Effect of light on the nucleotide composition of rRNA of wheat seedlings

Both qualitative and quantitative differences in the minor nucleotide constituents of rRNA from normally grown and from etiolated wheat plants (Triticum aestivum L.) were established. Using different degradation methods and separation techniques the 18S+26S RNA of 8-day-old wheat seedlings grown in the light was found to contain 5-methylcytidine, 3-methylcytidine, 5-methyluridine, 3-methyluridine, 5-carboxymethyluridine, 1-methyladenine, N-methyladenine, 5-hydroxymethylcytidine, O²-methyluridine, O²-methylcytidine, pseudouridine, O²-methylpseudouridine, N²,N²-dimethylguanine, 1-methylguanine, ribothymidine and some unknown minor constituents. On the other hand, there were only a few minor nucleotides in the rRNA of etiolated wheat seedlings. Cycloheximide, a cytoplasmic protein synthesis inhibitor, simulated etiolation in that it reduced the number of minor nucleotides in rRNA, whereas chloramphenicol, a chloroplast protein synthesis inhibitor, had no significant effect on the minor nucleotide content of rRNA. This finding suggests that illumination may cause de novo synthesis of cytoplasmic modifying enzymes leading to the formation of highly modified rRNAs.Abbreviations m⁶A N⁶-methyladenine - m¹A 1-methyladenine - 5hmc 5-hydroxymethylcytidine - Cm O²-methylcytidine - m⁵C 5-methylcytidine - m³C 3-methylcytidine - m¹G 1-methylguanine - m ₂ ² G N², N²-dimethylguanine - pseudouridine - m O²-methylpseudouridine - Um O²-methyluridine - m³U 3-methyluridine - m⁵U 5-methyluridine - cm⁵U 5-carboxymethyluridine - rT ribothymidine - Pur purine - Pyr pyrimidine - RNase ribonuclease - UV ultra violet - p phosphate     

Nucleotide sequence of the 17S–25S spacer region from rice rDNA

Summary The nucleotide sequence of a spacer region between rice 17S and 25S rRNA genes (rDNAs) has been determined. The coding regions for the mature 17S, 5.8S and 25S rRNAs were identified by sequencing terminal regions of these rRNAs. The first internal transcribed spacer (ITS1), between 17S and 5.8S rDNAs, is 194–195 bp long. The second internal transcribed spacer (ITS2), between 5.8S and 25S rDNAs, is 233 bp long. Both spacers are very rich in G+C, 72.7% for ITS1 and 77.3% for ITS2. The 5.8S rDNA is 163–164 bp long and similar in primary and secondary structures to other eukaryotic 5.8S rDNAs. The 5.8S rDNA is capable of interacting with the 5′ terminal region of 25S rDNA.     

Arrangement of the Template 3′ of the A-Site Codon on the Human 80S Ribosome

The arrangement of the template sequence 3′ of the A-site codon on the 80S ribosome was studied using mRNA analogs containing Phe codon UUU at the 5′ end and a photoreactive perfluoroarylazido group linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNA^Phe, which directed the UUU codon to the P site, bringing a modified nucleotide to position +9 or +12 relative to the first nucleotide of the P-site codon. Upon mild UV irradiation of ribosome complexes, the analogs of both types crosslinked to the 18S rRNA and proteins of the 40S subunit. Comparisons were made with the crosslinking patterns of complexes in which an mRNA analog contained a modified nucleotide in position +7 (the crosslinking to 18S rRNA in such complexes has been studied previously). The efficiency of crosslinking to ribosomal components depended on the nature of the modified nucleotide of an mRNA analog and its position on the ribosome. The extent of crosslinking to the 18S rRNA drastically decreased as the modified nucleotide was transferred from position +7 to position +12. The 18S rRNA nucleotides involved in crosslinking were identified. A modified nucleotide in position +9 crosslinked to the invariant dinucleotide A1824/A1825 and variable A1823 in the 3′ minidomain of the 18S rRNA and to S15. The same ribosomal components have earlier been shown to crosslink to modified nucleotides in positions +4 to +7. In addition, all mRNA analogs crosslinked to invariant C1698 in the 3′ minidomain and to conserved region 605–620, which closes helix 18 in the 5′ domain.     

