Transcriptional organization of the 5.8S ribosomal RNA cistron in Xenopus laevis ribosomal DNA.

Hybridization of purified, 32p-labeled 5.8S ribosomal RNA from Xenopus laevis to fragments generated from X. laevis rDNA by the restriction endonuclease, EcoRI, demonstrates that the 5.8S rRNA cistron lies within the transcribed region that links the 18S and 28S rRNA cistrons.     

Effect of point mutations on 5.8S ribosomal ribonucleic acid secondary structure and the 5.8S--28S ribosomal ribonucleic acid junction

Naturally occurring differences in the nucleotide sequences of 5.8S ribosomal ribonucleic acids (rRNAs) from a variety of organisms have been used to study the role of specific nucleotides in the secondary structure and intermolecular interactions of this RNA. Significant differences in the electrophoretic mobilities of free 5.8S RNAs and the thermal stabilities of 5.8S--28S rRNA complexes were observed even in such closely related sequences as those of man, rat, turtle, and chicken. A single base transition from a guanylic acid residue in position 2 in mammalian 5.8S rRNA to an adenylic acid residue in turtle and chicken 5.8S rRNA results both in a more open molecular conformation and in a 5.8S--28S rRNA junction which is 3.5 degrees C more stable to thermal denaturation. Other changes such as the deletion of single nucleotides from either the 5' or the 3' terminals have no detectable effect on these features. The results support secondary structure models for free 5.8S rRNA in which the termini interact to various degrees and 5.8S--28S rRNA junctions in which both termini of the 5.8S molecule interact with the cognate high molecular weight RNA component.     

High evolutionary divergence of the 5.8S ribosomal DNA in Mimulus glaucescens (Scrophulariaceae)

Ribosomal DNA sequences for the ITS 1, 5.8S, ITS 2 and adjoining regions of the 18S and 25S were obtained from Mimulus glaucescens (Scrophulariaceae) via cloned PCR products. The spacer sequences were completely unrelated to other plant taxa, although spacer lengths were approximately the same. Interestingly, the Mimulus 5.8S sequence was much more divergent than other higher-plant rDNA sequences. Consideration of the secondary structure of the 5.8S rRNA shows that most of the changes in Mimulus are compensatory and preserve the basic secondary structure of the mature RNA molecule.     

Chemical modification studies and the secondary structure of HeLa cell 5.8S rRNA.

Various secondary structure models have been proposed for 5.8 S rRNA. In this paper HeLa cell 5.8 S rRNA is shown to possess several sites that are reactive to carbodiimide at 25 degrees, and other regions that are unreactive. Previous work has established the distribution of reactive and unreactive cytidine residues along the primary structure (11). The secondary structure model of Nazar et al. (7) is fully compatible with the chemical reactivity data whereas other models are partly incompatible. We conclude that the model of Nazar et al. provides the best approximation so far available to the conformation of isolated 5.8 S rRNA. Findings on the effect temperature on the chemical reactivity of different parts of the structure are summarized. The findings described in this paper should provide a basis for examining the specific interaction of 5.8 S rRNA with 28 s rRNA.     

Higher-order structure of the 5.8 S rRNA sequence within the yeast 35 S precursor ribosomal RNA synthesized in vitro

Dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene-sulfonate, RNase T1 and RNase V1 have been used as structure-sensitive probes to examine the higher-order structure of the 5.8 S rRNA sequence within the yeast 35 S precursor ribosomal RNA molecule. Data produced have been used to evaluate several theoretical structure models for the 5.8 S rRNA sequence within the precursor rRNA. These models are generated by minimum free energy calculations. A model is proposed that accommodates 83% of the residues experimentally shown to be in either base-paired or single-stranded structure in the correct configuration. Several alternative suboptimal secondary structures have been evaluated. Moreover, the chemical reactivities of several residues within the 5.8 S rRNA sequence in the precursor rRNA molecule differ from those of the corresponding residues in the mature rRNA molecule. This finding provides experimental evidence to support the notion that the 5.8 S rRNA sequence within the precursor rRNA undergoes structural reorganization following rRNA processing.     

