Nucleotide sequence encoding the 5'' end of Xenopus laevis 18S rRNA.

We have sequenced a region of cloned Xenopus laevis ribosomal DNA encompassing the last 24 nucleotides of the external transcribed spacer and the first 275 nucleotides of the 18S gene. The start of the 18S gene was identified by correlating the results obtained from RNA hybridization and fingerprinting with the DNA sequence. This 5' region of 18S rRNA contains five 2'-O-methyl groups and at least six pseudouridine residues. Several of these modified nucleotides are clustered into a relatively short region from nucleotides 99-124. Nucleotides 227-250 constitute a distinctive sequence of 24 consecutive G and C residues. Comparison with the first 160 nucleotides of a yeast 18S gene (25) reveals three blocks of high sequence homology separated by two short tracts where homology is low or absent. The external transcribed spacer sequences diverge widely from within a few nucleotides of the start of the 18S gene.     

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.     

Xenopus borealis and Xenopus laevis 28S ribosomal DNA and the complete 40S ribosomal precursor RNA coding units of both species.

We have determined the nucleotide sequence of Xenopus borealis 28S ribosomal DNA (rDNA) and have revised the sequence of Xenopus laevis 28S rDNA (Ware et al., Nucl. Acids Res. 11, 7795-7817 (1983)). In the regions encoding the conserved structural core of 28S rRNA (2490 nucleotides) there are only four differences between the two species, each difference being a base substitution. In the variable regions, also called eukaryotic expansion segments (ca. 1630 nucleotides) there are some 61 differences, due to substitutions, mini-insertions and mini-deletions. Thus, evolutionary divergence in the variable regions has been at least 20-fold more rapid than in the conserved core. A search for intraspecies sequence variation has revealed minimal heterogeneity in X. laevis and none in X. borealis. At three out of four sites where heterogeneity was found in X. laevis (all in variable regions) the minority variant corresponded to the standard form in X. borealis. Intraspecies heterogeneity and interspecies divergence in the 28S variable regions are much less extensive than in the transcribed spacers. The 28S sequences are from the same clones that were used previously for sequencing the 18S genes and transcribed spacers. The complete sequences of the 40S precursor regions of the two reference clones are given.     

Internal transcribed spacer 1 of the yeast precursor ribosomal RNA. Higher order structure and common structural motifs

The higher order structure of the first internal transcribed spacer between the 18S and the 5.8S rRNA sequences in the Saccharomyces cerevisiae precursor ribosomal RNA has been investigated. Sites of potential base pairing in the RNA region have been determined by using a combination of enzymatic and chemical structure sensitive probes. Data generated have been used to evaluate secondary structure models predicted by minimum free energy calculations. Several alternative suboptimal structures were also evaluated. The derived model contains several stable hairpins. Theoretical secondary structural models for the corresponding RNA region from S. carlsbergensis, S. pombe, N. crassa, X. laevis, and mung bean have also been derived from identical calculations and assumptions. Certain structural motifs appear to be conserved despite extensive divergence in the base sequence. The yeast model should be a useful prototype for investigation of structure and function of precursor ribosomal RNA molecules.     

Characterization of two xenopus somatic 5S DNAs and one minor oocyte-specific 5S DNA

The somatic 5S DNA from X. borealis (Xbs 5S DNA) and X. laevis (Xis 5S DNA) and a minor oocyte-specific 5S DNA from X. laevis (Xit 5S DNA) have been purified, and individual repeating units have been cloned and sequenced. The two somatic 5S DNAs differ from the major oocyte 5S DNAs in having GC-rich spacers, homogeneous repeat lengths and no "pseudogenes." The somatic 5S DNAs from the two species have similar spacer sequences with differences due to single base changes and insertions/deletions. The spacer of the minor oocyte-specific 5S DNA (Xit) has the AT-rich sequence characteristic of the major oocyte 5S DNAs from X. laevis and X. borealis, and contains one duplication that has diverged approximately 40%. Like the somatic 5S DNAs, Xit 5S DNA has a homogeneous length repeat and a unique nucleotide sequence in its spacer. The presence of variable-length spacer regions in a multigene family correlates with variables numbers of a simple sequence in the spacer regions.     

DNaseI-hypersensitive sites at promoter-like sequences in the spacer of Xenopus laevis and Xenopus borealis ribosomal DNA.

We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X. laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAaseI. Unlike DNAaseI, Endo R. MspI digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this "proximal domain" is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function.     

Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.