Some characteristics of processing sites in ribosomal precursor RNA of yeast.

The DNA sequences of the intergenic region between the 17S and 5.8S rRNA genes of the ribosomal RNA operon in yeast has been determined. In this region the 37S ribosomal precursor RNA is specifically cleaved at a number of sites in the course of the maturation process. The exact position of these processing sites has been established by sequence analysis of the terminal fragments of the respective RNA species. There appears to be no significant complementarity between the sequences surrounding the two termini of the 18S secondary precursor RNA nor between those surrounding the two termini of 17S mature rRNA. This finding implies that the processing of yeast 37S ribosomal precursor RNA is not directed by a double-strand specific ribonuclease previously shown to be involved in the processing of E. coli ribosomal precursor RNA [see Refs 1,2]. The processing sites of yeast ribosomal precursor RNA described in the present paper are all flanked at one side by a very [A+T]-rich sequence. In addition, sequence repeats are found around the processing sites in this precursor RNA. Finally, sequence homologies are present at the 3'-termini [6 nucleotides] and the 5'-termini [13 nucleotides] of a number of mature rRNA products and intermediate ribosomal RNA precursors. These structural features are discussed in terms of possible recognition sites for the processing enzymes.     

Alternative pathways in the processing of ribosomal RNA precursor in Drosophila melanogaster

The size of RNA molecules that are intermediates in the processing of ribosomal RNA in Drosophila melanogaster has been determined by gel electrophoresis under fully denaturing conditions. These molecules have been characterized by transfer from agarose gels to diazobenzyloxymethyl-paper and hybridization with restriction fragments derived from cloned ribosomal DNA. Five cleavage sites leading to the production of 18 S and 28 S RNA have been mapped in the precursor. The first cleavage in the precursor molecule occurs at one of two different sites. Therefore, we propose two alternative pathways for the processing of D. melanogaster ribosomal RNA. A precursor molecule to 2 S and 5.8 S ribosomal RNA has been identified in nuclear RNA.     

Strict conservation of the ITS regions of the ribosomal RNA genes in Atlantic cod (Gadus morhua L.).

The nucleotide sequence of the internal transcribed region (ITS) of ribosomal RNA genes from Atlantic cod (Gadus morhua L.) was determined. The complete ITS region spanned approximately 1113 base pairs. The ITS1 region comprised 532 base pairs, the 5.8S region 159, and the ITS2 region contained 422 base pairs. Sequence data were obtained from a total of 12 samples, one pool from six cod and 11 individuals. The sequencing was carried out in two separate experimental periods employing slightly different methodology. The samples were from two different cod stocks, Norwegian costal cod and North East Arctic cod. The sequence analysis showed that in the 12 samples, the ITS region, including the 5.8S RNA, was identical. The ITS region is thus totally conserved in these two cod stocks. The extreme conservation of the ITS regions in the cod rDNA could reflect the small genome size of cod and/or indicate a specific critical role in the processing of the ribosomal units in cold-adapted species.     

Study of a cloned ribosomal operon region coding for 5.8S rRNA in the loach Misgurnus fossilis L

A fragment of the loach (Misgurnus fossilis L.) ribosomal operon containing 5.8S rDNA and adjacent regions of the internal transcribed spacer (ITS-1, and ITS-2) was sequenced. The 5'-terminal sequencing in 5.8S rDNA was corrected by analysing the primary structure of the loach 5.8S rRNA. This RNA was shown to be presented by three types of molecules; one of these was shorter by 4 nucleotides at the 5'-end because of the processing site being shifted in the rRNA precursor. The two other types differed in the 5'-terminal nucleotide (UMP or AMP). In the cloned fragment under study, the sequence of 5.8S rDNA has TMP at the 5'-terminus. The known nucleotide sequences of 5.8S rRNAs were compared in eukaryotes; as a result, conservative regions were revealed at the sites of molecule modification. All the 5.8S rRNAs of the vertebrates studied were found to have coincidences in the localization of nucleotide substitutions and other mutations (inversions and deletions). The authors propose a model for the secondary structure of ITS-1 and ITS-2 in the region of 5.8S rRNA processing.     