Structural organization of ribosomal RNAs from Novikoff hepatoma. II. Characterization of possible binding sites of 5 S rRNA and 5.8 S rRNA to 28 S rRNA

Interrelationships among 5 S, 5.8 S, and 28 S rRNA were probed by methods employed in the accompanying report (Choi, Y. C. (1985) J. Biol. Chem. 260, 12769-12772). Two complexes were isolated from 20 S ribonucleoprotein (RNP) fraction and 60 S subunit. The 20 S RNP fraction was found to contain the 3'-340 nucleotide fragment (domain VII) in association with 5 S rRNA. The 60 S subunit contained a stable complex consisting of the 5'-upstream portion (4220-4462, domain VI and VII), the 3'-downstream portion (4463-4802, domain VII) of 3'-583 nucleotides fragment, and 5.8 S rRNA. By computer analysis and hybridization, the 5'-upstream portion was found to contain the 5.8 S rRNA contact site. By affinity chromatography, the 3'-downstream portion was found to contain the 5 S rRNA association site. Furthermore, by comparison with the secondary structure of 28 S rRNA proposed by Hadjiolov et al. (Hadjiolov, A. A., Georgiev, O. I., Nosikov, V. V., and Yavachev, L. P. (1984) Nucleic Acids Res. 12, 3677-3693), it was found that domain VII is capable of binding 5.8 S rRNA and 5 S rRNA juxtaposed to each other. Accordingly, a model was proposed to indicate that a possible contact site for 5.8 S rRNA is within the region surrounding the alpha-sarcin site (4333-4350) and is a possible association site of 5 S rRNA within the 3'-downstream portion (4463-4802) of the 3'-583 nucleotide fragment (4220-4802).     

Primary and secondary structure of 5.8S rRNA from the silkgland of Bombyx mori.

Nucleotide sequence of 5.8S rRNA of the silkworm, Bombyx mori has been determined by gel sequencing methods. The 5.8S rRNA was the longest so far reported, with the 5'-terminal sequence several nucleotides longer than those of the other organisms. Upon constructing the secondary structure in accordance with the "burp gun" model (12), the Bombyx 5.8S rRNA formed a wide-open "muzzle" due to several unpaired bases at the ends. The overall structure also appeared less stable with less G . C pairs and more unpaired bases than that of the HeLa 5.8S rRNA. These structural features may be essential for those 5.8S rRNAs which interact with 28S rRNAs containing the hidden break to form a stable complex.     

Molecular systematics of dictyostelids: 5.8S ribosomal DNA and internal transcribed spacer region analyses

The variability and adaptability of the amoebae from the class Dictyosteliomycetes greatly complicate their systematics. The nucleotide sequences of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene have been determined for 28 isolates, and their utility to discriminate between different species and genera has been shown.     

Nucleotide sequences of the 5.8S rRNAs of a mollusc and a porifer, and considerations regarding the secondary structure of 5.8S rRNA and its interaction with 28S rRNA

We report the primary structures of the 5.8 S ribosomal RNAs isolated from the sponge Hymeniacidon sanguinea and the snail Arion rufus. We had previously proposed (Ursi et al., Nucl. Acids Res. 10, 3517-3530 (1982)) a secondary structure model on the basis of a comparison of twelve 5.8 S RNA sequences then known, and a matching model for the interaction of 5.8 S RNA with 26 S RNA in yeast. Here we show that the secondary structure model can be extended to the 25 sequences presently available, and that the interaction model can be extended to the binding of 5.8 S RNA to the 5'-terminal domain of 28 S (26 S) RNA in three species.     

Sequence and secondary structure of mouse 28S rRNA 5''terminal domain. Organisation of the 5.8S-28S rRNA complex.