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.     

The primary structure of lupin seed 5.8 S ribosomal RNA

The lack of colinearity between nucleotide sequence of the lupin 5.8 S rDNA gene (Rafalski, A.J., Wiewiórowski, M. and Soll, D. (1983) FEBS Lett. 152, 241-246) and 5.8 S rRNA of other plants (Erdmann, V.A. and Wolters, J. (1986) Nucleic Acids Res. 14, r1-r59.) prompted us to clarify this point by sequencing the native lupin 5.8 S rRNA. The sequence analysis was carried out using enzymatic and chemical methods. Lupin seed 5.8 S rRNA contains 164 nucleotides, including four modified ones: two residues of 2'-O-methylguanosine, one pseudouridine and one 2'-O-methyladenosine. The nucleotide sequence homology with the other plant 5.8 S rRNAs is approx. 88-96%.     

Primary structure of the 5.8S gene of rRNA and internal transcribed spacers of rDNA in diploid wheat Triticum urartu thum. ex. gandil

Nucleotide sequences of 5.8S rRNA gene and rDNA internal transcribed spacers ITS-1 and ITS-2 were determined in diploid wheat Triticum urartu. It was shown that 5.8S rRNA gene of this wheat species consists of 163 base pairs and GC-content is 59.5%. When comparing 5.8S rRNA sequences in diploid wheat, rice and lupine and also 5.8S rRNA in hexaploid wheat and horse beans a high evolutional conservatism of its structure was revealed. The size of ITS-1 and ITS-2 in Tr. urartu is 219 and 225 base pairs long correspondingly. While comparing structures of similar rDNA regions of Tr. urartu, rice and maize a high level of homology was found only between nucleotides adjoining genes of high molecular rRNAs. In ITS-1 of Tr. urartu an insertion of 5'-GACGACGACATTGTCCGTC-3' was found, which is absent in maize and rice.     

Ciliate evolution: The ribosomal phylogenies of the tetrahymenine ciliates

Summary We have assembled and analyzed nucleotide sequences for several different rRNA components from tetrahymenine ciliates. These include previously published and some new 5S and 5.8S rRNAs for a total of 18 species. We also report sequences for some 30 species obtained by primer extension analysis of a region near the 5′ end of the 23S rRNAs (region 580). Phylogenetic trees have been constructed for these species, utilizing heuristics (shifting ditypic site analysis) described in a companion paper. The trees based on these sequences are consistent with each other and with those based on longer sequences of the 17S rRNA. They show the tetrahymenines to consist of a number of distinctive clusters of species. The clusters (ribosets) are homogeneous with respect to certain life history characteristics, especially the mode of mating type determination, but are inhomogeneous with respect to some morphological and life history features, such as cyst formation and adaptations to parasitism or carnivory. Using the same molecular data, we also begin to explore the relationships of the tetrahymenines to some other ciliate taxa and to some other protists.     

Location of 2(')-O-methyl nucleotides in 26S rRNA and methylation guide snoRNAs in Caenorhabditis elegans

Many nucleotides in rRNAs are modified. We devised a method to locate 2(')-O-methyl nucleotide residues using a conventional DNA sequencer. We found 38 2(')-O-methyl nucleotides in the 26S rRNA of Caenorhabditis elegans using this method. Fourteen of the 38 residues are conserved in both human and yeast rRNAs and 14 residues are conserved in either human or yeast rRNA. The remaining 10 nucleotides are uniquely methylated in C. elegans 26S rRNA. We searched the C. elegans genomic sequence for small nucleolar RNAs (snoRNAs), which guide the methylation of ribose residues, and predicted 18 snoRNA sequences that are expected to guide the methylation of some of these nucleotide residues.     