Ribosomal RNA of the primitive eukaryote Giardia lamblia: large subunit domain I and potential processing signals

The cytoplasmic ribosomal RNA (rRNA) from the intestinal protozoan, Giardia lamblia, is unusually short; the large subunit (LS) and small subunit RNA and the 5.8S RNA are only 70-80% of the length found in typical protozoa, and are even smaller than most of their prokaryotic counterparts. Flanking regulatory DNA and processed rRNA sequences are similarly compact in size. To shed light on the origins and implications of this 'minimal' rRNA, the nucleotide sequence encoding the 5.8S RNA and domain I of LS RNA was determined. Secondary structure analysis revealed that an evolutionarily variable internal hairpin is partially 'deleted' in G. lamblia 5.8S RNA; the 3'-terminal pairing with LS RNA is conserved. Previously characterized eukaryotic 'expansion' regions are extensively shortened within the LS RNA; in one case, a hairpin is precisely 'deleted'. The short sequences flanking the mature 5.8S RNA that are removed by RNA processing (ITS1 and ITS2) are C-rich; our analysis suggests that the sequence GCGCCCC, in a hairpin configuration, may function as the processing signal.     

Topography of 5.8 S rRNA in rat liver ribosomes. Identification of diethyl pyrocarbonate-reactive sites

The topography of 5.8 rRNA in rat liver ribosomes has been examined by comparing diethyl pyrocarbonate-reactive sites in free 5.8 S RNA, the 5.8 S-28 rRNA complex, 60 S subunits, and whole ribosomes. The ribosomal components were treated with diethyl pyrocarbonate under salt and temperature conditions which allow cell-free protein synthesis; the 5.8 S rRNA was extracted, labeled in vitro, chemically cleaved with aniline, and the fragments were analyzed by rapid gel-sequencing techniques. Differences in the cleavage patterns of free and 28 S or ribosome-associated 5.8 S rRNA suggest that conformational changes occur when this molecule is assembled into ribosomes. In whole ribosomes, the reactive sites were largely restricted to the "AU-rich" stem and an increased reactivity at some of the nucleotides suggested that a major change occurs in this region when the RNA interacts with ribosomal proteins. The reactivity was generally much less restricted in 60 S subunits but increased reactivity in some residues was also observed. The results further indicate that in rat ribosomes, the two -G-A-A-C- sequences, putative binding sites for tRNA, are accessible in 60 S subunits but not in whole ribosomes and suggest that part of the molecule may be located in the ribosomal interface. When compared to 5 S rRNA, the free 5.8 S RNA molecule appears to be generally more reactive with diethyl pyrocarbonate and the cleavage patterns suggest that the 5 S RNA molecule is completely restricted or buried in whole ribosomes.     

Ribosomal RNA genes of Trypanosoma brucei: mapping the regions specifying the six small ribosomal RNAs

There are six small ribosomal RNAs in trypanosome ribosomes. sRNA3 and sRNA5 of Trypanosoma brucei brucei have been partially sequenced. Sequence homologies indicate that sRNA3 is 5.8S RNA and sRNA5 is 5S RNA of T. b. brucei. The regions specifying these two, and the remaining four small RNAs, have been identified within clones of rRNA genes and in the genome. Five of the small RNAs, 1, 2, 3, 4 and 6, hybridise exclusively within the major rRNA gene repeat. A map of the regions specifying these small RNAs is presented. sRNA3 (5.8S RNA) hybridises to a region corresponding to the transcribed spacer of other eukaryotes. sRNA1 hybridises to a region between sequences specifying the two large subunit RNA molecules of 2.3 kb and 1.8 kb. Sequences specifying sRNAs 2 and 4 are present near the sequence specifying sRNA1, while sRNA6 appears to be specified 3' to the sequence specifying the 1.8-kb RNA sequence. In addition regions of secondary hybridisation for small RNAs 2, 3, 4 and 6 have also been identified. Though sRNA5 (5S RNA) hybridises within the major rRNA repeat, a separate 5S RNA gene repeat with unit size of 760 bp is also present. It is 10 to 20 times more abundant than the major rRNA gene repeat.     