We present the sequence of the 5' terminal 585 nucleotides of mouse 28S rRNA as inferred from the DNA sequence of a cloned gene fragment. The comparison of mouse 28S rRNA sequence with its yeast homolog, the only known complete sequence of eukaryotic nucleus-encoded large rRNA (see ref. 1, 2) reveals the strong conservation of two large stretches which are interspersed with completely divergent sequences. These two blocks of homology span the two segments which have been recently proposed to participate directly in the 5.8S-large rRNA complex in yeast (see ref. 1) through base-pairing with both termini of 5.8S rRNA. The validity of the proposed structural model for 5.8S-28S rRNA complex in eukaryotes is strongly supported by comparative analysis of mouse and yeast sequences: despite a number of mutations in 28S and 5.8S rRNA sequences in interacting regions, the secondary structure that can be proposed for mouse complex is perfectly identical with yeast's, with all the 41 base-pairings between the two molecules maintained through 11 pairs of compensatory base changes. The other regions of the mouse 28S rRNA 5'terminal domain, which have extensively diverged in primary sequence, can nevertheless be folded in a secondary structure pattern highly reminiscent of their yeast' homolog. A minor revision is proposed for mouse 5.8S rRNA sequence.     

The sequence of the 5.8 S ribosomal RNA of the crustacean Artemia salina. With a proposal for a general secondary structure model for 5.8 S ribosomal RNA.

We report the primary structure of 5.8 S rRNA from the crustacean Artemia salina. The preparation shows length heterogeneity at the 5'-terminus, but consists of uninterrupted RNA chains, in contrast to some insect 5.8 S rRNAs, which consist of two chains of unequal length separated in the gene by a short spacer. The sequence was aligned with those of 11 other 5.8 S rRNAs and a general secondary structure model derived. It has four helical regions in common with the model of Nazar et al. (J. Biol. Chem. 250, 8591-8597 (1975)), but for a fifth helix a different base pairing scheme was found preferable, and the terminal sequences are presumed to bind to 28 S rRNA instead of binding to each other. In the case of yeast, where both the 5.8 S and 26 S rRNA sequences are known, the existence of five helices in 5.8 S rRNA is shown to be compatible with a 5.8 S - 26 S rRNA interaction model.     

Primary and secondary structures of Tetrahymena and aphid 5.8S rRNAs: structural features of 5.8S rRNA which interacts with the 28S rRNA containing the hidden break

The Tetrahymena 5.8S rRNA is 154 nucleotides long, the shortest so far reported except for the split 5.8S rRNAs of Diptera (m5.8S plus 2S rRNA). In this molecule several nucleotides are deleted in the helix e (GC-rich stem) region. Upon constructing the secondary structure in accordance with "burp-gun" model, the Tetrahymena 5.8S rRNA forms a wide-open "muzzle" of the terminal regions due to both extra nucleotides and several unpaired bases. The aphid 5.8S rRNA consists of 161 nucleotides and can form stable helices in both terminal and helix e regions. As a whole, the secondary structure of Tetrahymena 5.8S rRNA resembles that of Bombyx 5.8S molecule while the aphid 5.8S rRNA shares several structural features with the HeLa 5.8S molecule. Likely, the 5.8S rRNA attached to the 28S rRNA with the hidden break differs in structure from those interacting with the 28S partners without the break. Nucleotide sequences of 5.8S rRNA in insects as well as in protozoans are not so conservative evolutionarily as in vertebrates.     

Telomeric DNA localization on dinoflagellate chromosomes: structural and evolutionary implications

Dinoflagellates are eukaryotic microalgae with distinct chromosomes throughout the cell cycle which lack histones and nucleosomes. The molecular organization of these chromosomes is still poorly understood. We have analysed the presence of telomeres in two evolutionarily distant and heterogeneous dinoflagellate species (Prorocentrum micans and Amphidinium carterae) by FISH with a probe containing the Arabidopsis consensus telomeric sequence. Telomere structures were identified at the chromosome ends of both species during interphase and mitosis and were frequently associated with the nuclear envelope. These results identify for the first time telomere structures in dinoflagellate chromosomes, which are formed in the absence of histones. The presence of telomeres supports the linear nature of dinoflagellate chromosomes.     

