, and in plants it is transcribed polycistronically from four different locations. In addition, U49 has two copies in two different introns of the RpL14 gene in Drosophila. The results indicate a substantial degree of duplication and translocation of the U49 gene in evolution. In light of its variable organization we discuss which of the two proposed mechanisms of rearrangement has acted upon the U49 snoRNA gene: chromosomal duplication or transposition through an RNA intermediate.     

Structure analysis at the ends of the intervening DNA sequences in the chloroplast 23S ribosomal genes of C. reinhardii

All of the chloroplast 23S ribosomal genes of C. reinhardii are interrupted by a 0.87 kb sequence (Rochaix and Malnoë, 1978). We have sequenced the DNA across the two ends of this intervening element. In parallel, we have examined the nucleotide sequences in the corresponding part of the 23S ribosomal RNA. This allowed us to locate precisely the boundaries between the coding (that is, transcribed into mature 23S rRNA) and the noncoding DNA. The results show that the intervening sequence is flanked by two identical sets of 3 bp (5′-CGT) oriented as direct repeats. In addition, a sequence of 5 bp (5′-CGTGA) lies exactly next to one end and is found very close (16 bp) to the other end, in the coding part of the gene. These two sets are also oriented as direct repeats. Finally, sequences near one end of the intervening element are found with a few alterations near the other end, but in an inverted orientation. Possible interpretations of these results are discussed.     

Secondary structure maps of ribosomal RNA. II. Processing of mouse L-cell ribosomal RNA and variations in the processing pathway

Secondary structure mapping in the electron microscope was applied to ribosomal RNA and precusor ribosomal RNA molecules isolated from nucleoli and the cytoplasm of mouse L-cells. Highly reproducible loop patterns were observed in these molecules. The polarity of L-cell rRNA was determined by partial digestion with 3′-exonuclease. The 28 S region is located at the 5′-end of the 45 S rRNA precursor. Together with earlier experiments on labeling kinetics, these observations established a processing pathway for L-cell rRNA. The 45 S rRNA precursor is cleaved at the 3′-end of the 18 S RNA sequence to produce a 41 S molecule and a spacer-containing fragment (24 S RNA). The 41 S rRNA is cleaved forming mature 18 S rRNA and a 36 S molecule. The 36 S molecule is processed through a 32 S intermediate to the mature 28 S rRNA. This pathway is similar to that found in HeLa cells, except that in L-cells a 36 S molecule occurs in the major pathway and no 20 S precusor to 18 S RNA is found. The processing pathway and its intermediates in L-cells are analogous to those in Xenopus laevis, except for a considerable size difference in all rRNAs except 18 S rRNA.The arrangement of gene and transcribed spacer regions and of secondary structure loops, as well as the shape of the major loops were compared in L-cells, HeLa cell and Xenopus rRNA. The over-all arrangement of regions and loop patterns is very similar in the RNA from these three organisms. The shapes of loops in mature 28 S RNA are also highly conserved in evolution, but the shapes of loops in the transcribed spacer regions vary greatly. These observations suggest that the sequence complementarity that gives rise to this highly conserved secondary structure pattern may have some functional importance.