Identification of the locations of the methyl groups in 18 S ribosomal RNA from Xenopus laevis and man

The 18 S ribosomal RNA from a variety of vertebrate species contains some 40 to 47 methyl groups. The majority of these are 2'-O-ribose substituents; the remaining few are on bases. Several lines of evidence have permitted the identification of the precise locations of the methyl groups in the primary structure of 18 S ribosomal RNA of Xenopus laevis and man. Digestion of RNA with T1 ribonuclease, followed by analysis of the methylated oligonucleotides yielded data on sequences immediately surrounding the methyl groups. Preparative hybridization of X. laevis 18 S ribosomal RNA restriction fragments of ribosomal DNA, followed by fingerprinting analysis on RNA recovered from the hybrids, allowed each methylated oligonucleotide to be mapped to a specific region within 18 S ribosomal RNA. The data on RNA oligonucleotides were correlated with Xenopus ribosomal DNA sequence data in the regions defined by the mapping experiments to identify the precise locations of most of the methyl groups in the X. laevis 18 S RNA sequence. The remaining uncertainties in Xenopus were solved with the aid of data from ribonuclease A fingerprints and, in a few instances, relevant oligonucleotide or sequence data from other laboratories. The locations of most of the methyl groups in human 18 S ribosomal RNA were deduced from the high degree of correspondence between methylated oligonucleotides from human and X. laevis 18 S RNA, together with knowledge of the human 18 S ribosomal DNA sequence. The remaining methylation sites in human 18 S RNA were located with assistance from relevant published comparative data. In the aligned sequences, human and other mammalian 18 S RNA are methylated at all the same positions as in X. laevis, and there are seven additional 2'-O-methylation sites in mammalian 18 S RNA. Further features of the methyl group distribution are briefly reviewed.     

Characterization of cloned rat ribosomal DNA fragments

Summary Two Charon 4A lambda bacteriophage clones were characterized which contain all and part of the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA region were in the same places in mouse and rat DNA but the restriction sites in the 5-spacer regions were different.     

Creation of a data base for sequences of ribosomal nucleic acids and detection of conserved restriction endonucleases sites through computerized processing

As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed.     

Ribosomal RNA genes of Saccharomyces cerevisiae. II. Physical map and nucleotide sequence of the 5 S ribosomal RNA gene and adjacent intergenic regions.

A DNA fragment containing the structural gene for the 5 S ribosomal RNA and intergenic regions before and after the 35 S ribosomal RNA precursor gene of Saccharomyces cerevisiae has been amplified in a bacterial plasmid and physically mapped by restriction endonuclease cleavage and hybridization to purified yeast 5 S ribosomal RNA. The nucleotide sequence of the DNA fragments carrying the 5 S ribosomal RNA gene and adjacent regions has been determined. The sequence unambiguously identifies the 5 S ribosomal RNA gene, determines its polarity within the ribosomal DNA repeating unit, and reveals the structure of its promoter and termination regions. Partial DNA sequence of the regions near the beginning and end of the 35 S ribosomal RNA gene has also been determined as a preliminary step in establishing the structure of promoter and termination regions for the 35 S ribosomal RNA gene.     

Ribosomal RNA genes of Saccharomyces cerevisiae. I. Physical map of the repeating unit and location of the regions coding for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs.

The organization of the ribosomal DNA repeating unit from Saccharomyces cerevisiae has been analyzed. A cloned ribosomal DNA repeating unit has been mapped with the restriction enzymes Xma 1, Kpn 1, HindIII, Xba 1, Bgl I + II, and EcoRI. The locations of the sequences which code for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs have been determined by hybridization of the purified RNA species with restriction endonuclease generated fragments of the repeating unit. The position of the 5.8 S ribosomal DNA sequences within the repeat was also established by sequencing the DNA which codes for 83 nucleotides at the 5' end of 5.8 S ribosomal RNA. The polarity of the 35 S ribosomal RNA precursor has been established by a combination of hybridization analysis and DNA sequence determination and is 5'-18 S, 5.8 S, 25 S-3'.