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%.     

Altered features in the secondary structure of Vicia faba 5.8s rRNA.

We have re-examined the nucleotide sequence of Vicia faba (broad bean) 5.8S rRNA using partial chemical degradation and a new approach to high temperature (65-80 degrees C) sequencing gels. The results indicate that the secondary structure was not completely disrupted in previous studies (Tanaka, Y., Dyer, T.A. and Brownlee, G.G. (1980) Nucleic Acid Res. 8, 1259-1272) and explain ambiguities between the nucleotide sequence and T1 ribonuclease digests. Despite this revision, estimates in the secondary structure suggest that this 5.8S rRNA differs from previously examined examples in two respects, more open conformations in both the "GC-rich" and "AU-rich" stems. The secondary structure was probed under a variety of ionic conditions using limited pancreatic and T1 ribonuclease digestion and rapid gel sequencing techniques. These studies and theoretical considerations generally supported the "burp gun" model previously proposed for all 5.8S rRNAs and were inconsistent with the recently suggested "cloverleaf" configuration. More importantly, they were also consistent with more open stem structures in this higher plant.     

Effect of 2''-O-methylation on the structure of mammalian 5.8S rRNAs and the 5.8S-28S rRNA junction

The mammalian 5.8S rRNA contains a partially 2'-O-methylated uridylic acid residue at position 14 which is largely or entirely methylated in the cytoplasm (Nazar, R.N., Sitz, T.O. and Sommers, K.D. (1980) J. Mol. Biol. 142, 117-121). The effect of this methylation on the 5.8S RNA structure and 5.8-28S rRNA junction was investigated using both chemical and physical approaches. Electrophoretic studies indicated that the free 5.8S rRNA can take on at least two different conformations and that the 2'-O-methylation at U14 restricts the molecule to the more hydrodynamically open form. Structural studies using limited pancreatic or T1 ribonuclease digestion indicated that the methylated conformation was more susceptible to digestion, consistent with a more open tertiary structure. Modification-exclusion studies indicated that the first 29 nucleotides at the 5' end and residues 140 through 158 at the 3' end affect the 5.8S-28S rRNA interaction, supporting previous suggestions that the 5.8S RNA interacts with its cognate high molecular weight component through its termini. These results also suggested that the 2'-O-methylated uridylic acid residue plays a role in the 5.8S-28S rRNA interaction and thermal denaturation studies confirmed this by showing that methylation destabilizes the 5.8S-28S rRNA junction. The 5.8-28S rRNA interaction appears to be more complex than previously believed.     

Binding of proteins from the large ribosomal subunits to 5.8 S rRNA of Saccharomyces cerevisiae

Specific binding of purified proteins from the large ribosomal subunits of Saccharomyces cerevisiae to 5.8 S rRNA was examined by three different methods: nitrocellulose membrane filtration, sucrose density gradient centrifugation, and RNA-Sepharose column chromatography. RNA-protein complex formation was proportional to the amount of proteins added to the reaction mixture. The binding of proteins to the RNA could be saturated. Such RNA-protein complexes were isolated on sucrose density gradients. Protein species present in these complexes were isolated, iodinated, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Eleven proteins, L13, L14, L17, L19, L21, L24, L25, L29, L30, L33, and L39, were identified. By comparison, only six proteins interacted with the 5.8 S rRNA-Sepharose under similar ionic conditions. They were proteins L14, L21, L24, L27, L29, and L30. To better characterize these binding proteins, the interaction of individual proteins with 5.8 S rRNA was studied by nitrocellulose membrane filtration. Proteins L14, L19, L21, L29, L33, and L39 were observed to bind individually with 5.8 S rRNA. Binding of each protein to the RNA could be saturated. The apparent association constants (K'a), measured at 4 degrees C and in 30 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 330 mM KCl, and 6 mM beta-mercaptoethanol, ranged from 1.05 to 3.70 X 10(6) M-1.