Construction of genomic phage libraries of the arbuscular mycorrhizal fungi Glomus mosseae and Scutellospora castanea and isolation of ribosomal RNA genes

 Genomic phage libraries of arbuscular mycorrhizal fungi were constructed for the first time, and clones containing ribosomal RNA (rRNA) genes isolated for Glomus mosseae and Scutellospora castanea. The number of rDNA clones per library indicates that these libraries can be also used to isolate genes with low copy numbers. Sequences of the 18S rRNA gene, of the internal transcribed spacer and of the 5.8S rRNA gene were analysed and compared. Differences between the 18S and the 5.8S rRNA genes were few and in the range of variation found for other fungi. In contrast, the internal transcribed spacers of G. mosseae and S. castanea were highly variable, showing the potential of this region for the identification of different species or isolates. Interestingly, nucleotide exchanges were found in this region when the sequence for G. mosseae was compared to those of two other clones of the same isolate. Accepted: 10 November 1995     

Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp: Nucleotide sequence analysis and evolution of the 5.8 S rRNA gene region and its flanking nucleotides

A detailed restriction endonuclease map was prepared for the cloned 5.8 S ribosomal RNA (rRNA) gene region of the brine shrimp Artemia. The nucleotide sequence of the 5.8 S rRNA gene and its flanking nucleotides was determined. This sequence differs in two positions from that of the previously reported 5.8 S rRNA. The primary structure of the Artemia 5.8 S rRNA gene, which, unlike in dipteran insects, is shown to contain no insertion sequence, is conserved according to the relatedness of the species compared. The 5.8 S rRNA gene flanking nucleotides, which were sequenced 176 nucleotide pairs upstream and 70 nucleotide pairs downstream from the gene, show no evidence of sequence conservation between evolutionarily diverse species by computer analysis. Direct nucleotide repeats are present within the flanking sequences at both ends of the gene at about the same distance upstream and downstream, which could serve as processing signals.     

Changes in the content of modified nucleotides of total transfer RNA of wheat seedlings during greening

The contents in minor nucleotides of total transfer RNA (tRNA) of etiolated and light-grown wheat (Triticum aestivum L.) seedlings and of seedlings illuminated for 24 or 48 h were examined. The total tRNA of seedlings illuminated 24 h contained more, and that from seedlings illuminated 48 h still more modified nucleotides than that from etiolated ones. Thus, the appearance of the characteristic minor nucleotides of tRNA of light-grown wheat seedlings needs a rather long greening period, of at least 48 h.     

A Conserved Motif in the 5.8S Ribosomal RNA (rRNA) Gene is a Useful Diagnostic Marker for Plant Internal Transcribed Spacer (ITS) Sequences

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has become an important nuclear locus for molecular systematic investigations of angiosperms at the intergenic and interspecific levels. Universal PCR primers are positioned on the conserved rRNA genes (18S, 5.8S, 26S) to amplify the entire ITS spacer region. Recent reports of fungal and algal contaminants, first described as plant ITS sequences, stress the need for diagnostic markers specific for the angiosperm ITS region. This report describes a conserved 14 base pair (bp) motif in the 5.8S rRNA gene that can be used to differentiate between flowering plants, bryophytes, and several orders of algae and fungi, including common plant pathogenic and non-pathogenic fungi. A variant of the motif (found in fungi and algae) contains a convenient EcoRI restriction site that has several applications for eliminating problematic contaminants from plant ITS preparations.     

Sequence and secondary structure of Drosophila melanogaster 5.8S and 2S rRNAs and of the processing site between them.

Drosophila melanogaster 5.8S and 2S rRNAs were end-labeled with 32p at either the 5' or 3' end and were sequenced. 5.8S rRNA is 123 nucleotides long and homologous to the 5' part of sequenced 5.8S molecules from other species. 2S rRNA is 30 nucleotides long and homologous to the 3' part of other 5.8S molecules. The 3' end of the 5.8S molecule is able to base-pair with the 5' end of the 2S rRNA to generate a helical region equivalent in position to the "GC-rich hairpin" found in all previously sequenced 5.8S molecules. Probing the structure of the labeled Drosophila 5.8S molecule with S1 nuclease in solution verifies its similarity to other 5.8S rRNAs. The 2S rRNA is shown to form a stable complex with both 5.8S and 26S rRNAs separately and together. 5.8S rRNA can also form either binary or ternary complexes with 2S and 26S rRNA. It is concluded that the 5.8S rRNA in Drosophila melanogaster is very similar both in sequence and structure to other 5.8 rRNAs but is split into two pieces, the 2S rRNA being the 3' part. 2S anchors the 5.8S and 26S rRNA. The order of the rRNA coding regions in the ribosomal DNA repeating unit is shown to be 18S - 5.8S - 2S - 26S. Direct sequencing of ribosomal DNA shows that the 5.8S and 2S regions are separated by a 28 nucleotide spacer which is A-T rich and is presumably removed by a specific processing event. A secondary structure model is proposed for the 26S-5.8S ternary complex and for the presumptive precursor molecule.     

Unusual ribosomal RNA of the intestinal parasite Giardia lamblia.

The anaerobic protozoan Giardia lamblia is a common intestinal parasite in humans, but is poorly defined at molecular and phylogenetic levels. We report here a structural characterization of the ribosomal RNA (rRNA) and rRNA genes of G. lamblia. Gel electrophoresis under native or non-denaturing conditions identified two high molecular weight rRNA species corresponding to the 16-18S and 23-28S rRNAs. Surprisingly, both species (1300 and 2300 nucleotides long, respectively) were considerably shorter than their counterparts from other protozoa (typically 1800 and 3400 nucleotides), and from bacteria as well (typically 1540 and 2900 nucleotides long). Denaturing polyacrylamide gel electrophoresis identified a major low molecular RNA of 127 nucleotides and several minor species, but no molecules with the typical lengths of 5.8S (160 nucleotides) and 5S (120 nucleotides) rRNA. The G. lamblia 1300, 2300, and 127 nucleotide RNAs are encoded within a 5.6 kilobase pair tandemly repeated DNA, as shown by Southern blot analysis and DNA cloning. Thus, the rRNA operon of this eukaryotic organism can be no longer than a typical bacterial operon. Sequence analysis identified the 127 nucleotide RNA as homologous to 5.8S RNA, but comparisons to archaebacterial rRNA suggest that Giardia derived from an early branch in eukaryotic evolution.     

5S and 5.8S ribosomal RNA evolution in the suborder tetrahymenina (Ciliophora: Hymenostomatida)

Summary The nucleotide sequences of the 5S and 5.8S rRNAs of eight strains of tetrahymenine ciliates have been determined. The sequences indicate a clear distinction betweenTetrahymena paravorax and its suggested conspecificT. vorax, but leave the taxonomic distinction betweenT. vorax andT. leucophrys in doubt. The rRNA sequences of sixTetrahymena species and of three other species of the suborder Tetrahymenina have been used to deduce evolutionary schemes in which ancestral rRNA sequences and changes are proposed. These schemes suggest the predominant acceptance of GA and CT transitions in the 5S rDNA during the evolution of the suborder.     

3'-Terminal sequence of wheat mitochondrial 18S ribosomal RNA: further evidence of a eubacterial evolutionary origin.

We have determined the sequences of the 3'-terminal approximately 100 nucleotides of [5' -32P]pCp-labeled wheat mitochondrial, wheat cytosol, and E. coli small sub-unit rRNAs. Sequence comparison demonstrates that within this region, there is a substantially greater degree of homology between wheat mitochondrial 18S and E. coli 16S rRNAs than between either of these and wheat cytosol 18S rRNA. Moreover, at a position occupied by 3-methyluridine in E. coli 16S rRNA, the same (or a very similar) modified nucleoside is present in wheat mitochondrial 18S rRNA but not in wheat cytosol 18S rRNA. Further, E. coli 16S and 23S rRNAs hybridize extensively to wheat mitochondrial 18S and 26S rRNA genes, respectively, but wheat cytosol 18S and 26S rRNAs do not. No other mitochondrial system studies to date has provided comparable evidence that a mitochondrial rRNA is more closely related to its eubacterial homolog than is its counterpart in the cytoplasmic compartment of the same cell. The results reported here provide additional support for the view that plant mitochondria are of endosymbiotic, specifically eubacterial, origin.